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Dong-Hyun Kang, Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376, USA. E-mail: email@example.com
Aims: To evaluate the efficacy of acidic electrolysed water (EW) in the presence of organic matter (bovine serum) on the inoculated surfaces of lettuce and spinach.
Materials and Results: Lettuce and spinach leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes and treated with deionized water, acidic EW and acidic EW containing bovine serum (5, 10, 15 and 20 ml l−1) for 15 s, 30 s, 1 min, 3 min and 5 min at room temperature (22 ± 2°C). In the absence of bovine serum, acidic EW treatment reduced levels of cells below the detection limit (0·7 log) in 5 min. In the presence of bovine serum, bactericidal activity of acidic EW decreased with increasing serum concentration.
Conclusions: Organic matter reduces the effectiveness of acidic EW for reducing pathogens on the surfaces of lettuce and spinach.
Significance and Impact of the Study: From a practical standpoint, organic matter reduces the efficacy of acidic EW. This study was conducted to confirm the effect of organic matter on the properties of acidic EW in the inactivation of foodborne pathogens on the surface of vegetables.
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Consumption of minimally processed and fresh-cut vegetables is on the rise in quantity and variety in recent years because of their convenience and importance. However, raw fruits and vegetables can be contaminated by pathogenic bacteria through wash water, rodents, or insects in the production plant, shredders or slicers with poor sanitization, and infected workers (Beuchat and Brackett 1990; Lee and Kang 2001). Furthermore, pathogenic bacteria can grow on fresh fruits and vegetables during transportation and storage (Beuchat and Brackett 1990). These organisms have been implicated in outbreaks of foodborne illnesses and recalls (IFPA 2000; Beuchat 2002; FDA News 2006a,b). Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes are major foodborne pathogens linked to consumption of contaminated fresh vegetables worldwide (Beuchat 1998).
A variety of sanitizers, including chlorine, chlorine dioxide, ozone, hydrogen peroxide, UV light, calcinated water, organic acids and acidic electrolysed water, have been evaluated for their ability to reduce levels of pathogenic micro-organisms on fresh produce (Zhang and Farber 1996; Bari et al. 1999; Kim et al. 2000a). Chlorinated water (50–200 ppm) is widely used to reduce levels of micro-organisms (Beuchat 1998). However, most sanitizers showed a minimal microbial reduction of less 2 log CFU g−1 on the inoculated fresh fruits and vegetables, similar to that of chlorinated water (Beuchat 1999; Taormina and Beuchat 1999). A problem with chlorine treatment is that the efficacy of chlorinated water is reduced in the presence of organic materials (Gelinas and Goulet 1983).
Electrolysed water (EW) is generated from electrolysis of a 0·1% NaCl solution through an electrolysing chamber. Using a two-cell chamber containing anode and cathode, acidic electrolysed water (acidic EW) and alkaline electrolysed water (alkaline EW) is produced in anode and cathode compartments, respectively. Acidic EW has a low pH (<2·5) and a strong oxidizing potential (approx. 1100 mV). Conversely, alkaline EW has a high pH (>11·0) and a strong reducing potential (approx. 800 mV) (Anon 1997). Acidic EW is reported to have strong bactericidal activity against most pathogenic micro-organisms (Izumi 1999; Kim et al. 2000a). The decontaminative effects of acidic EW on the surfaces of lettuce, tomatoes, strawberries, cucumbers and spinach have been reported (Koseki et al. 2001, 2004; Bari et al. 2003; Guentzel et al. 2008). Acidic EW effectively inactivated E. coli O157:H7, Salmonella, L. monocytogenes and Bacillus cereus on the surfaces of vegetables (Venkitanarayanan et al. 1999; Kim et al. 2000b; Park et al. 2001). In general, aerobic bacteria grow in a pH range of 4–9, and at an ORP range of + 200 to 800 mV. Low pH may reduce the intensity of the bacterial outer membrane and allow entry of chlorine compounds through the inner membrane. Some mechanisms of bactericidal activity of chlorine compounds that have been proposed include killing the bacteria through glucose oxidation, disruption of protein synthesis, or destruction of a key enzyme (Huang et al. 2008). Liao et al. (2007) proposed a bactericidal theory based on the high oxidation potential of acidic EW causing damage to cell membranes. Therefore, the bactericidal activities of acidic EW may be dependent on the kinds of bacterial species and can be seen by using a scanning electron microscope (Liao et al. 2007). Wrinkled cell membranes are not an easy target for attack by acidic EW compared to smooth ones (Osafune et al. 2006). Park et al. (2004) demonstrated that acidic EW is very effective for inactivating and killing E. coli O157:H7 and L. monocytogenes.
