Journal of Agronomy and Agricultural Aspects (ISSN: 2574-2914)

research article

Effect of Ozone Exposure on Microbial Flora and Quality Attributes of Papaya (Carica Papaya L) Fruit

Ong Mei Kying1,2*, Asgar Ali3

1Department of Agricultural and Food Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak D.R, Malaysia

2Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak D.R, Malaysia

3Centre of Excellence for Postharvest Biotechnology (CEPB), School of Biosciences, The University of Nottingham Malaysia Campus,Jalan Broga, 43500 Semenyih,  Selangor D.E, Malaysia

*Corresponding author: Ong Mei Kying, Department of Agricultural and Food Science, Faculty of Science, Universiti- Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak DR, Malaysia. Tel: +605-4688888; ext. 4701; Fax: +605-4661313; Email: ongmk@utar.edu.my

Received Date: 23 November, 2016; Accepted Date: 10 December, 2016; Published Date: 17 December, 2016

Ozone, or triatomic oxygen, is a powerful disinfectant and oxidizing agent.  Despite the fact that the application of ozone for the disinfection of fresh fruits and vegetables has been studied widely, comparatively little information is presented on the potential of ozone to reduce microbial populations in papaya fruit.  In this study, ozone was applied in gaseous form to papaya fruit at five concentrations (0.05, 0.5, 2.0, 3.5 and 5.8 ppm) for five different periods (0.5, 1, 3, 5 and 24 h) and the reduction in the total bacterial count (coliforms, yeasts and moulds) was assessed.  The effect of ozone on weight loss, firmness, fruit peel colour, soluble solids concentration and titratable acidity were also determined. Total mesophilic bacteria on papaya fruit were reduced by 83.3 to 99.7% after exposure to 0.05 to 5.8 ppm ozone for 24 h. Ozone treatments did not affect peel colour and titratable acidity, but significantly reduced weight loss, retained firmness and increased soluble solids concentration during ambient storage. In summary, ozone treatments can be useful for maintaining quality attributes and reducing microbial flora in papaya fruit.

Keywords: Weight Loss; Ambient Storage; Fresh Fruits; Disinfecting Agent; Food Safety

In fresh produce industry, the number of illnesses associated with fresh produce is a safety challenges.  Fresh produces is often consumed raw or with only minimal processing. Microorganisms, especially bacteria, occur normally on fresh fruit and vegetable tissues.  They include mesophilic bacteria, lactic acid bacteria, coliforms, yeasts and moulds (Nguyen-The and Carlin, 1994).  Estimated numbers of microbial counts reported on fresh fruits and vegetables are usually within the range 101-109 cfug-1, depending upon the fruit or vegetable. The native microbial flora of fruits is mostly composed of moulds and yeasts.  Fungi, such as Botrytis cinereaand Aspergillusniger, and yeasts, such as species of CandidaCryptococcusFabosporaSaccharomycesand Zygosaccharomyces,are present in most fresh fruits (Chen, 2002).

Microbial spoilage appears to be one of the major reasons for quality loss in fresh fruits and vegetables by development of off-flavours, fermented aromas and tissue decay. The basic principle of shelf-life prediction involves the quantification of populations of microbes present on the food product (Zhuanget al., 2003).  In the search for useful disinfectant treatments for fresh vegetables and fruits, decontamination methods include physical (Ukukuet al., 2006; Ozkanet al., 2011; Tzortzakiset al., 2008), chemical (Wang et al., 2007; Maqboolet al., 2011) and biological processes (Rouse et al., 2008; Spadaroet al., 2008).  The most versatile treatment is processing with ionizing radiation.

It is a common sanitizing practice of applying cheap and effective synthetic fungicides on papaya, used in combination of pre-pack sanitation treatments employing chlorine- (or bromine-) based disinfectants (Eckert and Ogawa, 1988).  Although chemical sanitizer has been used for years, there is much controversial because the residues of it may be harmful to consumers (Hewajuligeet al., 2009).  Therefore, it is necessary to find a novel way to improve food safety by reducing or eliminating food-related microorganisms during the shelf life of food products without leaving any toxic residues.  Sanitation of food with ionizing radiation, such as ozone, is safe, efficient, environmentally clean and energy-efficient.   Recently, there has been a high level of interest in postharvest applications of ozone for decay control and as a potential sanitizer against human pathogens because ozone has been affirmed the status of Generally Recognized As Safe (GRAS) as a food processing aid and as compliant with EPA Disinfection by Products Rule (US FDA, 1997).

