- Email: [email protected]
Disinfection effect of slightly acidic electrolyzed water on celery and cilantro
Food Control 69 (2016) 147e152
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Disinfection effect of slightly acidic electrolyzed water on celery and cilantro Chunling Zhang a, Wei Cao a, Yen-Con Hung b, Baoming Li a, * a Key Laboratory of Agricultural Engineering in Structure and Environment, Ministry of Agriculture, China Agricultural University, P.O. Box 67, Beijing, 100083, PR China b Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2016 Received in revised form 10 April 2016 Accepted 22 April 2016 Available online 27 April 2016
This study was designed to evaluate the efficacy of slightly acidic electrolyzed water (SAEW) to reduce natural microbiota on celery and cilantro at different available chlorine concentrations (ACC), different treatment time and temperatures. Additionally, SAEW treated celery and cilantro were stored at 4 and 20 C for 6 days and population of total aerobic bacteria and yeast and mold were also determined at day 0, 2, 4 and 6, separately. Results showed that log reduction of total aerobic bacteria and yeast and mold significantly increased with increasing ACC and treatment time, respectively (p < 0.05). Celery and cilantro treated with SAEW at 30 mg/L ACC for 5 min and 25 mg/L for 7 min reduced yeast and mold to non-detectable level. No significant difference was observed for disinfection efficacy of SAEW on celery and cilantro at different temperatures (4, 20 and 37 C) (p > 0.05). The microbial population on celery and cilantro maintained at a low level during storage at 4 and 20 C after SAEW treatment (total aerobic bacteria: 3.3e4.1 log CFU/g, yeast and mold: 2.2e3.5 log CFU/g). The microbial inactivation effect as well as the absence of any sensory alterations on treated celery and cilantro rendered SAEW a promising disinfectant, which can be applied in fresh produce wash to control natural microbiota. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Slightly acidic electrolyzed water Celery and cilantro Total aerobic bacteria Yeast and mold Inactivation
1. Introduction Celery and cilantro are usually consumed raw or uncooked. Celery adapts easily to minimal process and is part of an array of fresh-cut or ready-to-use produce in the market. Young leaves of cilantro are usually used as fresh herbs, spice, garnishes, in chutneys and sauces (Sahib et al., 2013). Recent years, demand for consumption of minimally processed fruits and vegetables has increased rapidly, but fresh produce, in particular, leafy greens which are generally consumed raw, were identified as source for numerous foodborne pathogens outbreaks (Lynch, Tauxe, & Hedberg, 2009). Microbiological safety issues for fresh produce or fresh-cut produce are important, since these products are susceptible to be contaminated with bacteria, especially pathogens deriving from soil, water, hands of workers and process environment (Lu, Yu, Gao, Lu, & Zhang, 2005). Due to the absence of a
* Corresponding author. College of Water Resources and Civil Engineering, China Agricultural University, 17 Qinghua East Road, Haidian District, Beijing, 100083, PR China. E-mail address: [email protected] (B. Li). http://dx.doi.org/10.1016/j.foodcont.2016.04.039 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
sufficient step for inactivating pathogens before consumption, such as heating, fresh-cut vegetable is prone to an increased risk of public health (Beuchat, 2004). Therefore, disinfection of fresh-cut produce is desirable to control spoilage bacteria and fungi to extend shelf life and eliminate pathogens for food safety (McKellar et al., 2004; Qiang, Demirkol, Ercal, & Adams, 2005). The microbial contamination of fresh-cut vegetables during production and process is generally controlled by chemicals application, such as sodium hypochlorite, organic acid, ozone, chlorine and chlorine-based products, electrolyzed oxidizing (EO) water (Akbas & Olmez, 2007; Alexopoulos et al., 2013; Alwi & Ali, 2014; Hao, Li, Wan, & Liu, 2015; Raiputta, Setha, & Suthiluk, 2013; Wang, Feng, & Luo, 2004). Although many types of chemical are used for decontamination, more effective, appropriate products and methods are still searched by fresh-cut fruit and vegetable industry to ensure the safety of the fresh produce. Slightly acidic electrolyzed water (SAEW), a novel disinfectant, has been increasingly used to decontaminate fresh produce due to its quick on-site production and economic cost (Bosilevac, Shackelford, Brichta, & Koohmaraie, 2005; Koide, Shitanda, Note, & Cao, 2011). SAEW with a pH of 5.0e6.5 and a high concentration of hypochlorous acid is generated by electrolysis of dilute NaCl
148
C. Zhang et al. / Food Control 69 (2016) 147e152
and/or HCl solution in a non-membrane electrolytic chamber (Jadeja, Hung, & Bosilevac, 2013). SAEW was proved contains largely hypochlorous acid, an incredibly effective form of the chlorine, which is greater than that of hypochlorite ion for inactivating bacteria at a same free chlorine concentration and treatment time. The strong bactericidal, fungicidal, sporicidal and virucidal ability of SAEW have been reported in numerous previous studies (Cao, Zhu, Shi, Wang, & Li, 2009; Deza, Araujo, & Garrido, 2007; Hao et al., 2015; Hao et al., 2013; Jadeja & Hung, 2014; Koide et al., 2011; Nan et al., 2010; Rahman, Park, Song, Al-Harbi, & Oh, 2012; Zhang, Li, Jadeja, & Hung, 2016; Zheng et al., 2013). In addition, the disinfection efficacy of SAEW in reducing and eliminating pathogenic microorganisms on fresh produce has been reported (Abadias, Usall, Oliveira, Alegre, & Vinas, 2008; Hao et al., 2015; Koide, Takeda, Shi, Shono, & Atungulu, 2009; Pangloli & Hung, 2011; Zhang, Cao, Hung, & Li, 2016; Zhang et al., 2011). The objectives of this study were to evaluate the effects of different available chlorine concentrations (ACC, 15, 20, 25 and 30 mg/L), treatment time (1, 3, 5 and 7 min) and temperatures (4, 20 and 37 C) of SAEW on reducing natural microbiota (total aerobic bacteria and yeast and mold) on fresh celery and cilantro. Finally, variation of microbiota population on SAEW treated celery and cilantro during storage at 4 and 20 C was also investigated. 2. Materials and methods 2.1. Preparation of slightly acidic electrolyzed water Slightly acidic electrolyzed water (SAEW) was produced by electrolysis of 9% HCl solution using a SAEW generator (Purester MP-600T, Morinaga Engineering Co., Ltd., Tokyo, Japan) with a nonmembrane electrolytic chamber. SAEW with pH of 5.76e6.05, oxidation reduction potential (ORP) of 879.3e923.6 mV and ACC of 15, 20, 25 and 30 mg/L were produced at ambient temperature prior to experiment. The physicochemical properties of SAEW were measured immediately after preparation. The pH and ORP values were measured using a dual scale pH/ORP meter (HM-30R, DKKTOA Corp., Tokyo, Japan) with a pH electrode (GST-5741C) ranged from 0.00 to 14.00 or an ORP electrode (PST-5721C) ranged from 0.0 to 2000.0 mV. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan). All chemicals used in the experiment were of analysis grade. 2.2. Sample preparation and disinfection experiments Fresh celery and cilantro used in this study were purchased from a local market in Beijing, China on the day of the experiment and used for experiment within 24 h. Soil, wilted leaf and foreign matter on the surface were removed and celery and cilantro were subsequently aseptically cut into about 5-cm long before each experiment, respectively. Each sample with a similar amount of leaves and stems was prepared for bactericidal analysis. 2.2.1. Effect of SAEW with different ACC on microbiota inactivation SAEW with ACC of 15, 20, 25 and 30 mg/L at room temperature were used in this study. Each 25 g of sample was individually soaked with 225 mL of SAEW in a sterilized beaker flask and agitated vigorously at 150 rpm (COS-110 50, Shanghai Zollo Instrument Co., Ltd., China) for desired contact time. At the end of treatment time, 10 mL of treatment solution was immediately taken into 90 mL of sterilized neutralizing buffer (0.5% sodium thiosulphate þ 0.03 M phosphate buffer solution, pH 7) and mixed completely to terminate the residue chlorine reaction. Counts of viable total aerobic bacteria were obtained by plating 0.1 mL of 10-