Several factors such as the variety vegetable surfaces, kinds of contaminating micro-organisms, and soils present on vegetables, affect the inactivating effect of sanitizers on pathogens during food processing. Organic matter reduces the efficacy of sanitizers on pathogens in fresh produce. The presence of organic matter reduced the bactericidal effect of acidic EW. Nutrient broth, proteose peptone, glycine, glucose, sucrose, corn oil and chicken serum react with free available chlorine, reducing the effect of acidic EW (Oomori et al. 2000; Ayebah et al. 2005). Protein matter reacts with chlorine to form organochloramine and could reduce the oxidizing effects of acidic EW (White 1999). Since there are currently no reports on the efficacy of acidic EW against foodborne pathogens on the surfaces of fresh vegetables in the presence of organic matter, it must be established in order to optimize sanitization procedures. In practical usage, the effect of organic matter on the sanitizing effect of acidic EW is the challenge to the food processing industry. Other researchers reported that proteins such as proteose peptone and chicken serum reduced the effectiveness of acidic EW (Oomori et al. 2000; Ayebah et al. 2005). Therefore, bovine serum was chosen to simulate organic matter contamination of produce since it can be easily controlled and quantified, whereas natural soiling would be difficult to replicate and quantify under laboratory conditions.
In the present study we examined the effects of organic matter, such as bovine serum, on the properties of acidic EW and efficacy of acidic EW in the inactivation of foodborne pathogens such as E. coli O157:H7, S. Typhimurium, and L. monocytogenes on the surfaces of lettuce and spinach.
Materials and methods
Three strains each of E. coli O157:H7 (ATCC 35150, ATCC 43889 and ATCC 43890), S. Typhimurium (ATCC 19585, ATCC 13311 and ATCC 14028), and L. monocytogenes (ATCC 19113, ATCC 19114 and ATCC 7644) were obtained from the Food Science and Human Nutrition culture collection at Washington State University (Pullman, WA, USA). These strains are of both human and veterinary clinical origin. All strains of E. coli O157:H7, S. Typhimurium or L. monocytogenes were grown in 9 ml Tryptic Soy Broth (TSB; Difco, Chicago, IL, USA) at 37°C for 24 h, collected by centrifugation at 4000 g for 30 min at 4°C, washed three times with 5 ml of Buffered Peptone Water (BPW; Difco), and resuspended to 1/10 the original volume in 5 ml BPW (Difco), corresponding to approx. 108–109 CFU ml l−1. To inoculate lettuce and spinach, all pathogen strains were combined to construct culture cocktails and maintained at 22 ± 2°C. These culture cocktails were used in subsequent experiments within 1 h of preparation.
Preparation of inoculated sample
Iceberg lettuce and baby spinach were purchased at a local supermarket (Pullman, WA, USA). Lettuce and spinach leaves were trimmed to 10 g, washed for 1 min with deionized water, drained, separated and placed on sterile aluminium foil in a laminar flow biosafety hood. For inoculation, 0·1 ml of each pathogen cocktail was applied as 20 droplets onto leaf surfaces with a micropipettor. Inoculated leaves were air dried in the hood for 3 h with the fan running for bacteria to attach to leaf surfaces. To determine the initial number of the three pathogens inoculated onto surfaces of lettuce and spinach (0·1 ml), cell concentrations of culture cocktails were enumerated by conventional plating on selective media.
Preparation of acidic EW
Acidic EW was generated using a Super Oxide Series Π electrolysed water generator (Proton Lab., Portland, OR, USA). This apparatus generates electrolysed water by the electrolysis of a 0·1% sodium chloride solution for 15 min. The initial available chlorine content and pH of the mixture were determined by chlorine test kit (Bio-Lab Co., GA, USA) and a Corning Instruments pH meter (Corning, NY, USA), respectively. Acidic EW was prepared on the day of experiments and used within 1 h of production. The temperature of acidic EW was 24 ± 2°C for the entire experiment.
