Original papers
Efficacy of Slightly Acidic Electrolyzed Water for Reduction of Foodborne Pathogens and Natural Microflora on Shell Eggs
2014 年 20 巻 1 号 p. 93-100
詳細
2014 年 20 巻 1 号 p. 93-100
The efficacy of slightly acidic electrolyzed water (SAEW) to inactivate foodborne pathogens and indigenous microbiota on shell eggs was evaluated and compared to chlorine dioxide (CD), acidic electrolyzed water (AEW) and NaClO solution. The eggs were artificially inoculated with S. enteritidis, E. coli O157:H7 and S. aureus and sprayed or immersed with SAEW, alkaline electrolyzed water (AlEW) followed by SAEW (AlEW+SAEW), CD, AEW and NaClO solution, respectively. The effect of SAEW on the natural microbiota of shell eggs was also determined. Spraying shell eggs with SAEW, CD and NaClO solution at an ACC of 60 mg/L had no significant bactericidal difference for foodborne pathogens and indigenous microbiota on shell eggs, and the difference of disinfection effect between SAEW and AEW was not significant, whereas the bactericidal activity of SAEW for E. coli O157:H7, S. aureus, total aerobic bacteria and moulds and yeasts was significantly higher than that of CD and NaClO solution at ACCs of 80 or 100 mg/L. SAEW was found to be more effective when used in conjunction with AlEW, and higher reductions were obtained with the immersion treatment. Results indicate that the disinfectant efficiency of SAEW is equivalent to or higher than that of chlorine dioxide and NaClO solution and therefore SAEW shows the potential to be used for sanitization of egg shells as an environmentally friendly disinfection agent.
There has been increased concern about the safety of foods, especially products consumed fresh or slightly cooked. Salmonella spp, Escherichia coli O157:H7 and Staphylococcus aureus were reported to be the common foodborne pathogens that can cause human illness and death. The most frequent source of infection is raw or poorly cooked foods, including eggs, poultry, meat, fruits and vegetables (Hajmeera et al., 2006; Betancor et al., 2010). Several chemical disinfectants such as sodium hypochlorite, chlorine dioxide, hydrogen peroxide, organic acids and ozone have been available in food industries, and chlorinated water of 50 - 200 mg/L is the most commonly used sanitizer (Beuchat et al., 1998). However, chlorine is inactivated by organic materials and can also lead to the formation of potentially carcinogenic and teratogenic trihalomethanes and haloacetic acids, haloketones and chloropicrin (Allende et al., 2009; Gil et al., 2009; Keskinen et al., 2009). Therefore, developing effective methods for reducing or eliminating pathogens is crucial for food safety and human health.
Acidic electrolyzed water (AEW) and slightly acidic electrolyzed water (SAEW) with low chlorine concentrations have been developed as an alternative and a novel sanitizer in agriculture and food industries. AEW is generated by electrolysis of a dilute sodium chloride solution in an electrolytic cell where the anode and cathode are separated by a membrane and obtained from the anode side, and alkaline electrolyzed water (AlEW) is yielded from the cathode side. AEW has been reported to eliminate Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritidis and S. aureus on shell eggs and did not significantly affect albumen height or egg shell strength (Russell, 2003; Bialka et al., 2004). On the other hand, AlEW was reported to have an antioxidative effect on highly unsaturated fat and oils and have the superoxide dismutase-like and catalase-like activities (Miyashita et al.,1999; Shirahata et al., 1997). Additionally, eggs are usually washed in an alkaline detergent and then rinsed with a chlorine solution to reduce dirt, debris, and microbial load during commercial processing (Bialka et al., 2004), thus the high pH of AlEW may make it a substitute for the high pH detergents used to wash eggs. Sato et al. (2000) reported that the most effective use of electrolyzed water was washing with alkaline water to remove protein and other organisms followed by sanitizing with acidic water. Park et al. (2005) also demonstrated that a combination of AlEW and AEW wash is equivalent to 200 mg/L of chlorinated water wash for reducing populations of S.enteritidis and L.monocytogenes on shell eggs. Fasenko et al. (2009) found that the ability of AEW to reduce eggshell microbial load without negatively affecting hatchability or chick quality might make it a useful product for hatching egg sanitation. However, the use of AEW has limited potential for long-term applications because of its strong acidity (pH < 2.7), which may cause the corrosion of equipment and rapidly chlorine loss due to volatilization (Guentzel et al., 2008; Cao et al., 2009).
