Use of electrolyzed oxidizing water for the control of microbial contamination of date fruits

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1 International Journal of Microbiology and Immunology Research Vol.2(3), pp , March, 2013 Available online at ISSN Apex Journal International Full Length Research Paper Use of electrolyzed oxidizing water for the control of microbial contamination of date fruits Siddig H. Hamad 1 * and Salah M. Aleid 2 1 College of Agric. and Food Sciences, King Faisal University, P.O. Box 400, Alahsa, 31982, Saudi Arabia, 2 Date Palm Research Center of Excellence, King Faisal University, P.O. Box 400, Alahsa, 31982, Saudi Arabia. Accepted 26 February, 2013 The effect of treating date fruits with electrolyzed oxidizing water for 5, 15 and 30 min on the general microbial contamination and on the added contamination with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Aspergillus fumigates was investigated. Soaking for 15 min was found enough for the reduction of general contamination with mesophilic aerobic bacteria, coliforms, molds and yeasts to levels that meet the requirements of Saudi Standards. The treatment was also found as the most effective for the reduction of added contamination with S. aureus, P. aeruginosa, E. coli and A. fumigates. Synthesis normal microbial contamination of date fruits can be successfully controlled by treatment with electrolyzed oxidizing water. Key words: Date fruits, electrolyzed oxidizing water, microbial contamination, preservation. INTRODUCTION Dates, like any other agricultural produce, are subject to microbial contamination in the field and during handling processes. Studies conducted so far showed that date fruits in the rutab and Tamr stages are contaminated with many types of microorganisms (Abu-Zinada and Ali, 1982; Aidoo et al., 1996; El-Sherbeenyet al., 1985; Nussinovitch et al., 1989; Hamad, 2008). Potential spoilage microorganisms that contaminate date fruits include molds, yeasts and lactic acid bacteria, whereas potential pathogens include bacteria such as Staphylococcus aureus and yeasts such as Candida pelliculosa. Microbial contamination can lead to considerable losses in the crop especially at the rutab stage while contamination of the fruit at the tamr stage limits export chances, especially to the markets of the industrial countries. Treatments designed to control microbial contamination in date fruit processing factories in Saudi Arabia depend on washing with pure or chlorinated water. A survey conducted by the authors in a factory for date fruit processing that depend on washing with pure and chlorinated water showed that this treatment was not quite effective. In some cases the processed dates had higher microbial loads than the raw fruits. The aim of this study was to investigate the effect of treatment with electrolyzed oxidizing water on the microbial contamination of date fruits. Collected samples were treated with electrolyzed oxidizing water by soaking for 5, 15 and 30 min. The effect of these treatments on the microbial load of the fruits was determined. MATERIALS AND METHODS s collection Date fruit samples from Khalas variety were purchased from one shop in Hofuf City, Saudi Arabia. They were collected in sterile containers and transported to the laboratory for analysis and treatment. *Corresponding author. siddighamad@yahoo.com. Tel: Electrolyzed water system ROX-20 from E-water Systems (Port Melbourne, Victoria,

2 030 Int. J. Microbiol. Immunol. Res. 3207, Australia) was used. The system includes adjustable flow rates and production volumes, and flexible nozzles to dispense water where needed. treatment Electrolyzed oxidizing water used had the following composition: 50 ppm residual chlorine, about mv oxidation reduction potential and ph about 3.0. Treatment with electrolyzed oxidizing water was performed by soaking 10 g sample in the electrolyzed water for 5, 15 and 30 min and then determining the microbial load. In a similar way 10 g samples were soaked in sterile tap water to determine the effect of washing. Microbiological analysis Date fruit samples (10 g) were weighed into sterile stomacher bags, 90 ml sterile peptone water (Oxoid, CM0009) added, homogenized in a stomacher (Lab- Blender 400, Seward Medical, England) for 45 s and aliquots (1.0 or 0.1 ml) plated out in duplicate as 10-fold dilutions in peptone water. Aerobic mesophilic bacteria were counted on Plate