The application of ozone at the postharvest stage has been studied by many researchers, especially for the prevention of fungal decay (Palou et al.,2002; Perez et al., 1999; Onget al., 2012), inactivation of bacteria (Achen and Yousef, 2001; Kim and Yousef, 2000; Sharma et al., 2002; Xu, 1999), destruction of pesticides and chemical residues (Onget al., 1996; Hwang et al., 2001) and the control of storage pests (Kellset al., 2001; Mendez et al.,2002).

The study of the effect of ozone on papaya fruit is of great importance to determine the potential of ozone as a disinfecting agent and, most importantly, to enhance food safety by not affecting the sensory or nutrient quality.  The aim of this work was to evaluate the efficacy of ozone in gaseous form on microbial flora disinfection and quality of papaya fruits.

 Materials and methods

Plant material

Mature green papaya cv ‘Sekaki’ of colour index 2 (green with trace of yellow) were obtained from a local fruit wholesaler at PasarBorong Selangor, Malaysia on the day of their harvesting.  Fruit of uniform size (800-1200g), shape, maturity and free from any indication of mechanical injury, insect or pathogenic infection were selected for the experiment.   They were washed with distilled water and air-dried at ambient temperature (25-28°C) before exposure to ozone.

Ozone fumigation

The fumigation system, comprised of chambers constructed from 5 mm thick polycarbonate (112.0 cm length x 47.0 cm width x 43.0 cm height) equipped with four 12V fans positioned directly below the sample platform to ensure a well-mixed atmosphere, was set up in the postharvest laboratory, School of Biosciences, University of Nottingham Malaysia Campus.  Ozone was introduced to each chamber by an ozone generator (Model MedKlinn Professional Series, MedKlinn International Sdn. Bhd., Malaysia) and the ozone concentration was controlled manually using a sensor (Eco-Sensor, Model OEM-2, MedKlinn International Sdn. Bhd., Malaysia). The ozone concentration in each chamber was recorded via an ozone analyzer (Model IN-2000 L2-LC, IN USA Incorporated, USA). The chambers were maintained at 25 ± 3°C and 70 ± 5 % relative humidity (RH), monitored by temperature/humidity sensors (Model U14-001, HOBO LCD Data Logger, Onset Computer Corporation, USA).

Microbial evaluation

Fruit were exposed to ozone enriched atmospheres of 0.05, 0.5, 2.0, 3.5 or 5.8 ppm for durations of 0, 0.5, 1, 3, 5 and 24 h.The experiments were conducted in a completely randomized design and a sample of 48 fruits in each treatment was used.  Four fruits from each treatment were used for the microbiological analysis.

Standard plate counts

Microbiological analysis of papaya samples was carried out aseptically according to APHA (1992) procedures by mixing 25 g of papaya fruit skin with 225 ml sterile Ringer solution in a sterile stomacher bag.  The samples were massaged by hand for 1 min.  The pour plate method was used for total mesophilic bacteria count (Plate count agar, Merck) at 35°C for 24 h.  Microbial counts were expressed as log cfug-1.

Yeast and mould counts

The surface spread method was used for yeast andmould enumeration (Yeast Extract Glucose Chloramphenicol Agar, YGC, Merck) at 25 °C for 3 to 5 days.  Microbial counts were expressed as log cfug-1.

Total coliform counts

The surface spread method was used for coliform bacteria (Mcconkey agar, Merck) at 37 °C for 48 hours.  Microbial counts were expressed as log cfug-1.

Quality evaluation

All treatments with 24 h exposure were kept for up to 14 days of ambient storage (25±3 °C, 70±5 %RH) during which the quality attributes of the fruit were evaluated.  All data were recorded at 2-day intervals. In each treatment, eight fruits were randomly selected for physicochemical analysis.

Physical quality parameters

Fruit in each replication were marked and weighed (using a digital balance, EK-600H, Japan) before storage for weight loss determination.  The fruits were then weighed at the 2-day intervals over the storage period.  The results were expressed as percentage of initial weight loss.