Slightly acidic electrolyzed water (SAEW) is a novel disinfectant with a pH value of 5.0 - 6.5, which is generated by electrolysis of a dilute hydrochloric acid and/or NaCl solution in a chamber without a membrane. At a pH of 5.0 - 6.5, the effective form of chlorine compounds in SAEW is mainly the hypochlorous acid (HOCl) having strong antimicrobial activity (Cao et al., 2009; Koide et al., 2009). With a near neutral pH, SAEW application seems promising as it minimizes human health and safety issues from Cl2 off-gassing, reduces corrosion of surfaces and limits phototoxic side effects (Guentzel et al., 2008). SAEW has been proved as an effective antimicrobial agent for inactivating E.coli, S.aureus and Salmonella spp in vitro (Issa-Zacharia et al., 2010). However, little information is available on the efficacy of SAEW to inactivate natural microflora on shell eggs. Therefore, the objective of this work was to evaluate the efficiency of SAEW to inactivate foodborne pathogens and indigenous microbiota on shell eggs and to compare the efficiency of SAEW with chlorine dioxide, AEW and NaClO solution.
Bacterial cultures Freeze-dried pure cultures of Escherichia coli O157:H7 ATCC35150 (human feces isolate) and Staphylococcus aureus ATCC6538 (raw milk isolate) were obtained from the China Veterinary Culture Collection (CVCC, Beijing, China), and the strain of Salmonella enteritidis used was SE596 (egg isolate), which was isolated by one of the authors. Each bacterium was hydrated according to manufacturer's directions and cultured in tryptic soy broth supplemented with 0.6% (w/v) yeast extract (TSBYE, CVCC, Beijing, China) at 37°C for 24 h. Cells of each culture were separately centrifuged at 3,000×g for 10 min at 4°C (3K15, Sigma, Germany) and the supernatants were discarded. The cell pellets were washed twice with 0.1% peptone water and then re-suspended in 10 mL of the same solution (Nan et al., 2010). Counts of viable bacteria were obtained by plating 0.1 mL tenfold serial dilution of broth cultures onto sterile tryptic soy agar supplemented with 0.6% (w/v) yeast extract (TSAYE, CVCC, Beijing, China) and incubating the plates at 37°C for 24 h. The population of S.enteritidis, E.coli O157:H7 and S.aureus in each culture was approximately 9.0 log10 CFU/mL.
Preparation and inoculation of shell eggs Eggs weighing 55 ± 2 g were collected from the 65-wk-old commercial layer chickens. Shell eggs were stored at 4°C for no more than 1 week before treatment. Shell eggs were equilibrated to room temperature before testing, then washed with tap water and a commercially available chlorine-based sanitizer at available chlorine concentration of 50 mg/L (Beijing Kelin Rongan Medical Technology Co. Ltd, Beijing, China) for 1 min, rinsed in sterile deionized water and dried for 1 h in a laminar flow safety hood. For inoculation, eggs were individually dipped into the inoculum prepared by placing 0.1 mL of approximately 109 CFU/mL S.enteritidis, E.coli O157:H7 and S.aureus suspension into 200 mL of sterile 0.1% peptone water for 10 min, respectively, and allowed to dry under a laminar flow safety hood for 1 h at a room temperature of 20 ± 2°C to provide time for the bacteria attaching.
Preparation of treatment solutions Slightly acidic electrolyzed water was generated using a SAEW generator (Beijing Zhouji Ziyuan Huanbao Technology Company Ltd., Beijing, China) that basically consists of a non-membrane electrolytic cell with anode and cathode electrodes. SAEW with the available chlorine concentration (ACC) of 60, 80 and 100 mg/L was produced by electrolysis of 10% NaCl (w/v) in the SAEW generator and the current was set at 8, 10 and 12 A, respectively. SAEW was collected from the anode compartment, and AlEW was obtained from the cathode side of the generator (Part of the product formed at the anode was redirected into the cathode chamber and mixed with the product formed at the cathode).
Acidic electrolyzed water was produced by electrolysis of a 0.1% NaCl solution in a chamber with a separating membrane between anode and cathode using an AEW generator (Beijing Zhouji Ziyuan Huanbao Technology Company Ltd., Beijing, China) following the manufacturer's instructions, and AEW was collected from the anode side of the AEW generator. Chlorine dioxide (CD) solution was diluted in sterile deionized water to obtain the same ACCs with SAEW. Sodium hypochlorite (NaClO) solution was prepared by diluting 8% NaClO solution using deionized water to obtain the final different concentrations, and deionized water was used as control for this experiment.
The ACC was determined by a colorimetric method with a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan), of which the detection limit is 0 - 300 mg/L. The pH and oxidation-reduction potential (ORP) of different solutions were measured using a dual scale pH/ORP meter (HM-30R, DKK-TOA Corporation, Tokyo, Japan) with a pH electrode(GST- 5741C) or an ORP electrode (PST-5721C).