Count Agar dishes (PCA Oxoid, CM0325) incubated at 30 C for 2 to 3 days, coliforms on Violet Red Bile Agar plates (VRBA Oxoid, CM0107) at 37 C for 24 h, yeasts and molds on PDA plates at 20 to 30 C for 3 to 7 days. Escherichia coli were plated on L- EMB Agar (CM0069, Oxoid), S. aureus on Baird-Parker Agar Base (CM 0961, Oxoid), Pseudomonas aeruginosa on Pseudomonas Agar Base (CM0559) and Aspergillus fumigatus on Potato Dextrose Agar (CM0139, Oxoid). Contamination of fruits with selected microorganisms was performed as follows: the microorganisms were grown for 2 days in Petri dishes containing appropriate media. Then suspensions of the individual microorganisms were made in bottles containing 50 ml sterile peptone water by transferring 3 loopfuls from the Petri dishes to each bottle. Ten grams of each sample were then contaminated with the selected microorganisms by immersing in these suspensions for few seconds; the resulting contamination was 10 6 to 10 8 cfu/g. Three portions from each date fruit sample (10 g each) were contaminated with the selected microorganism. One portion was treated with electrolyzed water by soaking for 5, 15 or 30 min, the second portion treated by soaking in sterile tap water for the same period of time, and the third portion analyzed as untreated sample. The microbial loads of the treated and untreated portions were then determined. The selected microorganisms were S. aureus ATCC as representative of potential pathogens, P. aeruginosa ATCC as representative of potential spoilage bacteria, E. coli ATCC as representative of coliforms, and A. fumigatus ATCC as a representative of potential spoilage molds. Statistical analysis Analysis of variance was performed to detect differences in effects of applied treatments on microbial loads of samples. Duncan multiple ranges at 5% level of significance was used to compare between means. The analysis was carried out using the PROC ANOVA procedure of Statistical Analysis System (SAS, 1996). RESULTS AND DISCUSSION Effect of electrolyzed oxidizing water on mesophilic aerobic bacteria, coliforms, molds and yeasts Treatment for 5 min Five fruit samples tested were found contaminated with mesophilic aerobic bacteria at loads to cfu/g (Table 1). The washing effect of soaking in sterile water reduced this load by 0.65 to 1.11 log cycles, with an average of 0.87 log cycles, i.e. removed an average of about 78% of the bacterial population. Soaking in electrolyzed oxidizing water reduced loads of the range 10 2 cfu/g to non-detectable levels. s in loads of the range 10 3 to 10 5 cfu/g was 1.56 to 2.04 log cycles, with an average of 1.77 log cycles, i.e. removed an average of about 97% of the bacterial population or about 0.90 log cycles over the washing effect of water. Since mesophilic aerobic bacteria are not considered an important potential spoilage agent of date fruits, their presence is only regarded as an index for the hygienic status of the fruits. The 5 samples were also found contaminated with molds and yeasts at loads of 68 to cfu/g (Table 1), about 90% of which was molds. Soaking in sterile water reduced the contamination of 68 cfu/g to nondetectable level, while contaminations in the range 10 2 cfu/g and above were reduced by 0.67 to 1.06 log cycles. Soaking in the electrolyzed oxidizing water reduced the contamination in the range 10 2 cfu/g to non-detectable levels. The loads of the range 10 3 cfu/g were reduced by 1.75 to 2.12 log cycles, with an average of 1.94 log cycles, that is about 98.5% of the population was removed, and no yeasts were detected in the treated samples (result not shown). Molds and yeasts are considered important spoilage agents of date fruits. The Saudi standard for microbiological criteria of foods requires that the loads of yeasts in date fruits should not exceed 10 cfu/g in 3 out of 5 replicates of tested sample and that of molds not to exceed 10 2 cfu/g in 3 of 5 replicates of tested sample (SASO, 1998). This treatment 2