Fruit firmness was analysed using anInstron Universal Testing Machine (Model 5540, USA). Fruit in each replication were compressed using the 50 mm diameter probe, at a speed of 50 mm min-1.  The compression force measured at the maximum peak of the recorded force was expressed in Newtons (N).

Fruit peel colour was determined using a Minolta CR-300 Chroma Meter (Minolta Corp., Japan).  The colour determination made on papaya peel was expressed in chromaticity values of L*, C* and hº.  The values of lightness (L*) form the vertical axis with values ranging from 0 = black to 100 = white.Values for a* (red-green axis) and b*(yellow-blue axis) represent coordinates in the colour chart indirectly reflecting chroma (C*) and hue angle (hº).  The C* value which refers to the vividness of colour was computed from a* and b*, i.e. C* = (a*2 + b*2½, which represents the hypotenuse of a right angled triangle with values ranging from 0 = least intense, to 60 = most intense.  The hº value was referredto as colour, and was the angle of tangent-1 b*/a*, where 0º = red purple, 90º = yellow, 180º= bluish-green and 270º = blue.

Chemical quality parameters

During storage, the soluble solids concentration (SSC) and titratable acidity of the fruit pulp were determined.  Fruit samples from each replication (10 g) were homogenized using a kitchen blender with 40 ml of distilled water, and filtered through muslin cloth. The fruit pulp was sampled from the equatorial region of the whole piece of fruit.  A drop of the filtrate was then used to determine the SSC (°Brix) using a Palette Digital Refractometer (Model: PR-32α) from Atago Co, Ltd (Japan) calibrated with distilled water prior to taking readings.  The readings were multiplied by the dilution factor to obtain the original SSC (%) of the papaya pulp.

The titratable acidity (TA) was analysed using the titration method described by Ranganna (1999).  Ten grams of pulp tissue was homogenized with 40 ml of distilled water using a kitchen blender for two minutes.  The mixture was then filtered through muslin cloth.  Five ml of filtrate with one to two drops of 0.1% phenolphthalein as indicator was titrated against 0.1N sodium hydroxide (NaOH) to endpoint pink (10 seconds). Titratable acidity was expressed as percentage citric acid equivalents.

Statistical analysis

Treatments were arranged in a completely randomized design.  There were four replications for each ozone treatment.  A replicate of 12 fruits were placed in a single layer of the chamber. Each chamber was prepared as a replicate. Four fruits were withdrawn at different times to give the ‘duration’ treatment.  Experimental data were analyzed using analysis of variance (ANOVA) via SAS 9.1 software and means were separated using Duncan’s Mutiple Range Test (DMRT) at (p<0.05).  The entire experiment was repeated twice. The results presented in this study were the means of these experiments.

Results and discussion

Standard plate count

Treatment of fruit with ozone at 0.5, 2.0, 3.5 and 5.8 ppm for 24 h reduced the total mesophilic microorganism counts with initial values of 4.48 to 2.18 log cfug-1 (Table 1) (p<0.05 for both the concentration of ozone and the exposure time). These bacteria are commonly found on crop surfaces.  Many microbes are capable of colonizing the produce by producing pectin-degrading enzymes which enhance tissue softening and breakdown (Watadaet al., 1996).  Microbial load in fresh-cut produce is greater than that in intact produce because tissue damage caused by cutting breaks cells and promotes the release of nutrients used by microflora (Toivonen and DeEll, 2002). Ozone is an effective antimicrobial gas due to its oxidizing capacity to kill spoilage and human pathogenic bacteria, yeasts and moulds  (Guzel-Seydimet al., 2004).  Ozone destroys microorganisms by the progressive oxidation of vital cell components (Beuchat, 1992), preventing the microbial growth and extending the shelf-life of fruit.

Yeast and mould counts

The initial yeast and mould count was 3.48 log cfug-1.  It decreased significantly as ozone concentration increased.  One hour of ozone at 0.05, 0.5, 2.0, 3.5 and 5.8 ppm reduced the yeast and mould count to 3.16, 2.93, 2.98, 2.65 and 1.7 log cfug-1, respectively (Table 1).  A decrease in yeast and mould count with longer ozone treatment on dried figs has been reported by Oztekinet al. (2006).