Effect of organic matter on the pH and available free chlorine content of acidic EW
Different volumes (2, 4, 6, 8, 10, 15 and 20 ml l−1) of sterile bovine serum (60 mg ml l−1 protein, Sigma, St Louis, MO, USA) were added to acidic EW. These mixtures were stirred 5 min using a Corning Instruments magnetic stirrer (Corning, NY, USA). The residual available free chlorine content and pH of the mixture were measured using previously described methods.
Treatment of E. coli with acidic EW in the presence of organic matter
Different volumes (2, 4, 6, 8 and 10 ml l−1) of sterile filtered bovine serum (Sigma) were added to acidic EW, and mixed for 5 min as described previously. A volume of 0·1 ml of a three strain E. coli culture cocktail (109 CFU ml−1) was added to 9·9 ml of deionized water (DW, control), acidic EW and acidic EW containing different concentrations bovine serum for 30 s, 1 min, 3 min and 5 min. Samples (1 ml) were taken at treatment time intervals, neutralized with 9 ml of D/E broth (Difco). The neutralized mixture was serially diluted in 9 ml of sterile buffered peptone water (Difco), and 0·1 ml of sample or diluent was spread-plated onto each selective medium. All tests were performed at room temperature (22 ± 2°C).
Treatment of inoculated lettuce or spinach with acidic EW in the presence of organic matter
Inoculated lettuce or spinach leaves were immersed in 500 ml of DW, acidic EW, and acidic EW with different concentrations (5, 10, 15 and 20 ml l−1) of sterile filtered bovine serum, and agitated for <1s (momentary dispersal), followed by stationary exposure in the treatment solutions at one-minute intervals for 15 s, 30 s, 1 min, 3 min and 5 min. At the selected time intervals, lettuce or spinach leaves (10 g) were removed from treatment solutions and immediately placed in a stomacher bag containing 50 ml of neutralizing D/E broth (Difco) and homogenized for 2 min with a Seward stomacher (400 Circulator, Seward, London, UK). After homogenization, 1 ml of neutralized mixture was serially diluted in 9 ml of sterile buffered peptone water (Difco), and 0·1 ml of sample or diluent was spread-plated onto each selective medium. All tests were performed at room temperature (22 ± 2°C).
Escherichia coli O157:H7, S. Typhimurium, and L. monocytogenes were enumerated on Sorbitol MacConkey agar (SMAC; Difco), Xylose Lysine Desoxycholate agar (XLD; Difco), and modified Oxford Agar Base (OAB; Difco) with antimicrobic supplement (Bacto™ Oxford antimicrobic supplement, Difco), respectively. Where low levels of surviving cells were anticipated, 1 ml of undiluted aliquots was equally distributed between four plates of each selective medium and spread-plated. All inoculated enumeration media were incubated at 37°C for 24–48 h, and then the presence of typical colonies were enumerated.
Three replicate trials for each experiment were performed. The data were analysed by analysis of variance using the anova procedure of sas (SAS Institute, Cary, NC, USA). Means were separated using Duncan’s multiple range tests. Significant differences between mean values are presented at a level of P = 0·05.
The initial pH and free available chlorine concentration of acidic EW were 2·06 and 37·5 ± 2·5 mg l−1, respectively. The detection limit for free chlorine analysis was 5 mg l−1. Fig. 1 shows the changes of pH and free available chlorine of acidic EW mixed with different volumes of bovine serum. As the bovine serum concentration increased from 0 to 10 ml l−1, the free available chlorine disappeared from 35 to 0 mg l−1. The addition of bovine serum up to 20 ml l−1 did not significantly affect the pH of acidic EW.
The population of E. coli O157:H7 was reduced to below the detection limit (0·7 log) with acidic EW treatment in the presence of bovine serum from 0 to 4 ml l−1 after 15-s treatment. With bovine serum at a concentration of 6 and 8 ml l−1 in acidic EW, the survival of E. coli O157:H7 after treatment generally increased (Fig. 2). As bovine serum concentration increased from 10 up to 20 ml l−1, there were no significant reductions in the population of E. coli O157:H7 after treatment with acidic EW.