fold serial dilution of mixture onto sterile nutrient agar (Aiboxing Bioscience Inc., Beijing, China) and plates were incubated at 37 C for 48 h before enumeration. Rose Bengal medium (Aiboxing Bioscience Inc., Beijing, China) with chloramphenicol addition was used for determining the population of viable yeast and mold. Plates were incubated at 25 C for 5 days. Another 25 g of sample treated with 225 mL sterilized 0.85% saline solution was used as a control. The control sample was washed at 150 rpm agitation for 5 min at room temperature to obtain the initial population of natural microbiota on the surface of analyzed celery and cilantro. Ten plates were used to be separately spread by plating 0.1 mL of bacterial suspension and the detection limit of this method is 2.0 log CFU/g. The experiment was replicated in triplicate. 2.2.2. Effect of SAEW on microbiota inactivation for different treatment time Each 25 g of celery and cilantro sample was treated with 225 mL of SAEW with ACC of 25 mg/L at room temperature for 1, 3, 5 and 7 min, respectively. After treatment terminated by neutralizing buffer, viable colonies of total aerobic bacteria and yeast and mold were enumerated by plating count according to the method described in 2.2.1. 2.2.3. Effect of SAEW at different temperatures on microbiota inactivation Freshly prepared SAEW in sealed brown glass bottles were stored in 4, 20 and 37 C water bath for 1 h, separately, and properties (ACC, pH and ORP) of SAEW were measured before and after water bath. Each 25 g of celery and cilantro sample was treated with 225 mL of SAEW of 4, 20 and 37 C with 25 mg/L available chlorine for 5 min. The following procedure of viable colonies count was conducted according to the previous method described in 2.2.1. 2.2.4. Procedure of storage experiment Each 200 g of celery and cilantro sample (uncut) was treated using the method described above by 1800 mL of SAEW with 25 mg/L ACC at room temperature for 5 min. Treated samples were drained and dried in the bio-safety cabinet for 2 h, then packaged in sterile stomacher bags and stored at 4 and 20 C, respectively. A 25 g of sample was collected for bactericidal analysis at 0, 2, 4 and 6 days storage using the method described in 2.2.1. Samples treated with 0.85% saline solution were stored at 4 and 20 C as control (untreated). Appearance quality of celery and cilantro including overall quality, color, texture, skin integrity, and aroma were also taken into consideration. Three to five lab members were invited for this test and compared the appearance quality of test sample with the fresh one by naked eyes in this study. 2.3. Statistical analysis For each treatment, results presented were obtained from independent replicate trials and the means and the standard deviations were calculated. Statistical analysis was performed using SAS software 9.2 (Institute, Inc., Cary, NC, USA). Least significance difference of means test was used for multiple comparisons at the 0.05 probability level. 3. Results and discussion 3.1. Effect of SAEW with different ACC on reducing microbiota on celery and cilantro After treatments of various SAEW, the surviving population of total aerobic bacteria and yeast and mold on the surface of celery and cilantro are shown in Fig. 1. The initial population of total
C. Zhang et al. / Food Control 69 (2016) 147e152
aerobic bacteria, yeast and mold on celery in control were 7.61 and 6.17 log CFU/g (Fig. 1A), on cilantro in control were 7.35 and 5.86 log CFU/g (Fig. 1B), respectively. The surviving population of total aerobic bacteria and yeast and mold on celery was 5.31, 4.84, 3.82, 3.0 log CFU/g and 4.84, 3.20, 2.48 2.0 log CFU/g after treated with SAEW at ACC of 15, 20, 25 and 30 mg/L, respectively, while 5.57, 4.56, 3.48, 2.72 log CFU/g and 4.63, 3.13, 2.29, 2.0 log CFU/g was observed for total aerobic bacteria, and yeast and mold on cilantro treated with SAEW at the same ACC. The surviving population of total aerobic bacteria and yeast and mold decreased significantly with ACC of SAEW increased from 15 to 30 mg/L, in comparison with control for both celery and cilantro (p < 0.05). This result was in agreement with the previous study, in which SAEW was used to inactivate Escherichia coli O157:H7 and Salmonella enteritidis on mung beans that surviving population of these two types of microorganism was decreased with ACC of SAEW increased (Zhang et al., 2011). Celery and cilantro treated with SAEW at 30 mg/L ACC achieved surviving population of total aerobic bacteria by 3.0 and 2.72 log CFU/g, while surviving population of yeast and mold to non-detectable level (detection limit is 2.0 log CFU/g in this study). The reason that aerobic bacteria is more resistant to SAEW than yeast and mold could be that initial population of total aerobic bacteria is slightly higher than yeast and mold, in addition, aerobic bacteria population on vegetables which grown close to the soil surface, may include several spore-forming bacteria, which are known to be more resistant to sanitizers or extreme environment (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000). In previous study, E. coli O157:H7 and Bacillus subtilis vegetative cell in pure culture at the population level of 7 log could be completely inactivated by SAEW with ACC of 0.5 mg/L for 30 s, while in the present study higher ACC and longer treatment time required to achieve the same result (Zhang, Li, Jadeja, Fang & Hung, 2016). A possible explanation for this difference could be that microbiota on fresh produce was more resistant to SAEW than those in pure culture. When microorganism attached to fresh produce, they might be in more complicate environment contaminated with foreign matter including organic matter which was reported could neutralize free chlorine and protect cells from membrane permeabilization (Rule, Ebbett, & Vikesland, 2005; Virto, Manas, Alvarez, Condon, & Raso, 2005). Aerobic bacteria, and yeast and mold observed on the surface of celery and cilantro could affect the shelf-life of vegetables. Moreover, pathogens may also remain in these vegetables prior to use and could do harm to human health. In this study, SAEW showed highly effective in eliminating both types of microorganism from
9 8 7 6 5 4 3 2 1 0
Bb
B c
control
15
3.2. Effect of SAEW on reducing microbiota on celery and cilantro for different treatment time The surviving population of total aerobic bacteria, and yeast and mold on the surface of celery and cilantro after treated with SAEW at the ACC of 25 mg/L for 1, 3, 5 and 7 min are shown in Fig. 2. With the exception of treatment for 5 and 7 min in Fig. 2B, the surviving microbial population (total aerobic bacteria, yeast and mold) decreased significantly after celery and cilantro was treated with SAEW from 1 to 7 min (p < 0.05). The population of total aerobic bacteria, and yeast and mold on celery samples in control were 7.61 and 6.17 log CFU/g (Fig. 2A), on cilantro samples were 7.35 and 5.86 log CFU/g (Fig. 2B), however, after treatment with SAEW for 7 min, the surviving population of total aerobic bacteria was 2.98 and 2.69 log CFU/g on celery and cilantro, while yeast and mold were at nondetectable level. Therefore, celery and cilantro after SAEW treatment with ACC of 25 mg/L for 7 min could be used safely since microbial population was maintained at a low or non-detectable level. In previous study, surviving population of E. coli O157:H7, Listeria monocytogenes and Salmonella typhimurium natural on green onions and tomatoes exposed to acidic EO water decreased, in response to increasing exposure time from 0 to 5 min (Park, Alexander, Taylor, Costa, & Kang, 2009). The result of our study was in agreement with the previous study which also indicated more reduction could be obtained with elevated treatment time when fresh produces was treated with EO water (Hung, Tilly, & Kim, 2010; Pangloli & Hung, 2013; Sharma & Demirci, 2003; Udompijitkul, Daeschel, & Zhao, 2007; Zhang et al., 2011). Longtime exposure could allow EO water to exert more effective effect on decontamination of fresh-cut celery and cilantro, and no negative sensory quality was observed (data not shown). 3.3. Effect of SAEW at different temperatures on reducing microbiota on celery and cilantro Effects of SAEW with ACC of 25 mg/L on decontamination of natural microbiota on fresh celery and cilantro at temperature of 4, 20 and 37 C for 5 min were determined and the results are presented in Fig. 3. The surviving population of total aerobic bacteria, yeast and mold on celery after treated with SAEW of 4, 20 and 37 C were of 3.57, 3.28, 3.22 log CFU/g and 2.47, 2.31, 2.16 log CFU/g
yeast and mold
A a
fresh-cut celery and cilantro. Additionally, SAEW treatments with ACC varying from 15 to 30 mg/L did not adversely altered the visual appearance of celery and cilantro (data not shown).