The moulds commonly associated with the spoilage of fruits and vegetables include Botrytis cinereaand Aspergillusniger.  They produce a cocktail of enzymes such as hydrolases that are able to break down cell walls of produce, causing spoilage (Walton, 1994). The anthracnose symptom which commonly occurs in papaya is caused by the fungus Colletotrichumgloeosporioides.  The effectiveness of ozone treatment on fungus C. gloeosporioideshas been reported by Onget al. (2012).  The major factor determining spore resistance to biocidal agents appears to be the spore coat and ozone treatment may cause damage to the surface layer as well as to the outer and inner coats of fungus spores.  Such damage facilitates the action of ozone on the cortex, and finally causes spore inactivation through intracellular damage (Kim et al., 2003; Young and Setlow, 2004).

To reduce yeast and mould activity, ozone should be applied either for long periods at low concentration, or conversely for short periodsat higher concentrations (Najafi andKhodaparast, 2008). At 5.8 ppm, no yeasts and moulds were found after 1 h of exposure.  Likewise, no yeasts and moulds were seen in fruit samples exposed for 3 h to 2.0, 3.5 and 5.8 ppm.  Some yeasts are very effective at fermenting single sugars to produce alcohol and other volatiles that affect fruit quality (Barnett et al.,2000).The destruction of yeasts and moulds just after harvesting reduces the possibility of aflatoxin formation before the next processing steps (Ahmed and Ahmed, 1997; Ahmed and Robinson, 1999), which is a better preventive strategy than a subsequent complicated detoxification process.

Total coliform counts

The initial mean value of coliform bacteria was 4.13 log cfug-1 (Table 1).  After 5 h of ozone at 0.05, 0.5, 2.0, 3.5 and 5.8 ppm, coliform counts were reduced to 2.98, 2.65, 2.18, 2.93 and 1.7 log cfug-1, respectively. In addition,  there was no increase in coliform bacteria after 24 h at all concentrations of ozone.  Similarly, Achen and Yousef (2001) reported that the use of ozone-containing water for washing apples decreased the counts of E. coliO157:H7.

Physical quality parameters

Apart from food safety, it is necessary to investigate the quality of fruit is not negatively affected after ozone application.   There were significant differences in weight loss between treated and untreated fruits (Table 2). The percentage of weight loss was smaller in all the treated fruits (24.10-25.32 %) compared with the untreated controls (25.47 %) at day 14.  In this study, it showed that treated fruit maintained the full typical colour and reduced weight loss.  Rodoniet al. (2010) also found reduced weight loss for ozone-exposed tomatoes after 9 days storage at 20 °C. Papaya fruit treated with ozone were also significantly firmer than the untreated fruit (Table 2).  Fruit treated with 2 ppm ozone were the most firm (16.43 N) at day 14, when compared to other treated fruit (10.87- 14.32 N).  This result was in agreement with the findings of Aguayoet al. (2006) who reported that ozone treatments reduced softness in whole tomato fruit.  Other authors have also described that ozone exposure resulted in better firmness retention (Tzortzakiset al., 2007; Rodoniet al., 2010).  However, the visual quality of the fruit in terms of values of lightness (L*), hue angle (h°) and chroma (C*) was maintained after ozone treatments over the storage period and were not significantly different from untreated samples (Table 2).

Chemical quality parameters

The soluble solids concentration (SSC) of ‘Sekaki’ papaya fruits increased over the storage period.  However, a reduction in SSC was observed after 8 days and 10 days of storage, for untreated fruit and treated fruit respectively.  Highest SSC was obtained from fruits treated with 2.0 ppm ozone (11.02 %) at 10 days of storage (Figure 1).

Therefore, storage period and ozone concentration influenced the SSC changes in papaya fruit, which was consistent with the reported SSC increase in strawberry (Kuteet al., 1995) and papaya (Ali et al., 2014) fruits in response to ozone exposure.  Titratable acidity (TA) of treated and untreated papaya fruit showed no difference, ranging from 0.50 to 1.57 %, and neither storage period nor ozone concentration had a significant effect on this parameter (Table 3).  Similarly, no significant effect of ozone on TA was reported by Garcia et al. (1998) for navel oranges and Perez et al.(1999) for the storage of strawberry.