Reductions calculated are values exceeding those of the water control treatment observed at each exposure time. Shown in Figs 3–5 are the surviving cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes from lettuce leaves treated with DW, acidic EW, and acidic EW with various concentration of bovine serum (5, 10, 15 and 20 ml l−1), respectively. Treatment with only acidic EW reduced levels of E. coli O157:H7 on lettuce to below the detection limit (0·7 log) after 3 min (Fig. 3). The levels of surviving cells treated with acidic EW containing serum at 5 ml l−1 were significantly reduced by 1·27, 1·70, 2·10, 2·89 and 3·68 log CFU g−1 after 15 s, 30 s, 1 min, 3 min and 5 min treatment (P < 0·05), respectively, compared to the deionized water control. At the 10 ml l−1 containing serum, the numbers of E. coli O157:H7 reduced by 1·16, 1·59, 1·88, 2·42 and 3·07 log CFU g−1 at the selected time intervals, respectively (P < 0·05). However, there were no significant differences between levels of E. coli O157:H7 cells treated with acidic EW containing serum at 15 and 20 ml l−1 compared with the water control (DW). For S. Typhimurium (Fig. 4), there were no significant differences between treatments with DW and acidic EW containing serum at 10, 15 and 20 ml l−1 at all treatment times (P > 0·05). However, acidic EW treatment alone showed significant reduction of 4·53 log CFU g−1 after 30 s and more than 6·17 log CFU g−1 (detection limit 0·7 log) after 3 min treatment (P < 0·05). At 5 ml l−1 concentration, levels of S. Typhimurium were significantly reduced after 15 s, 30 s, and 1 min; however, there was no significant difference compared with DW after 3 min. Fig. 5 shows surviving cells of L. monocytogenes enumerated on OAB agar after treatment with test solutions. The number of L. monocytogenes cells experienced significant reductions of 2·49, 3·96, 4·66 and 4·98 log CFU g−1 after 15 s, 30 s, 1 min, and 3 min of treatment with acidic EW alone, respectively (P < 0·05). After 5 min of treatment, levels of cells were reduced to below the detection limit (0·7 log). At all treatment solution containing different volume of bovine serum, however, there was no statistically significant difference up to 3 min treatment (P > 0·05). After 5 min, the surviving cells of L. monocytogenes were significantly reduced by 3·08 log CFU g−1 at 5 ml l−1 serum containing acidic EW compared with DW (P < 0·05).
The surviving cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes after exposure to DW, acidic EW, and acidic EW containing bovine serum on spinach leaves are shown in Figs. 6, 7 and 8, respectively. Figure 6 shows the population of E. coli O157:H7 after 15 s, 30 s, 1 min, 3 min and 5 min treatment with selected solutions. In the absence of serum, the numbers of cells were reduced to below the detection limit (0·7 log) at 3 min exposure. With serum at a concentration of 5 ml l−1, 1·67, 2·15, 2·89, 3·66 and 4·80 log CFU g−1 significant reductions of E. coli O157:H7 were detected after 15 s, 3 s, 1 min, 3 min and 5 min treatment, respectively (P < 0·05). As the serum concentration increased to 10 ml l−1, the survival of E. coli O157:H7 after treatment with acidic EW containing 10 ml l−1 serum was reduced by 1·17, 1·88, 2·29, 3·26 and 3·89 log CFU g−1 after each exposure time, respectively. There were significant differences in numbers of surviving cells between DW and acidic EW containing 10 ml l−1 serum after 3 min treatment (P < 0·05). The populations of E. coli O157:H7 after treatment with acidic EW in the presence of 15 and 20 ml l−1 serum showed no significant differences compared to DW. Fig. 7 shows levels of surviving cells of S. Typhimurium after treatment with solutions. Acidic EW treatment alone reduced microbial levels by 2·22 and 4·82 log CFU g−1 after 15 s and 30 s, respectively. And after 1 min, this pathogen was reduced to below the detection limit (0·7 log). The survival of S. Typhimurium after treatments with acidic EW containing bovine serum is a result lowered bactericidal activity. No significant reductions were observed in acidic EW containing 15 and 20 ml l−1 serum compared with DW at all exposure times. L. monocytogenes (Fig. 8) experienced significant reductions of 2·64, 4·13 and 5·18 log CFU g−1 after 15 s, 30 s and 1 min of treatment, respectively, with acidic EW in the absence of serum (P < 0·05). After treatment with acidic EW containing serum at 5 ml l−1, levels of L. monocytogenes were significantly reduced by 2·16, 2·56, 3·68 and 4·26 log CFU g−1 after 30 s, 1 min, 3 min and 5 min, respectively (P < 0·05), compared to the deionized water control. However, no significant differences in survival were detected with acidic EW amended with serum at 10, 15 and 20 ml l−1 (P > 0·05).