C d
20 25 ACC (mg/L) A
D
e
30
total aerobic bacteria Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
149
8 7 6 5 4 3 2 1 0
yeast and mold
A a
B
b
C c
control
15
D d
20 25 ACC (mg/L) B
E
d
30
Fig. 1. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW at different ACC. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
150
C. Zhang et al. / Food Control 69 (2016) 147e152
9 8 7 6 5 4 3 2 1 0
yeast and mold
A a
B C
b
control
D
c
d
E
1 3 5 Treatment time (min) A
e
total aerobic bacteria 8 7 6 5 4 3 2 1 0
Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
7
yeast and mold
A B
a
C
b
D
c
d
control
D
1 3 5 Treatment time (min) B
d
7
Fig. 2. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW for different time. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
9 8 7 6 5 4 3 2 1 0
yeast and mold
A a B b
control
B
b
4 20 Temperature (°C) A
B
b
37
total aerobic bacteria Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
8 7 6 5 4 3 2 1 0
yeast and mold
A a B
B b
control
b
4 20 Temperature (°C) B
B
b
37
Fig. 3. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW at different temperature. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
(Fig. 3A), respectively, 3.32, 3.25, 2.92 log CFU/g and 2.27, 2.11, 2.0 log CFU/g (Fig. 3B) were observed on cilantro as well. Results showed that the surviving population of total aerobic bacteria and yeast and mold slightly decreased when samples were treated with SAEW at temperature of 4, 20 and 37 C, although there was no significant difference observed in celery and cilantro among SAEW of different temperatures (4, 20 and 37 C) (p > 0.05). This result was in agreement with the previous study, in which more reductions were achieved with increasing temperature of EO water from 4 to 50 C (Rahman, Ding, & Oh, 2010). They found that reductions of L. monocytogenes were 4.98, 5.0, 5.20, 6.20 and 7.42 logs CFU/ml after low concentration EO water treatment at 4, 15, 23, 35 and 50 C, respectively. On the other hand, similar reductions were found for another three types of bacteria (S. typhimurium, Staphylococcus aureus and E. coli O157:H7) after treated with the same EO water. Room temperature or higher could slightly improve the reaction of EO water and microorganism on the surface of fresh produce. It was reported that the population of total aerobic bacteria and yeast and mold in carrot samples was lower after treated with mildly heated (45 C) SAEW than that at the temperature of 18 C (Koide et al., 2011). In this study, available chlorine of SAEW was measured before and after SAEW was stored in water bath to reach desired temperature (4, 20 and 37 C) and no change was observed. It was also reported that the pH, ORP and residual chlorine of EO water was almost no changed at temperature 4 and 24 C
(Hung et al., 2010). No negative variation was observed on sensory quality of celery and cilantro treated with SAEW containing 25 mg/ L available chlorine for 5 min at 4, 20 and 37 C (data not shown). 3.4. Growth of natural microbiota on treated celery and cilantro by SAEW during storage Fresh celery and cilantro were treated with SAEW with ACC of 25 mg/L for 5 min at room temperature and stored at 4 and 20 C. The surviving population of total aerobic bacteria and yeast and mold on these two vegetables were detected at storage time of 0, 2, 4 and 6 days, respectively. Celery and cilantro treated with 0.85% saline were stored at 4 and 20 C as control (untreated). Results presented in Fig. 4 that the surviving population of total aerobic bacteria and yeast and mold on celery after treatment slowly increased when it was stored at 20 C. In comparison with day 0 at 20 C, the surviving population of total aerobic bacteria on treated celery and the control on day 6 achieved an additional 0.66 and 1.25 log CFU/g, while an additional 0.14 and 0.05 log CFU/g were observed on day 6 at 4 C. On the other hand, an increase of total aerobic bacteria of 1.20 log CFU/g was obtained when comparing untreated (control) sample stored at 20 C with that stored at 4 C, while 0.80 log CFU/g increase of total aerobic bacteria on treated samples. The appropriate temperature, 20 C, for example, could allow bacteria to grow on both treated sample and untreated
C. Zhang et al. / Food Control 69 (2016) 147e152
10 9 8 7 6 5 4 3 2 1 0 0
2
4 °C control 4 °C
20 °C control 20 °C
4
6
Yeaat and mold population (log CFU/g)
Total aerobic bacterial population (log CFU/g)
4 °C control 4 °C
151
20 °C control 20 °C
8 7 6 5 4 3 2 1 0 0
2
4
6
Time (day)
Time (day)
4 °C
20 °C
control 4 °C
control 20 °C Yeast and mold population (log CFU/g)
Total aerobic bacterial population (log CFU/g)
Fig. 4. Growth of natural microbiota on the surface of celery during storage at 4 and 20 C after treated with SAEW.
10 9 8 7 6 5 4 3 2 1 0 0
2
4
6
4 °C
20 °C
control 4 °C
control 20 °C
8 7 6 5 4 3 2 1 0 0
2
4
6
Time (day)
Time (day)
Fig. 5. Growth of natural microbiota on the surface of cilantro during storage at 4 and 20 C after treated with SAEW.
sample. However, the increase of microbiota population on stored celery samples was not very high. The reason could be that celery before storage was dry and no proper humidity for bacteria to grow and reproduce quickly. Similar trend of variation was observed on population of yeast and mold on celery. An additional 1.0 and 1.25 log CFU/g were observed on treated and untreated samples which were stored at 20 C for 6 days. Low temperature (4 C) may not allow yeast and mold to grow and reproduce, since only 0.09 and 0.01 log CFU/g additional counts were obtained on untreated and treated celery samples after stored for 6 days at 4 C. As can be seen in Fig. 4, the microbial population on treated samples increased very slowly or even no increase in the first two days storage at 20 C. In contrast, an obvious increase was observed on untreated samples during the same storage period. It could be that bacteria on sample which was treated with SAEW may be injured to some extent and it would take some time to recover and reproduce when storage at room temperature. The similar variation trend for microbial population was observed on cilantro which was treated and stored at the same condition (Fig. 5). It was reported that population of Salmonella inoculated on celery significantly decreased by 0.5e1.0 log CFU/g under 4 C storage and 2.0 log CFU/g increased under 22 C storage. In addition, the similar results were found for E. coli O157:H7 and L. monocytogenes inoculated on celery (Vandamm, Li, Harris, Schaffner, & Danyluk, 2013). In our study, the microbial population on both celery and cilantro was increased when stored at 20 C, which was in agreement with the previous study mentioned as above, whereas it did not agree with the result at 4 C since no
microbial population decreased when stored at 4 C in our study. The microbial population of total aerobic bacteria, yeast and mold were significantly reduced after SAEW treatment with 25 mg/ L available chlorine for 5 min at room temperature. Moreover, it could extend the shelf-life of celery and cilantro, because the microbial population grew slowly and maintained at a low level, which would do no harm to human health. The microbial inactivation results as well as the absence of any sensory alterations on the exposed fresh celery and cilantro render SAEW a promising disinfectant, which can be used in fresh produce against foodborne disease. 4. Conclusions The effect of SAEW on inactivation of natural microbiota on celery and cilantro significantly increased with increasing ACC and treatment time (P < 0.05), and was not significantly affected by temperature of SAEW. The microbial population grew slowly and maintained at a low level during storage after celery and cilantro treated with SAEW. The main finding of this study suggests that SAEW is highly effective in eliminating microbiota on the surface of celery and cilantro and can be used in fresh produce against natural microbiota. Acknowledgments This work was supported by Beijing Municipal Commission of Education, university discipline group construction (No.