Conclusions

Treatmentof papaya fruit with gaseous ozone reduced significantly the total count of mesophilic microorganisms, coliform bacteria, yeasts and moulds. Significant differences between exposure times were found for all treatments. It can be concluded that a minimum of 5 h of ozone treatment at 5.8 ppm could be successfully used for eliminating the coliforms, total mesophilic bacteria, yeasts and moulds.  However, longer exposure times (up to 24 h)were required to reduce total counts of mesophilic bacteria, coliform bacteria, yeasts and moulds for all the ozone concentrations tested.  Fumigation by ozone reduced the risk of microbial spoilage and maintained acceptable physical and chemical qualities of the fruit during 14 days of ambient storage.

Acknowledgements

We are grateful to MedKlinn International Sdn. Bhd. for providing financial and technical support for our research.

 

Figure 1: Effect of ozone concentration on soluble solids concentration of ‘Sekaki’ papaya after treated for 24 h and later stored up to 14 days at ambient storage (25±3ºC and 70±5% RH).  Each value is the mean of four replicates ± SE.

 

  Exposure time (hour)

 

Ozone concentration (ppm)

 

  0.05 0.5 2 3.5

5.8

Total coliforms 0 4.13Aa 4.13Aa 4.13Aa 4.13Aa

4.13Aa

0.5

4.01Bb 3.48Cb 3.33Db 3.02Eb 2.98Fb

1

3.56Bc 3.31Cc 2.54Fc 2.93Dc 2.88Ec

3

2.98Bd 2.81Dd 2.4Fd 2.93Cc 2.54Ed

5

2.98Bd 2.65De 2.18Ee 2.93Cc 1.7Fe

24

1.7Be 1.7Bf 1.7Bf 1.7Bd 1.7Be
 

 

         
Yeasts and moulds

0

3.48Aa 3.48Aa 3.48Aa 3.48Aa 3.48Aa

0.5

3.22Bb 3.16Cb 3.06Db 2.81Eb 2.54Fb

1

3.16Bc 2.93Dc 2.98Cc 2.65Ec 1.7Fc

3

2.88Bd 2.54Cd 1.7Dd 1.7Dd 1.7Dc

5

2.81Be 2.4Ce 1.7Dd 1.7Dd 1.7Dc

24

2.18Bf 1.7Cf 1.7Cd 1.7Cd 1.7Cc
 

 

         
Total mesophilic bacteria

0

4.48Aa 4.48Aa 4.48Aa 4.48Aa 4.48Aa

0.5

4.39Bb 3.94Cb 3.8Db 3.7Eb 3.55Fb

1

4.3Bc 3.75Cc 3.24Fc 3.61Dc 3.48Ec

3

4.0Bd 3.67Cd 3.19Ed 3.27Dd 2.98Fd

5

3.86Be 3.19Ce 3.13De 3.02Ee 2.18Fe

24

3.7Bf 2.18Cf 2.18Cf 2.18Cf 2.18Ce

*Data represent average value of three counts of colonies.

ABCDmeans within row with different letters indicate significant difference (p<0.05) between treatments.

abcdmeans within column with different letters indicate significant difference (p<0.05) between ozone exposure time.

 

Table 1: Effect of exposure time and ozone concentration on microorganism count on ‘Sekaki’ papaya fruit (log cfu/g)

 

Physical quality parameters Storage period (days)

 

 

Ozone concentration (ppm)

 

0

0.05 0.5 2 3.5 5.8
Weight Loss (%)

0

0a 0a 0a 0a 0a 0a

2

1.87a 1.76a 1.02b 0.94b 0.56c 0.61c

4

3.53a 2.22b 1.86b 1.34c 1.29c 1.38c

6

6.32a 5.88b 4.35c 3.82d 4.37c 4.30c

8

9.11a 8.24b 8.83b 6.46d 7.55c 7.19c

10

12.95a 12.11b 10.73c 11.05c 12.15b 11.12c

12

18.16a 17.65b 15.25c 15.37c 18.21a 17.39b

14

25.47a 25.32ab 25.03ab 24.51ab 24.10b 24.72ab
 

 

           
Firmness (N)