3 Hamad and Aleid 031 Table 1. Effect of soaking in tap water and electrolyzed oxidizing water for 5 min on the contamination of date fruits with mesophilic aerobic bacteria (MAB), molds and yeasts (M & Y) and coliforms. MAB M & Y Coliforms 1 (U) n.d. 1 (T) n.d. - 1 (E) n.d. - 2 (U) (T) n.d (E) n.d n.d (U) n.d. 3 (T) n.d. - 3 (E) n.d n.d n.d. - 4 (U) (T) n.d (E) n.d (U) n.d. 5 (T) n.d. - 5 (E) n.d n.d n.d. - U=untreated, T= treated with tap water, E=treated with electrolyzed water. reduced contamination to levels that meet Saudi standard for both molds and yeasts. Coliforms were detected in 2 of the 5 samples tested at loads of 84 and cfu/g Table 1). Soaking in water reduced the load of 84 cfu/g to non-detectable level and that of to 75 cfu/g. Soaking in electrolyzed oxidizing water reduced contamination of the 2 samples to non-detectable levels. Coliforms are not included in the Saudi standard for date fruits, but their presence is an index of possible recent contamination with human feces. In general, this treatment can be regarded as enough to control normal coliform contamination of date fruits. Treatment for 15 min The 5 samples tested were found contaminated with mesophilic aerobic bacteria at loads in the range10 2 to 10 5 cfu/g (Table 2). Soaking in sterile water reduced these loads by 0.79 to 1.18 log cycles, with an average of about 0.96 log cycles. Soaking in electrolyzed water reduced loads of 10 2 to 10 3 cfu/g to non-detectable levels. Load of 10 4 and 10 5 cfu/g were reduced by 2.43 and 2.71 log cycles, respectively, i.e. an average of 2.57 log cycles, or about 99.6% reduction (soaking for 5 minutes reduced loads by 1.77 log cycles or 97%). Thus, up to 10 3 cfu/g contamination loads of mesophilic aerobic 3 bacteria can be reduced to non-detectable level by soaking in electrolyzed oxidizing water for 15 min. All 5 samples tested were found contaminated with molds and yeasts at loads of less than 100 and up to 10 4 cfu/g (Table 2); again about 90% was molds. Soaking in sterile water reduced contaminations of up to 200 cfu/g to non-detectable levels, while contaminations above that were removed by an average of 0.95 log cycles. Soaking in the electrolyzed oxidizing water reduced contaminations up to 10 3 to non-detectable levels and contamination of the range 10 4 was reduced by 2.37 log cycles, still no yeasts detected in the treated samples (result not shown). This treatment is therefore enough for reducing loads of molds and yeasts of up to 10 4 cfu/g to levels that meet the Saudi standards. Coliforms were detected in only one of the 5 samples tested at a load of 78 cfu/g. As expected, soaking in sterile water and in electrolyzed oxidizing water reduced this load to non-detectable level. Actually, and as stated above, soaking for only 5 min was enough to control the normal contamination of date fruits with coliforms. Treatment for 30 min All 5 samples tested were found contaminated with mesophilic aerobic bacteria, molds and yeast (Table 3).

4 032 Int. J. Microbiol. Immunol. Res. Table 2. Effect of soaking in tap water and electrolyzed oxidizing water for 15 min on the contamination of date fruits with mesophilic aerobic bacteria (MAB), molds and yeasts (M & Y) and coliforms. MAB M & Y Coliforms 1 (U) n.d. 1 (T) n.d n.d. - 1 (E) n.d n.d. - 2 (U) n.d. 2 (T) n.d. - 2 (E) n.d. - 3 (U) (T) n.d (E) n.d n.d n.d (U) n.d. 4 (T) n.d n.d. - 4 (E) n.d n.d n.d. - 5 (U) n.d. 5 (T) n.d. - 5 (E) n.d n.d n.d. - U=untreated, T= treated with tap water, E=treated with electrolyzed water. Table 3. Effect of soaking in tap water and electrolyzed oxidizing water for 30 min on the contamination of date fruits with mesophilic aerobic bacteria (MAB), molds and yeasts (M & Y) and coliforms. MAB M & Y Coliforms 1 (U) n.d. 1 (T) n.d. - 1 (E) n.d n.d n.d. - 2 (U) n.d. 2 (T) n.d n.d. - 2 (E) n.d n.d. - 3 (U) n.d. 3 (T) n.d. - 3 (E) n.d n.d. - 4 (U) n.d. 4 (T) n.d n.d. - 4 (E) n.d n.d n.d. - 5 (U) (T) n.d (E) n.d n.d n.d U=untreated, T= treated with tap water, E=treated with electrolyzed water. 4

5 Hamad and Aleid 033 Table 4. Effect of soaking in tap water and electrolyzed oxidizing water for 5 min on the microbial load of date fruits contaminated with S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. coli ATCC and A. fumigatus ATCC S. aureus P. aeruginosa E. coli F. fumigatus 1 (T) (E) (T) (E) (T) (E) (T) (E) (T) (E) T= treated with tap water, E=treated with electrolyzed water. Contamination with mesophilic aerobic bacteria was in the range 10 2 to 10 4 cfu/g. Soaking in sterile water reduced these loads by an average of about 0.96 log cycles. Soaking in electrolyzed oxidizing water reduced loads of up to 10 3 to non-detectable levels while loads above that were reduced by an average of 3.0 log cycles that is about 99.9% compared to reduction of 2.57 log cycles or 99.6% for the 15 min treatment. Because of the relative insignificance