152
C. Zhang et al. / Food Control 69 (2016) 147e152
100190553). The author thanks Dr. Ravirajsinh Jadeja for specific comments on the preparation of this manuscript. References Abadias, M., Usall, J., Oliveira, M., Alegre, I., & Vinas, I. (2008). Efficacy of neutral electrolyzed water (NEW) for reducing microbial contamination on minimallyprocessed vegetables. International Journal of Food Microbiology, 123, 151e158. Akbas, M. Y., & Olmez, H. (2007). Effectiveness of organic acid, ozonated water and chlorine dippings on microbial reduction and storage quality of fresh-cut iceberg lettuce. Journal of the Science of Food and Agriculture, 87, 2609e2616. Alexopoulos, A., Plessas, S., Ceciu, S., Lazar, V., Mantzourani, I., Voidarou, C., et al. (2013). Evaluation of ozone efficacy on the reduction of microbial population of fresh cut lettuce (Lactuca sativa) and green bell pepper (Capsicum annuum). Food Control, 30, 491e496. Alwi, N. A., & Ali, A. (2014). Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Control, 46, 304e311. Beuchat, L. R. (2004). Difficulties in eliminating human pathogenic microorganisms on raw fruits and vegetables. Horticulture: Art and Science for Life, 642, 151e160. Bosilevac, J. M., Shackelford, S. D., Brichta, D. M., & Koohmaraie, M. (2005). Efficacy of ozonated and electrolyzed oxidative waters to decontaminate hides of cattle before slaughter. Journal of Food Protection, 68, 1393e1398. Cao, W., Zhu, Z. W., Shi, Z. X., Wang, C. Y., & Li, B. M. (2009). Efficiency of slightly acidic electrolyzed water for inactivation of Salmonella enteritidis and its contaminated shell eggs. International Journal of Food Microbiology, 130, 88e93. Deza, M. A., Araujo, M., & Garrido, M. J. (2007). Efficacy of neutral electrolyzed water to inactivate Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus on plastic and wooden kitchen cutting boards. Journal of Food Protection, 70, 102e108. Hao, J. X., Li, H. Y., Wan, Y. F., & Liu, H. J. (2015). Combined effect of acidic electrolyzed water (AcEW) and alkaline electrolyzed water (AlEW) on the microbial reduction of fresh-cut cilantro. Food Control, 50, 699e704. Hao, X. X., Shen, Z. Q., Wang, J. L., Zhang, Q., Li, B. M., Wang, C. Y., et al. (2013). In vitro inactivation of porcine reproductive and respiratory syndrome virus and pseudorabies virus by slightly acidic electrolyzed water. The Veterinary Journal, 197, 297e301. Hung, Y. C., Tilly, P., & Kim, C. (2010). Efficacy of electrolyzed oxidizing (Eo) water and chlorinated water for inactivation of Escherichia coli O157:H7 on strawberries and broccoli. Journal of Food Quality, 33, 559e577. Jadeja, R., & Hung, Y. C. (2014). Efficacy of near neutral and alkaline pH electrolyzed oxidizing waters to control Escherichia coli O157:H7 and Salmonella Typhimurium DT 104 from beef hides. Food Control, 41, 17e20. Jadeja, R., Hung, Y. C., & Bosilevac, J. M. (2013). Resistance of various shiga toxinproducing Escherichia coli to electrolyzed oxidizing water. Food Control, 30, 580e584. Koide, S., Shitanda, D., Note, M., & Cao, W. (2011). Effects of mildly heated, slightly acidic electrolyzed water on the disinfection and physicochemical properties of sliced carrot. Food Control, 22, 452e456. Koide, S., Takeda, J., Shi, J., Shono, H., & Atungulu, G. G. (2009). Disinfection efficacy of slightly acidic electrolyzed water on fresh cut cabbage. Food Control, 20, 294e297. Lu, Z. X., Yu, Z. F., Gao, X., Lu, F. X., & Zhang, L. K. (2005). Preservation effects of gamma irradiation on fresh-cut celery. Journal of Food Engineering, 67, 347e351. Lynch, M. F., Tauxe, R. V., & Hedberg, C. W. (2009). The growing burden of foodborne outbreaks due to contaminated fresh produce: Risks and opportunities. Epidemiology & Infection, 137, 307e315. McKellar, R. C., Odumeru, J., Zhou, T., Harrison, A., Mercer, D. G., Young, J. C., et al. (2004). Influence of a commercial warm chlorinated water treatment and packaging on the shelf-life of ready-to-use lettuce. Food Research International, 37, 343e354. Nan, S. J., Li, Y. Y., Li, B. M., Wang, C. Y., Cui, X. D., & Cao, W. (2010). Effect of slightly acidic electrolyzed water for inactivating Escherichia coli O157:H7 and Staphylococcus aureus analyzed by transmission electron microscopy. Journal of Food Protection, 73, 2211e2216.
Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J., & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and Molecular Biology Reviews, 64(3), 548e572. Pangloli, P., & Hung, Y. C. (2011). Efficacy of slightly acidic electrolyzed water in killing or reducing Escherichia coli O157:H7 on iceberg lettuce and tomatoes under simulated food service operation conditions. Journal of Food Science, 76, M361eM366. Pangloli, P., & Hung, Y. C. (2013). Reducing microbiological safety risk on blueberries through innovative washing technologies. Food Control, 32, 621e625. Park, E. J., Alexander, E., Taylor, G. A., Costa, R., & Kang, D. H. (2009). The decontaminative effects of acidic electrolyzed water for Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on green onions and tomatoes with differing organic demands. Food Microbiology, 26, 386e390. Qiang, Z., Demirkol, O., Ercal, N., & Adams, C. (2005). Impact of food disinfection on beneficial biothiol contents in vegetables. Journal of Agricultural and Food Chemistry, 53, 9830e9840. Rahman, S. M. E., Ding, T., & Oh, D. H. (2010). Effectiveness of low concentration electrolyzed water to inactivate foodborne pathogens under different environmental conditions. International Journal of Food Microbiology, 139, 147e153. Rahman, S. M., Park, J., Song, K. B., Al-Harbi, N. A., & Oh, D. H. (2012). Effects of slightly acidic low concentration electrolyzed water on microbiological, physicochemical, and sensory quality of fresh chicken breast meat. Journal of Food Science, 77, M35eM41. Raiputta, J., Setha, S., & Suthiluk, P. (2013). Microbial reduction and quality of freshcut 'phulae' pineapple (ananas comosus) treated with acidic electrolyzed water. In VII International Postharvest Symposium (Vol. 1012, pp. 1049e1055). Rule, K. L., Ebbett, V. R., & Vikesland, P. J. (2005). Formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan. Environmental Science & Technology, 39, 3176e3185. Sahib, N. G., Anwar, F., Gilani, A. H., Hamid, A. A., Saari, N., & Alkharfy, K. M. (2013). Coriander (Coriandrum sativum L.): a potential source of high-value components for functional foods and nutraceuticals - A review. Phytotherapy Research, 27, 1439e1456. Sharma, R. R., & Demirci, A. (2003). Treatment of Escherichia coli O157:H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water. International Journal of Food Microbiology, 86, 231e237. Udompijitkul, P., Daeschel, M. A., & Zhao, Y. (2007). Antimicrobial effect of electrolyzed oxidizing water against Escherichia coli O157:H7 and Listeria monocytogenes on fresh strawberries (Fragaria x ananassa). Journal of Food Science, 72, M397eM406. Vandamm, J. P., Li, D., Harris, L. J., Schaffner, D. W., & Danyluk, M. D. (2013). Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery. Food Microbiology, 34, 151e157. Virto, R., Manas, P., Alvarez, I., Condon, S., & Raso, J. (2005). Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorinedemanding substrate. Applied and Environmental Microbiology, 71, 5022e5028. Wang, H., Feng, H., & Luo, Y. G. (2004). Microbial reduction and storage quality of fresh-cut cilantro washed with acidic electrolyzed water and aqueous ozone. Food Research International, 37, 949e956. Zhang, C. L., Cao, W., Hung, Y. C., & Li, B. M. (2016a). Application of electrolyzed oxidizing water in production of radish sprouts to reduce natural microbiota. Food Control, 67, 177e182. Zhang, C. L., Li, B. M., Jadeja, R., Fang, J. L., & Hung, Y. C. (2016b). Effects of bacterial concentrations and centrifugations on susceptibility of Bacillus subtilis vegetative cells and Escherichia coli O157:H7 to various electrolyzed oxidizing water treatments. Food Control, 60, 440e446. Zhang, C. L., Li, B. M., Jadeja, R., & Hung, Y. C. (2016c). Effects of electrolyzed oxidizing water on inactivation of Bacillus subtilis and Bacillus cereus spores in suspension and on carriers. Journal of Food Science, 81(1), M144eM149. Zhang, C. L., Lu, Z. H., Li, Y. Y., Shang, Y. C., Zhang, G., & Cao, W. (2011). Reduction of Escherichia coli O157:H7 and Salmonella enteritidis on mung bean seeds and sprouts by slightly acidic electrolyzed water. Food Control, 22, 792e796. Zheng, W. C., Kang, R., Wang, H., Li, B. M., Xu, C., & Wang, S. (2013). Airborne bacterial reduction by spraying slightly acidic electrolyzed water in a laying-hen house. Journal of the Air & Waste Management Association, 63, 1205e1211.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Disinfection effect of slightly acidic electrolyzed water on celery and cilantro Chunling Zhang a, Wei Cao a, Yen-Con Hung b, Baoming Li a, * a Key Laboratory of Agricultural Engineering in Structure and Environment, Ministry of Agriculture, China Agricultural University, P.O. Box 67, Beijing, 100083, PR China b Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA, 30223, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2016 Received in revised form 10 April 2016 Accepted 22 April 2016 Available online 27 April 2016