0

95.04d 95.08d 96.28c 97.32b 98.07a 96.75c

2

67.74d 73.14c 80.76b 82.33a 82.83a 81.64b

4

24.71e 38.03c 40.54b 31.32d 42.80a 41.87a

6

18.83c 19.41bc 19.34bc 22.30a 20.24b 19.32bc

8

16.44d 16.63d 17.18c 22.11a 19.64b 16.45d

10

13.40d 14.64cd 15.01c 20.12a 18.76b 13.68d

12

11.25c 12.47bc 13.47b 18.36a 18.22a 12.35bc

14

10.87d 11.13cd 12.41c 16.43a 14.32b 11.36cd

 

           
Lightness (L*)

0

43.26a 42.36ab 42.29ab 41.58b 40.76b 44.52a

2

55.57a 54.77a 54.66a 52.86b 52.21b 52.34b

4

58.52a 58.34a 59.12a 57.21b 57.36b 56.54b

6

64.67a 63.34a 63.15a 61.17b 61.28b 61.15b

8

61.78a 61.72a 61.17a 59.23b 58.32b 58.38b

10

56.15b 56.25b 56.21b 61.26a 60.42a 59.19a

12

50.23b 52.12ab 53.61a 53.11a 53.12a 53.42a

14

46.21ab 47.32a 48.53a 45.76ab 47.23a 43.95b

 

           
Hue angle (h°)

0

122.10a 121.24ab 120.33b 123.12a 123.18a 120.23b

2

119.29a 120.25a 118.15b 118.26b 120.53a 118.46b

4

102.64a 102.45a 102.48a 102.11a 103.57a 103.65a

6

96.13b 96.72b 96.71b 98.06a 99.83a 98.33a

8

87.81ab 87.93ab 87.96ab 87.12b 88.54a 88.84a

10

79.34b 79.82b 80.45a 81.21a 81.72a 80.56a

12

73.93b 73.54b 74.42a 74.57a 75.01a 75.41a

14

67.56b 67.43b 67.78b 68.28a 68.98a 69.39a
 

 

           
Chroma (C*)

0

25.21a 26.22a 25.34a 25.43a 25.48a 26.12a

2

33.45a 32.23a 31.12b 31.57b 31.35b 31.36b

4

44.83a 44.67a 44.33a 42.65b 42.87b 42.39b

6

48.47a 47.86a 47.18a 37.77b 36.32b 36.67b

8

52.54a 51.76a 52.46a 48.34b 48.32b 47.45b

10

43.36b 42.71b 43.43b 45.71a 45.73a 44.92a

12

37.82a 38.53a 38.23a 39.54a 39.41a 38.43a

14

28.61a 28.56a 28.38a 29.34a 29.13a 28.33a

Values are means of three experiments with four replicates per experiment.

abcdmeans within row with different letters indicate significant difference (p<0.05) between treatments.

 

Table 2 :  Effect of ozone concentration on physical quality parameters of ‘Sekaki’ papaya after treated for 24 h and later stored up to 14 days at ambient storage (25±3ºC and 70±5% RH).

 

Chemical quality parameters Storage period (days)  

 

Ozone concentration (ppm)

 

0

0.05 0.5 2 3.5 5.8
Titratable acidity (%)

0

1.56a 1.54a 1.54a 1.55a 1.55a 1.56a

2

1.52a 1.51a 1.51a 1.46b 1.48b 1.53a

4

1.36b 1.38b 1.36b 1.40a 1.41a 1.42a

6

1.11b 1.13b 1.18b 1.23a 1.24a 1.25a

8

0.99b 1.01b 1.02b 1.06ab 1.10a 1.11a

10

0.76a 0.76a 0.75a 0.72b 0.73b 0.75a

12

0.67a 0.66a 0.66a 0.63b 0.64b 0.66a

14

0.52a 0.51a 0.51a 0.50a 0.51a 0.52a

Values are means of three experiments with four replicates per experiment.

abcdmeans within row with different letters indicate significant difference (p<0.05) between treatments.

 

Table 3:  Effect of ozone concentration on titratable acidity of ‘Sekaki’ papaya after treated for 24 h and later stored up to 14 days at ambient storage (25±3ºC and 70±5% RH).

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