of mesophilic aerobic bacteria in date fruit spoilage, this relatively slight increase in effectiveness may not justify soaking for 30 min in place of soaking for 15 min. Therefore it could be concluded that soaking in electrolyzed oxidizing water for 15 min would reduce contamination with mesophilic aerobic bacteria to acceptable levels. Soaking in electrolyzed oxidizing water reduced the contamination with molds and yeasts in all of the 5 samples tested to non-detectable levels (Table 3). The effect of this treatment gave similar results to the treatment of soaking for 15 min. As stated above, it can therefore be concluded that the treatment of soaking for 15 min is enough for reducing normal contamination levels of molds and yeasts to values that meet Saudi standard requirement for date fruits. Also and as expected, soaking in electrolyzed oxidizing water for 30 min reduced contamination with coliforms, which was found in only one sample, to non-detectable levels. It seems that such a treatment is not necessary for date fruits. A soaking treatment for only 5 min would be quite enough to reduce normal contamination of date fruits with coliforms to safe levels. Effect of electrolyzed oxidizing water on inoculated potential pathogenic and potential spoilage microorganisms Table 4 contains the results of soaking 5 date fruit samples in electrolyzed oxidizing water for 5 minutes. The levels of contamination of the untreated samples were 10 7 to 10 8 cfu/g for the bacteria and 10 6 to 10 7 cfu/g for the mold. Soaking in sterile tap water reduced the level of contamination in all tested samples by an average near one log cycle, which was the normal average value obtained for all other treatments discussed above. Soaking in electrolyzed oxidizing water for 5 minutes reduced contamination with S. aureus by an average of 1.87 log cycles, i.e. about 98% 0f the population was removed. This is an increase in effectiveness of about 0.92 log cycles over the effect of soaking in water. The effect of soaking in electrolyzed oxidizing water for 5 minutes on E. coli was similar to that on S. aureus, where contamination was reduced by an average of 1.83 log cycles, i.e. by about 97.5% compared to about 98% for S. aureus. Soaking in electrolyzed water for 5 minutes seems to be less effective on P. aeruginosa and A. fumigatus as compared to its effect on S. aureus and E. coli. Soaking in electrolyzed oxidizing water for 5 min reduced the contamination level of P. aeruginosa by 1.56 log cycles and that of A. fumigatus by 1.54 log cycles, that by about 95% and 94.9%, respectively compared to about 97.5% for E. coli and 98% for S. aureus. Soaking in sterile tap water reduced the levels of 5

6 034 Int. J. Microbiol. Immunol. Res. Table 5. Effect of soaking in tap water and electrolyzed oxidizing water for 15 min on the microbial load of date fruits contaminated with S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. coli ATCC and A. fumigatus ATCC S. aureus P. aeruginosa E. coli A. fumigatus 1 (T) (E) (T) (E) (T) (E) (T) (E) (T) (E) T= treated with tap water, E=treated with electrolyzed water. Table 6. Effect of soaking in tap water and electrolyzed oxidizing water for 30 min on the microbial load of date fruits contaminated with S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. coli ATCC and A. fumigatus ATCC S. aureus P. aeruginosa E. coli A. fumigatus 1 (T) (E) (T) (E) (T) (E) (T) (E) (T) (E) T= treated with tap water, E=treated with electrolyzed water. contamination by an average near one log cycle. Again S. aureus and E. coli were more affected by this treatment than P. aeruginosa and A. fumigatus. Contamination level of S. aureus was reduced by an average of 2.32 log cycles that of E. coli by 2.44 log cycle, while contamination of P. aeruginosa was reduced by an average of 2.07 log cycles and that of A. fumigatus by 2.04 log cycles. It is apparent that treatment for 15 min increased the killing effect of electrolyzed oxidizing water 6 over treatment for 5 min for all of the 4 microorganisms tested. Increases in the killing effect of soaking for 15 min over soaking for 5 minutes were 0.45, 0.50, 0.61 and 0.50 log cycles for S. aureus, P. aeruginosa, E. coli and A. fumigatus, respectively. The results of soaking the contaminated samples for 30 min are presented in Table 6. The levels of contamination with the 4 microorganisms and the amount removed by soaking in sterile tap water were similar to values