This study was designed to evaluate the efficacy of slightly acidic electrolyzed water (SAEW) to reduce natural microbiota on celery and cilantro at different available chlorine concentrations (ACC), different treatment time and temperatures. Additionally, SAEW treated celery and cilantro were stored at 4 and 20 C for 6 days and population of total aerobic bacteria and yeast and mold were also determined at day 0, 2, 4 and 6, separately. Results showed that log reduction of total aerobic bacteria and yeast and mold significantly increased with increasing ACC and treatment time, respectively (p < 0.05). Celery and cilantro treated with SAEW at 30 mg/L ACC for 5 min and 25 mg/L for 7 min reduced yeast and mold to non-detectable level. No significant difference was observed for disinfection efficacy of SAEW on celery and cilantro at different temperatures (4, 20 and 37 C) (p > 0.05). The microbial population on celery and cilantro maintained at a low level during storage at 4 and 20 C after SAEW treatment (total aerobic bacteria: 3.3e4.1 log CFU/g, yeast and mold: 2.2e3.5 log CFU/g). The microbial inactivation effect as well as the absence of any sensory alterations on treated celery and cilantro rendered SAEW a promising disinfectant, which can be applied in fresh produce wash to control natural microbiota. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Slightly acidic electrolyzed water Celery and cilantro Total aerobic bacteria Yeast and mold Inactivation
1. Introduction Celery and cilantro are usually consumed raw or uncooked. Celery adapts easily to minimal process and is part of an array of fresh-cut or ready-to-use produce in the market. Young leaves of cilantro are usually used as fresh herbs, spice, garnishes, in chutneys and sauces (Sahib et al., 2013). Recent years, demand for consumption of minimally processed fruits and vegetables has increased rapidly, but fresh produce, in particular, leafy greens which are generally consumed raw, were identified as source for numerous foodborne pathogens outbreaks (Lynch, Tauxe, & Hedberg, 2009). Microbiological safety issues for fresh produce or fresh-cut produce are important, since these products are susceptible to be contaminated with bacteria, especially pathogens deriving from soil, water, hands of workers and process environment (Lu, Yu, Gao, Lu, & Zhang, 2005). Due to the absence of a
* Corresponding author. College of Water Resources and Civil Engineering, China Agricultural University, 17 Qinghua East Road, Haidian District, Beijing, 100083, PR China. E-mail address: [email protected] (B. Li). http://dx.doi.org/10.1016/j.foodcont.2016.04.039 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
sufficient step for inactivating pathogens before consumption, such as heating, fresh-cut vegetable is prone to an increased risk of public health (Beuchat, 2004). Therefore, disinfection of fresh-cut produce is desirable to control spoilage bacteria and fungi to extend shelf life and eliminate pathogens for food safety (McKellar et al., 2004; Qiang, Demirkol, Ercal, & Adams, 2005). The microbial contamination of fresh-cut vegetables during production and process is generally controlled by chemicals application, such as sodium hypochlorite, organic acid, ozone, chlorine and chlorine-based products, electrolyzed oxidizing (EO) water (Akbas & Olmez, 2007; Alexopoulos et al., 2013; Alwi & Ali, 2014; Hao, Li, Wan, & Liu, 2015; Raiputta, Setha, & Suthiluk, 2013; Wang, Feng, & Luo, 2004). Although many types of chemical are used for decontamination, more effective, appropriate products and methods are still searched by fresh-cut fruit and vegetable industry to ensure the safety of the fresh produce. Slightly acidic electrolyzed water (SAEW), a novel disinfectant, has been increasingly used to decontaminate fresh produce due to its quick on-site production and economic cost (Bosilevac, Shackelford, Brichta, & Koohmaraie, 2005; Koide, Shitanda, Note, & Cao, 2011). SAEW with a pH of 5.0e6.5 and a high concentration of hypochlorous acid is generated by electrolysis of dilute NaCl
148
C. Zhang et al. / Food Control 69 (2016) 147e152
and/or HCl solution in a non-membrane electrolytic chamber (Jadeja, Hung, & Bosilevac, 2013). SAEW was proved contains largely hypochlorous acid, an incredibly effective form of the chlorine, which is greater than that of hypochlorite ion for inactivating bacteria at a same free chlorine concentration and treatment time. The strong bactericidal, fungicidal, sporicidal and virucidal ability of SAEW have been reported in numerous previous studies (Cao, Zhu, Shi, Wang, & Li, 2009; Deza, Araujo, & Garrido, 2007; Hao et al., 2015; Hao et al., 2013; Jadeja & Hung, 2014; Koide et al., 2011; Nan et al., 2010; Rahman, Park, Song, Al-Harbi, & Oh, 2012; Zhang, Li, Jadeja, & Hung, 2016; Zheng et al., 2013). In addition, the disinfection efficacy of SAEW in reducing and eliminating pathogenic microorganisms on fresh produce has been reported (Abadias, Usall, Oliveira, Alegre, & Vinas, 2008; Hao et al., 2015; Koide, Takeda, Shi, Shono, & Atungulu, 2009; Pangloli & Hung, 2011; Zhang, Cao, Hung, & Li, 2016; Zhang et al., 2011). The objectives of this study were to evaluate the effects of different available chlorine concentrations (ACC, 15, 20, 25 and 30 mg/L), treatment time (1, 3, 5 and 7 min) and temperatures (4, 20 and 37 C) of SAEW on reducing natural microbiota (total aerobic bacteria and yeast and mold) on fresh celery and cilantro. Finally, variation of microbiota population on SAEW treated celery and cilantro during storage at 4 and 20 C was also investigated. 2. Materials and methods 2.1. Preparation of slightly acidic electrolyzed water Slightly acidic electrolyzed water (SAEW) was produced by electrolysis of 9% HCl solution using a SAEW generator (Purester MP-600T, Morinaga Engineering Co., Ltd., Tokyo, Japan) with a nonmembrane electrolytic chamber. SAEW with pH of 5.76e6.05, oxidation reduction potential (ORP) of 879.3e923.6 mV and ACC of 15, 20, 25 and 30 mg/L were produced at ambient temperature prior to experiment. The physicochemical properties of SAEW were measured immediately after preparation. The pH and ORP values were measured using a dual scale pH/ORP meter (HM-30R, DKKTOA Corp., Tokyo, Japan) with a pH electrode (GST-5741C) ranged from 0.00 to 14.00 or an ORP electrode (PST-5721C) ranged from 0.0 to 2000.0 mV. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan). All chemicals used in the experiment were of analysis grade. 2.2. Sample preparation and disinfection experiments Fresh celery and cilantro used in this study were purchased from a local market in Beijing, China on the day of the experiment and used for experiment within 24 h. Soil, wilted leaf and foreign matter on the surface were removed and celery and cilantro were subsequently aseptically cut into about 5-cm long before each experiment, respectively. Each sample with a similar amount of leaves and stems was prepared for bactericidal analysis. 2.2.1. Effect of SAEW with different ACC on microbiota inactivation SAEW with ACC of 15, 20, 25 and 30 mg/L at room temperature were used in this study. Each 25 g of sample was individually soaked with 225 mL of SAEW in a sterilized beaker flask and agitated vigorously at 150 rpm (COS-110 50, Shanghai Zollo Instrument Co., Ltd., China) for desired contact time. At the end of treatment time, 10 mL of treatment solution was immediately taken into 90 mL of sterilized neutralizing buffer (0.5% sodium thiosulphate þ 0.03 M phosphate buffer solution, pH 7) and mixed completely to terminate the residue chlorine reaction. Counts of viable total aerobic bacteria were obtained by plating 0.1 mL of 10-