In this study, we evaluated the effects of acidic EW in reducing foodborne pathogens in the presence of organic matter. No significant reductions (<1 log) occurred when inoculated lettuce and spinach were subjected to the water control treatment. This may be the result of physical removal of bacterial cells from vegetable surfaces. Investigations of the bactericidal activity of acidic EW have been conducted for lettuce, tomatoes, cucumbers, and strawberries (Koseki et al. 2001; Park et al. 2001; Bari et al. 2003). However, there were differences in removal of surface micro-organisms depending on the vegetable studied. Because lettuce and tomatoes have relatively smoother surfaces than cucumbers and strawberries, acidic EW may more easily contact surface micro-organisms on those vegetables (Koseki et al. 2004). In this study, there were no significant difference in acidic EW activity between lettuce and spinach. Perhaps the smoother surface of spinach facilitated better contact with acidic EW. The bactericidal effects of acidic EW on foodborne pathogens are well documented by numerous studies. Koseki et al. (2003) investigated the effect of spot inoculated lettuce leaves treated with acidic EW containing 40 ppm free chlorine for 3 min. They observed an approximately 4·6 and 4·4 log CFU g−1 reduction of E. coli O157:H7 and S. Typhimurium, respectively. Ayebah et al. (2005) showed that acidic EW produced a > 6 log CFU ml−1 reduction in L. monocytogenes after a 1 min treatment. These results show that acidic EW is effective at killing pathogens on vegetables and resulted in above 4·0 log CFU g−1 reduction for 1 min treatment. However, there are several studies that showed that acidic EW was less effective at killing pathogens on vegetables than previous studies. Park et al. (2001) demonstrated that a 1 min treatment of acidic EW containing 45 ppm free chlorine significantly reduced E. coli O157:H7 and L. monocytogenes by 2·41 and 2·61 log CFU g−1 on lettuce, respectively. Izumi (1999) reported that immersing lettuce in acidic EW containing 45 ppm free chlorine for 1 min results in reduction of 2·78 and 2·38 log CFU g−1 of E. coli O157:H7 and L. monocytogenes, respectively. In practical usage, however, acidic EW generally is used in the presence of organic matter, such as soils or vegetable debris. In our study, treatment with acidic EW containing bovine serum as a form of organic matter resulted in a lower bactericidal effect on foodborne pathogens, including E. coli O157:H7, S. Typhimurium, and L. monocytogenes. In the absence of serum, acidic EW completely inactivated all pathogens within 5 min. As the serum concentration increased, the free available chlorine content decreased rapidly. The effect of organic matter on chlorinated water has been demonstrated by several researchers. White (1999) demonstrated that proteins can react with chlorine and results in formation of organichloramines. The neutralization of chlorine in acidic EW may due to the addition of organic matter (Ayebah et al. 2005). El-Kest and Marth (1988) reported that as organic matter present increased, free available chlorine content decreased. Tanaka et al. (1996) reported that organic materials convert free available chlorine in acidic EW to the combined form. Also, Oomori et al. (2000) concluded that the bactericidal activity of acidic EW declines in the presence of organic materials, such as amino acids and proteins. In their study, materials containing amino acids or proteins were quickly transformed from free available chlorine into N-chloro compounds (combined available chlorine). They also observed that free available chlorine in acidic EW was removed through the reaction of oxidation–reduction with corn oil, vitamins, minerals or lipids. Ayebah et al. (2005) studied that the effect of acidic EW on the inactivation of planktonic cells and biofilms of L. monocytogenes in the presence of chicken serum. They reported that acidic EW treatment with 5 and 10 ml l−1 serum for 5 min reduced the population of L. monocytogenes by only 0·46 and 0·11 log CFU g−1 ml−1, respectively, compared to deionized water. Also, the populations of L. monocytogenes biofilms exposed to acidic EW alone and acidic EW with increasing serum concentration up to 7·5 ml l−1 for 60 s experienced reductions of 4·37 and 2·95 log CFU g−1, respectively. The results of our study show that acidic EW is an effective and convenient sanitizer for reduction of foodborne pathogens. However, several factors affect the bactericidal activity of acidic EW, including the type of food product and degree of organic matter contamination. Thus, for effective usage in the food processing industry, it is suggested that a pretreatment or preremoval step of organic debris, before use of sanitizers, is need to improve their activity.