7 Hamad and Aleid 035 Table 7. Effect of soaking in electrolyzed oxidizing water for 5, 15 and 30 min on contamination of date fruit samples with some microorganisms. Time (min) Amount of reduction S. aureus P. aeruginosa E. coli A. fumigatus b 1.56 b 1.83 b 1.53 b b 2.07 a 2.44 a 2.02 a a 2.20 a 2.75 a 2.19 a Means within columns having same letter are not significantly different (P 0.05). reported above. Soaking for 30 min in electrolyzed oxidizing water reduced the levels of contamination by 2.89, 2.21, 2.76 and 2.18 log cycles for S. aureus, P. aeruginosa, E. coli and A. fumigatus, respectively. The killing effect of soaking for 30 minutes increased over the killing effect of soaking for 15 min for all of the 4 microorganisms. The increase in killing effect was 0.56, 0.14, 0.23 and 0.15 log cycles for S. aureus, P. aeruginosa, E. coli and A. fumigatus, respectively, with S. aureus and E. coli still more sensitive to electrolyzed oxidizing water. There was no significant difference in the effect of soaking date fruits in electrolyzed oxidizing water for 5 and 15 min on contamination with S. aureus (Table 7). However, the amount of reduction in contamination with this bacterium increased significantly after soaking for 30 min compared to soaking for 5 and 15 min in electrolyzed oxidizing water. Soaking for 30 min was the most effective treatment for the control of S. aureus, but since contamination of date fruits with pathogenic bacteria is normally low, treatment for 15 min would be enough for their control. In case of contamination of date fruits with P. aeruginosa, E. coli and A. fumigatus, the effect of treatment for 15 min was significantly higher than that of treatment for 5 min but not significantly different from that of 30 min (Table 7). This means that soaking in electrolyzed oxidizing water for 15 min can be regarded as the most suitable treatment for the control of contamination of date fruits with these microorganisms. After immersion of different test surfaces in electrolyzed oxidizing water for 5 minutes populations of Enterobacter aerogenes and S. aureus (6.1 log cfu/cm 2 ) were reduced by 2.2 to 2.5 log cycles and by 1.7 to 1.9 log cycles, respectively (Al-Haq et al., 2005). Treatment of wheat seed for 20 minutes with electrolyzed oxidizing water eliminated contamination by fungi such as Aspergillus, Cladosporium and Penicillium (Al-Haq et al., 2005). Dip (10 min.) treatment of spinach in electrolyzed oxidizing water at 100 and 120 ppm total residual chlorine resulted in a 4.0 to 5.0 log cfu/ml reduction in the counts of E. coli, Salmonella Typhimurium, S. aureus, Listeria monocytogenes and Enterococcus faecalis (Guentzel et al., 2008). Dipping (10 min.) of lettuce at 100 and 120 ppm total residual chlorine reduced counts of E. coli by to 2.5 log cfu/ml and reduced the counts of Salmonella Typhimurium, S. aureus, L. monocytogenes and E. faecalis by 2.43 to 3.81 log cfu/ml (Guentzel et al. 2008). Treatment of fresh-cut vegetables with electrolyzed oxidizing water (20 ppm residual chlorine) for 3 min reduced the total microbial count by 0.6 to 2.6 log cycles (Rivera, 2005). Conclusion Contamination of date fruits with mesophilic aerobic bacteria was mostly in the range 10 2 to 10 4, with molds and yeasts in the range 10 2 to 10 3 and with coliforms less than 10 2 cfu/g. Treatment with electrolyzed oxidizing water for 15 minutes reduced these levels of contamination by more than 2 log cycles which was enough to meet Saudi standard requirement. Hence normal microbial contamination of date fruits can be successfully controlled by treatment with electrolyzed oxidizing water. ACKNOWLEDGMENTS The authors would like to thank the Ministry of Higher Education, Saudi Arabia for funding the project No. PR-1, through a generous financial support to the Date Palm Research Center of Excellence, King Faisal University, Alahsa, Saudi Arabia. REFERENCES Abu-Zinada, A.H., Ali, M.I. (1982). Fungi associated with dates in Saudi Arabia. J. food. Protection. 45(9): Aidoo, K.E., Tester, R.F., Morrison, J.E., MacFarlane, D. (1996). The composition and microbial quality of prepacked dates purchased in Greater Glasgow. Int. J. food. Sci. Technol. 31: Al-Haq, M.I., Sugichi, J., Isobe, S. (2005). Application of electrolyzed water in agriculture and food industries. Food. Sci. Technol. Res., 11 (2):

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