fold serial dilution of mixture onto sterile nutrient agar (Aiboxing Bioscience Inc., Beijing, China) and plates were incubated at 37 C for 48 h before enumeration. Rose Bengal medium (Aiboxing Bioscience Inc., Beijing, China) with chloramphenicol addition was used for determining the population of viable yeast and mold. Plates were incubated at 25 C for 5 days. Another 25 g of sample treated with 225 mL sterilized 0.85% saline solution was used as a control. The control sample was washed at 150 rpm agitation for 5 min at room temperature to obtain the initial population of natural microbiota on the surface of analyzed celery and cilantro. Ten plates were used to be separately spread by plating 0.1 mL of bacterial suspension and the detection limit of this method is 2.0 log CFU/g. The experiment was replicated in triplicate. 2.2.2. Effect of SAEW on microbiota inactivation for different treatment time Each 25 g of celery and cilantro sample was treated with 225 mL of SAEW with ACC of 25 mg/L at room temperature for 1, 3, 5 and 7 min, respectively. After treatment terminated by neutralizing buffer, viable colonies of total aerobic bacteria and yeast and mold were enumerated by plating count according to the method described in 2.2.1. 2.2.3. Effect of SAEW at different temperatures on microbiota inactivation Freshly prepared SAEW in sealed brown glass bottles were stored in 4, 20 and 37 C water bath for 1 h, separately, and properties (ACC, pH and ORP) of SAEW were measured before and after water bath. Each 25 g of celery and cilantro sample was treated with 225 mL of SAEW of 4, 20 and 37 C with 25 mg/L available chlorine for 5 min. The following procedure of viable colonies count was conducted according to the previous method described in 2.2.1. 2.2.4. Procedure of storage experiment Each 200 g of celery and cilantro sample (uncut) was treated using the method described above by 1800 mL of SAEW with 25 mg/L ACC at room temperature for 5 min. Treated samples were drained and dried in the bio-safety cabinet for 2 h, then packaged in sterile stomacher bags and stored at 4 and 20 C, respectively. A 25 g of sample was collected for bactericidal analysis at 0, 2, 4 and 6 days storage using the method described in 2.2.1. Samples treated with 0.85% saline solution were stored at 4 and 20 C as control (untreated). Appearance quality of celery and cilantro including overall quality, color, texture, skin integrity, and aroma were also taken into consideration. Three to five lab members were invited for this test and compared the appearance quality of test sample with the fresh one by naked eyes in this study. 2.3. Statistical analysis For each treatment, results presented were obtained from independent replicate trials and the means and the standard deviations were calculated. Statistical analysis was performed using SAS software 9.2 (Institute, Inc., Cary, NC, USA). Least significance difference of means test was used for multiple comparisons at the 0.05 probability level. 3. Results and discussion 3.1. Effect of SAEW with different ACC on reducing microbiota on celery and cilantro After treatments of various SAEW, the surviving population of total aerobic bacteria and yeast and mold on the surface of celery and cilantro are shown in Fig. 1. The initial population of total
C. Zhang et al. / Food Control 69 (2016) 147e152
aerobic bacteria, yeast and mold on celery in control were 7.61 and 6.17 log CFU/g (Fig. 1A), on cilantro in control were 7.35 and 5.86 log CFU/g (Fig. 1B), respectively. The surviving population of total aerobic bacteria and yeast and mold on celery was 5.31, 4.84, 3.82, 3.0 log CFU/g and 4.84, 3.20, 2.48 2.0 log CFU/g after treated with SAEW at ACC of 15, 20, 25 and 30 mg/L, respectively, while 5.57, 4.56, 3.48, 2.72 log CFU/g and 4.63, 3.13, 2.29, 2.0 log CFU/g was observed for total aerobic bacteria, and yeast and mold on cilantro treated with SAEW at the same ACC. The surviving population of total aerobic bacteria and yeast and mold decreased significantly with ACC of SAEW increased from 15 to 30 mg/L, in comparison with control for both celery and cilantro (p < 0.05). This result was in agreement with the previous study, in which SAEW was used to inactivate Escherichia coli O157:H7 and Salmonella enteritidis on mung beans that surviving population of these two types of microorganism was decreased with ACC of SAEW increased (Zhang et al., 2011). Celery and cilantro treated with SAEW at 30 mg/L ACC achieved surviving population of total aerobic bacteria by 3.0 and 2.72 log CFU/g, while surviving population of yeast and mold to non-detectable level (detection limit is 2.0 log CFU/g in this study). The reason that aerobic bacteria is more resistant to SAEW than yeast and mold could be that initial population of total aerobic bacteria is slightly higher than yeast and mold, in addition, aerobic bacteria population on vegetables which grown close to the soil surface, may include several spore-forming bacteria, which are known to be more resistant to sanitizers or extreme environment (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000). In previous study, E. coli O157:H7 and Bacillus subtilis vegetative cell in pure culture at the population level of 7 log could be completely inactivated by SAEW with ACC of 0.5 mg/L for 30 s, while in the present study higher ACC and longer treatment time required to achieve the same result (Zhang, Li, Jadeja, Fang & Hung, 2016). A possible explanation for this difference could be that microbiota on fresh produce was more resistant to SAEW than those in pure culture. When microorganism attached to fresh produce, they might be in more complicate environment contaminated with foreign matter including organic matter which was reported could neutralize free chlorine and protect cells from membrane permeabilization (Rule, Ebbett, & Vikesland, 2005; Virto, Manas, Alvarez, Condon, & Raso, 2005). Aerobic bacteria, and yeast and mold observed on the surface of celery and cilantro could affect the shelf-life of vegetables. Moreover, pathogens may also remain in these vegetables prior to use and could do harm to human health. In this study, SAEW showed highly effective in eliminating both types of microorganism from
9 8 7 6 5 4 3 2 1 0
Bb
B c
control
15
3.2. Effect of SAEW on reducing microbiota on celery and cilantro for different treatment time The surviving population of total aerobic bacteria, and yeast and mold on the surface of celery and cilantro after treated with SAEW at the ACC of 25 mg/L for 1, 3, 5 and 7 min are shown in Fig. 2. With the exception of treatment for 5 and 7 min in Fig. 2B, the surviving microbial population (total aerobic bacteria, yeast and mold) decreased significantly after celery and cilantro was treated with SAEW from 1 to 7 min (p < 0.05). The population of total aerobic bacteria, and yeast and mold on celery samples in control were 7.61 and 6.17 log CFU/g (Fig. 2A), on cilantro samples were 7.35 and 5.86 log CFU/g (Fig. 2B), however, after treatment with SAEW for 7 min, the surviving population of total aerobic bacteria was 2.98 and 2.69 log CFU/g on celery and cilantro, while yeast and mold were at nondetectable level. Therefore, celery and cilantro after SAEW treatment with ACC of 25 mg/L for 7 min could be used safely since microbial population was maintained at a low or non-detectable level. In previous study, surviving population of E. coli O157:H7, Listeria monocytogenes and Salmonella typhimurium natural on green onions and tomatoes exposed to acidic EO water decreased, in response to increasing exposure time from 0 to 5 min (Park, Alexander, Taylor, Costa, & Kang, 2009). The result of our study was in agreement with the previous study which also indicated more reduction could be obtained with elevated treatment time when fresh produces was treated with EO water (Hung, Tilly, & Kim, 2010; Pangloli & Hung, 2013; Sharma & Demirci, 2003; Udompijitkul, Daeschel, & Zhao, 2007; Zhang et al., 2011). Longtime exposure could allow EO water to exert more effective effect on decontamination of fresh-cut celery and cilantro, and no negative sensory quality was observed (data not shown). 3.3. Effect of SAEW at different temperatures on reducing microbiota on celery and cilantro Effects of SAEW with ACC of 25 mg/L on decontamination of natural microbiota on fresh celery and cilantro at temperature of 4, 20 and 37 C for 5 min were determined and the results are presented in Fig. 3. The surviving population of total aerobic bacteria, yeast and mold on celery after treated with SAEW of 4, 20 and 37 C were of 3.57, 3.28, 3.22 log CFU/g and 2.47, 2.31, 2.16 log CFU/g
yeast and mold
A a
fresh-cut celery and cilantro. Additionally, SAEW treatments with ACC varying from 15 to 30 mg/L did not adversely altered the visual appearance of celery and cilantro (data not shown).
C d
20 25 ACC (mg/L) A
D
e
30
total aerobic bacteria Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
149
8 7 6 5 4 3 2 1 0
yeast and mold
A a
B
b
C c
control
15
D d
20 25 ACC (mg/L) B
E
d
30
Fig. 1. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW at different ACC. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
150
C. Zhang et al. / Food Control 69 (2016) 147e152
9 8 7 6 5 4 3 2 1 0
yeast and mold
A a
B C
b
control
D
c
d
E
1 3 5 Treatment time (min) A
e
total aerobic bacteria 8 7 6 5 4 3 2 1 0
Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
7
yeast and mold
A B
a
C
b
D
c
d
control
D
1 3 5 Treatment time (min) B
d
7
Fig. 2. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW for different time. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
9 8 7 6 5 4 3 2 1 0
yeast and mold
A a B b
control
B
b
4 20 Temperature (°C) A
B
b
37
total aerobic bacteria Surviving population (log CFU/g)
Surviving population (log CFU/g)
total aerobic bacteria
8 7 6 5 4 3 2 1 0
yeast and mold
A a B
B b
control
b
4 20 Temperature (°C) B
B
b
37
Fig. 3. The surviving population of natural microbiota on the surface of celery (A) and cilantro (B) after treated with SAEW at different temperature. Bars labeled with no common uppercase letters in the population of total aerobic bacteria are significantly different. Bars labeled with no common lowercase letters in the population of yeast and mold are significantly different (p < 0.05).
(Fig. 3A), respectively, 3.32, 3.25, 2.92 log CFU/g and 2.27, 2.11, 2.0 log CFU/g (Fig. 3B) were observed on cilantro as well. Results showed that the surviving population of total aerobic bacteria and yeast and mold slightly decreased when samples were treated with SAEW at temperature of 4, 20 and 37 C, although there was no significant difference observed in celery and cilantro among SAEW of different temperatures (4, 20 and 37 C) (p > 0.05). This result was in agreement with the previous study, in which more reductions were achieved with increasing temperature of EO water from 4 to 50 C (Rahman, Ding, & Oh, 2010). They found that reductions of L. monocytogenes were 4.98, 5.0, 5.20, 6.20 and 7.42 logs CFU/ml after low concentration EO water treatment at 4, 15, 23, 35 and 50 C, respectively. On the other hand, similar reductions were found for another three types of bacteria (S. typhimurium, Staphylococcus aureus and E. coli O157:H7) after treated with the same EO water. Room temperature or higher could slightly improve the reaction of EO water and microorganism on the surface of fresh produce. It was reported that the population of total aerobic bacteria and yeast and mold in carrot samples was lower after treated with mildly heated (45 C) SAEW than that at the temperature of 18 C (Koide et al., 2011). In this study, available chlorine of SAEW was measured before and after SAEW was stored in water bath to reach desired temperature (4, 20 and 37 C) and no change was observed. It was also reported that the pH, ORP and residual chlorine of EO water was almost no changed at temperature 4 and 24 C
(Hung et al., 2010). No negative variation was observed on sensory quality of celery and cilantro treated with SAEW containing 25 mg/ L available chlorine for 5 min at 4, 20 and 37 C (data not shown). 3.4. Growth of natural microbiota on treated celery and cilantro by SAEW during storage Fresh celery and cilantro were treated with SAEW with ACC of 25 mg/L for 5 min at room temperature and stored at 4 and 20 C. The surviving population of total aerobic bacteria and yeast and mold on these two vegetables were detected at storage time of 0, 2, 4 and 6 days, respectively. Celery and cilantro treated with 0.85% saline were stored at 4 and 20 C as control (untreated). Results presented in Fig. 4 that the surviving population of total aerobic bacteria and yeast and mold on celery after treatment slowly increased when it was stored at 20 C. In comparison with day 0 at 20 C, the surviving population of total aerobic bacteria on treated celery and the control on day 6 achieved an additional 0.66 and 1.25 log CFU/g, while an additional 0.14 and 0.05 log CFU/g were observed on day 6 at 4 C. On the other hand, an increase of total aerobic bacteria of 1.20 log CFU/g was obtained when comparing untreated (control) sample stored at 20 C with that stored at 4 C, while 0.80 log CFU/g increase of total aerobic bacteria on treated samples. The appropriate temperature, 20 C, for example, could allow bacteria to grow on both treated sample and untreated
C. Zhang et al. / Food Control 69 (2016) 147e152
10 9 8 7 6 5 4 3 2 1 0 0
2
4 °C control 4 °C
20 °C control 20 °C
4
6
Yeaat and mold population (log CFU/g)
Total aerobic bacterial population (log CFU/g)
4 °C control 4 °C
151
20 °C control 20 °C
8 7 6 5 4 3 2 1 0 0
2
4
6
Time (day)
Time (day)
4 °C
20 °C
control 4 °C
control 20 °C Yeast and mold population (log CFU/g)
Total aerobic bacterial population (log CFU/g)
Fig. 4. Growth of natural microbiota on the surface of celery during storage at 4 and 20 C after treated with SAEW.
10 9 8 7 6 5 4 3 2 1 0 0
2
4
6
4 °C
20 °C
control 4 °C
control 20 °C
8 7 6 5 4 3 2 1 0 0
2
4
6
Time (day)
Time (day)
Fig. 5. Growth of natural microbiota on the surface of cilantro during storage at 4 and 20 C after treated with SAEW.
sample. However, the increase of microbiota population on stored celery samples was not very high. The reason could be that celery before storage was dry and no proper humidity for bacteria to grow and reproduce quickly. Similar trend of variation was observed on population of yeast and mold on celery. An additional 1.0 and 1.25 log CFU/g were observed on treated and untreated samples which were stored at 20 C for 6 days. Low temperature (4 C) may not allow yeast and mold to grow and reproduce, since only 0.09 and 0.01 log CFU/g additional counts were obtained on untreated and treated celery samples after stored for 6 days at 4 C. As can be seen in Fig. 4, the microbial population on treated samples increased very slowly or even no increase in the first two days storage at 20 C. In contrast, an obvious increase was observed on untreated samples during the same storage period. It could be that bacteria on sample which was treated with SAEW may be injured to some extent and it would take some time to recover and reproduce when storage at room temperature. The similar variation trend for microbial population was observed on cilantro which was treated and stored at the same condition (Fig. 5). It was reported that population of Salmonella inoculated on celery significantly decreased by 0.5e1.0 log CFU/g under 4 C storage and 2.0 log CFU/g increased under 22 C storage. In addition, the similar results were found for E. coli O157:H7 and L. monocytogenes inoculated on celery (Vandamm, Li, Harris, Schaffner, & Danyluk, 2013). In our study, the microbial population on both celery and cilantro was increased when stored at 20 C, which was in agreement with the previous study mentioned as above, whereas it did not agree with the result at 4 C since no
microbial population decreased when stored at 4 C in our study. The microbial population of total aerobic bacteria, yeast and mold were significantly reduced after SAEW treatment with 25 mg/ L available chlorine for 5 min at room temperature. Moreover, it could extend the shelf-life of celery and cilantro, because the microbial population grew slowly and maintained at a low level, which would do no harm to human health. The microbial inactivation results as well as the absence of any sensory alterations on the exposed fresh celery and cilantro render SAEW a promising disinfectant, which can be used in fresh produce against foodborne disease. 4. Conclusions The effect of SAEW on inactivation of natural microbiota on celery and cilantro significantly increased with increasing ACC and treatment time (P < 0.05), and was not significantly affected by temperature of SAEW. The microbial population grew slowly and maintained at a low level during storage after celery and cilantro treated with SAEW. The main finding of this study suggests that SAEW is highly effective in eliminating microbiota on the surface of celery and cilantro and can be used in fresh produce against natural microbiota. Acknowledgments This work was supported by Beijing Municipal Commission of Education, university discipline group construction (No.
152
C. Zhang et al. / Food Control 69 (2016) 147e152
100190553). The author thanks Dr. Ravirajsinh Jadeja for specific comments on the preparation of this manuscript. References Abadias, M., Usall, J., Oliveira, M., Alegre, I., & Vinas, I. (2008). Efficacy of neutral electrolyzed water (NEW) for reducing microbial contamination on minimallyprocessed vegetables. International Journal of Food Microbiology, 123, 151e158. Akbas, M. Y., & Olmez, H. (2007). Effectiveness of organic acid, ozonated water and chlorine dippings on microbial reduction and storage quality of fresh-cut iceberg lettuce. Journal of the Science of Food and Agriculture, 87, 2609e2616. Alexopoulos, A., Plessas, S., Ceciu, S., Lazar, V., Mantzourani, I., Voidarou, C., et al. (2013). Evaluation of ozone efficacy on the reduction of microbial population of fresh cut lettuce (Lactuca sativa) and green bell pepper (Capsicum annuum). Food Control, 30, 491e496. Alwi, N. A., & Ali, A. (2014). Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Control, 46, 304e311. Beuchat, L. R. (2004). Difficulties in eliminating human pathogenic microorganisms on raw fruits and vegetables. Horticulture: Art and Science for Life, 642, 151e160. Bosilevac, J. M., Shackelford, S. D., Brichta, D. M., & Koohmaraie, M. (2005). Efficacy of ozonated and electrolyzed oxidative waters to decontaminate hides of cattle before slaughter. Journal of Food Protection, 68, 1393e1398. Cao, W., Zhu, Z. W., Shi, Z. X., Wang, C. Y., & Li, B. M. (2009). Efficiency of slightly acidic electrolyzed water for inactivation of Salmonella enteritidis and its contaminated shell eggs. International Journal of Food Microbiology, 130, 88e93. Deza, M. A., Araujo, M., & Garrido, M. J. (2007). Efficacy of neutral electrolyzed water to inactivate Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus on plastic and wooden kitchen cutting boards. Journal of Food Protection, 70, 102e108. Hao, J. X., Li, H. Y., Wan, Y. F., & Liu, H. J. (2015). Combined effect of acidic electrolyzed water (AcEW) and alkaline electrolyzed water (AlEW) on the microbial reduction of fresh-cut cilantro. Food Control, 50, 699e704. Hao, X. X., Shen, Z. Q., Wang, J. L., Zhang, Q., Li, B. M., Wang, C. Y., et al. (2013). In vitro inactivation of porcine reproductive and respiratory syndrome virus and pseudorabies virus by slightly acidic electrolyzed water. The Veterinary Journal, 197, 297e301. Hung, Y. C., Tilly, P., & Kim, C. (2010). Efficacy of electrolyzed oxidizing (Eo) water and chlorinated water for inactivation of Escherichia coli O157:H7 on strawberries and broccoli. Journal of Food Quality, 33, 559e577. Jadeja, R., & Hung, Y. C. (2014). Efficacy of near neutral and alkaline pH electrolyzed oxidizing waters to control Escherichia coli O157:H7 and Salmonella Typhimurium DT 104 from beef hides. Food Control, 41, 17e20. Jadeja, R., Hung, Y. C., & Bosilevac, J. M. (2013). Resistance of various shiga toxinproducing Escherichia coli to electrolyzed oxidizing water. Food Control, 30, 580e584. Koide, S., Shitanda, D., Note, M., & Cao, W. (2011). Effects of mildly heated, slightly acidic electrolyzed water on the disinfection and physicochemical properties of sliced carrot. Food Control, 22, 452e456. Koide, S., Takeda, J., Shi, J., Shono, H., & Atungulu, G. G. (2009). Disinfection efficacy of slightly acidic electrolyzed water on fresh cut cabbage. Food Control, 20, 294e297. Lu, Z. X., Yu, Z. F., Gao, X., Lu, F. X., & Zhang, L. K. (2005). Preservation effects of gamma irradiation on fresh-cut celery. Journal of Food Engineering, 67, 347e351. Lynch, M. F., Tauxe, R. V., & Hedberg, C. W. (2009). The growing burden of foodborne outbreaks due to contaminated fresh produce: Risks and opportunities. Epidemiology & Infection, 137, 307e315. McKellar, R. C., Odumeru, J., Zhou, T., Harrison, A., Mercer, D. G., Young, J. C., et al. (2004). Influence of a commercial warm chlorinated water treatment and packaging on the shelf-life of ready-to-use lettuce. Food Research International, 37, 343e354. Nan, S. J., Li, Y. Y., Li, B. M., Wang, C. Y., Cui, X. D., & Cao, W. (2010). Effect of slightly acidic electrolyzed water for inactivating Escherichia coli O157:H7 and Staphylococcus aureus analyzed by transmission electron microscopy. Journal of Food Protection, 73, 2211e2216.
Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J., & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and Molecular Biology Reviews, 64(3), 548e572. Pangloli, P., & Hung, Y. C. (2011). Efficacy of slightly acidic electrolyzed water in killing or reducing Escherichia coli O157:H7 on iceberg lettuce and tomatoes under simulated food service operation conditions. Journal of Food Science, 76, M361eM366. Pangloli, P., & Hung, Y. C. (2013). Reducing microbiological safety risk on blueberries through innovative washing technologies. Food Control, 32, 621e625. Park, E. J., Alexander, E., Taylor, G. A., Costa, R., & Kang, D. H. (2009). The decontaminative effects of acidic electrolyzed water for Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on green onions and tomatoes with differing organic demands. Food Microbiology, 26, 386e390. Qiang, Z., Demirkol, O., Ercal, N., & Adams, C. (2005). Impact of food disinfection on beneficial biothiol contents in vegetables. Journal of Agricultural and Food Chemistry, 53, 9830e9840. Rahman, S. M. E., Ding, T., & Oh, D. H. (2010). Effectiveness of low concentration electrolyzed water to inactivate foodborne pathogens under different environmental conditions. International Journal of Food Microbiology, 139, 147e153. Rahman, S. M., Park, J., Song, K. B., Al-Harbi, N. A., & Oh, D. H. (2012). Effects of slightly acidic low concentration electrolyzed water on microbiological, physicochemical, and sensory quality of fresh chicken breast meat. Journal of Food Science, 77, M35eM41. Raiputta, J., Setha, S., & Suthiluk, P. (2013). Microbial reduction and quality of freshcut 'phulae' pineapple (ananas comosus) treated with acidic electrolyzed water. In VII International Postharvest Symposium (Vol. 1012, pp. 1049e1055). Rule, K. L., Ebbett, V. R., & Vikesland, P. J. (2005). Formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan. Environmental Science & Technology, 39, 3176e3185. Sahib, N. G., Anwar, F., Gilani, A. H., Hamid, A. A., Saari, N., & Alkharfy, K. M. (2013). Coriander (Coriandrum sativum L.): a potential source of high-value components for functional foods and nutraceuticals - A review. Phytotherapy Research, 27, 1439e1456. Sharma, R. R., & Demirci, A. (2003). Treatment of Escherichia coli O157:H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water. International Journal of Food Microbiology, 86, 231e237. Udompijitkul, P., Daeschel, M. A., & Zhao, Y. (2007). Antimicrobial effect of electrolyzed oxidizing water against Escherichia coli O157:H7 and Listeria monocytogenes on fresh strawberries (Fragaria x ananassa). Journal of Food Science, 72, M397eM406. Vandamm, J. P., Li, D., Harris, L. J., Schaffner, D. W., & Danyluk, M. D. (2013). Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery. Food Microbiology, 34, 151e157. Virto, R., Manas, P., Alvarez, I., Condon, S., & Raso, J. (2005). Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorinedemanding substrate. Applied and Environmental Microbiology, 71, 5022e5028. Wang, H., Feng, H., & Luo, Y. G. (2004). Microbial reduction and storage quality of fresh-cut cilantro washed with acidic electrolyzed water and aqueous ozone. Food Research International, 37, 949e956. Zhang, C. L., Cao, W., Hung, Y. C., & Li, B. M. (2016a). Application of electrolyzed oxidizing water in production of radish sprouts to reduce natural microbiota. Food Control, 67, 177e182. Zhang, C. L., Li, B. M., Jadeja, R., Fang, J. L., & Hung, Y. C. (2016b). Effects of bacterial concentrations and centrifugations on susceptibility of Bacillus subtilis vegetative cells and Escherichia coli O157:H7 to various electrolyzed oxidizing water treatments. Food Control, 60, 440e446. Zhang, C. L., Li, B. M., Jadeja, R., & Hung, Y. C. (2016c). Effects of electrolyzed oxidizing water on inactivation of Bacillus subtilis and Bacillus cereus spores in suspension and on carriers. Journal of Food Science, 81(1), M144eM149. Zhang, C. L., Lu, Z. H., Li, Y. Y., Shang, Y. C., Zhang, G., & Cao, W. (2011). Reduction of Escherichia coli O157:H7 and Salmonella enteritidis on mung bean seeds and sprouts by slightly acidic electrolyzed water. Food Control, 22, 792e796. Zheng, W. C., Kang, R., Wang, H., Li, B. M., Xu, C., & Wang, S. (2013). Airborne bacterial reduction by spraying slightly acidic electrolyzed water in a laying-hen house. Journal of the Air & Waste Management Association, 63, 1205e1211.