Treatment of shell eggs with disinfectants Shell eggs individually inoculated with S.enteritidis, E.coli O157:H7 and S. aureus were placed in a sterile plastic egg plate, and then randomly divided into 18 treatment groups, each consisting of 5 eggs. The samples were treated by SAEW, AlEW+SAEW (alkaline electrolyzed water followed by slightly acidic electrolyzed water), CD, AEW and NaClO solution at 60, 80 and 100 mg/L of available chlorine, AlEW (Alkaline electrolyzed water) and deionized water, respectively. Spray disinfection was performed by spray the treatment solutions on the inoculated or uninoculated egg samples for 10 s (approximately 15 mL solution per egg) using a hand-operated manually sprayer. Immersion treatments were carried out by immersing the egg samples in each solution for 1 min. After treatment, the samples were allowed to dry for 30 min and then subjected to bacterial assay. The eggs with no treatment were used as the control and placed directly into sterile plastic bags for bacterial enumeration.
Microbiological analysis After the treatment, egg samples were aseptically transferred into a sterile plastic bag containing 25 mL of sterile neutralizing buffer solution (0.5% sodium thiosulphate + 0.03 M phosphate buffer solution, pH 7.2 - 7.4) and gently rubbed by hand for 1 min. The egg was then removed, and the neutralizing buffer solution was serially diluted in sterile 0.1% peptone water. The viable bacterial populations of S. enteritidis, E. coli O157:H7 and S. aureus were enumerated by plating 0.1 mL of the appropriate dilutions in triplicate on TSAYE plates and incubating at 37°C for 48 h before counting.
Total aerobic bacterial and staphylococci populations were determined by plating 0.1 mL of the appropriate dilutions on TSAYE plates and Baird-Parker Agar, respectively, and incubated at 37°C for 48 h before enumeration. Enterobacteriaceae counts were obtained by placing 1 mL of each dilution in violet red bile glucose agar pour plates, and incubated at 37°C for 48 h prior to counting. Moulds and yeasts count were enumerated by plating 0.1 mL of each solution onto potato dextrose agar plates, and incubated at 28°C for 3 - 5 days and examined. The weight of the shell was measured to determine the colony-forming unit of per gram of eggshell + membrane (CFU/g) by the method reported by Bialka et al. (2004). All microbial counts were expressed as log colony forming units per gram (log10 CFU/g). All analyses were made in triplicates at temperatures of 20 ± 2°C.
Statistical analysis For each treatment, data from independent replicate trials were collected and the means and standard deviations were calculated. All data were subjected to the general linear model procedure of SPSS 17.0 software (SPSS Inc, Chicago, IL). Duncan's multiple range test was used to separate means using a level of significance of P < 0.05.
Physicochemical properties of treatment solutions The values of pH, ORP and ACC of treatment solutions are shown in Table 1. Alkaline electrolyzed water (AlEW) had the highest pH value and lowest ORP, while acidic electrolyzed water (AEW) at an ACC of 100 mg/L had the lowest pH and highest ORP. At the same ACC, both chlorine dioxide (CD) and AEW had lower pH values than slightly acidic electrolyzed water (SAEW), AlEW and sodium hypochlorite (NaClO) solution. The ORP values of SAEW, AEW and CD solution were higher than those of AlEW, NaClO and deionized water, indicating that they contained more active oxidizers.
| Solutions | Available chlorine (mg/L) | pH values | ORP a(mV) |
|---|---|---|---|
| Deionized water | 0 | 6.15 ± 0.01 | 400 ± 5 |
| Alkaline electrolyzed water | 28 | 10.50 ± 0.01 | − 755 ± 5 |
| Slightly acidic electrolyzed water | 60 | 5.74 ± 0.02 | 902 ± 5 |
| 80 | 5.66 ± 0.01 | 912 ± 2 | |
| 100 | 5.67 ± 0.01 | 926 ± 4 | |
| Chlorine dioxide | 60 | 2.24 ± 0.03 | 840 ± 6 |
| 80 | 2.07 ± 0.01 | 853 ± 2 | |
| 100 | 1.95 ± 0.01 | 841 ± 2 | |
| Acidic electrolyzed water | 60 | 2.12 ± 0.02 | 1170 ± 3 |
| 80 | 1.99 ± 0.01 | 1182 ± 2 | |
| 100 | 1.88 ± 0.05 | 1187 ± 2 | |
| Sodium hypochlorite | 60 | 10.20 ± 0.06 | 501 ± 6 |
| 80 | 10.37 ± 0.03 | 477 ± 4 | |
| 100 | 10.50 ± 0.02 | 475 ± 3 |
Values are means ± standard deviation, n = 3
a Oxidation reduction potential
Efficiency of SAEW for inactivation of S.enteritidis, E.coli O157:H7 or S.aureus inoculated on shell eggs The bactericidal activity of SAEW for inactivating S.enteritidis, E.coli O157:H7 and S.aureus on shell eggs are presented in Fig.1. The initial viable cells recovered after 1 h of inoculation on shell eggs were 6.33, 6.45 and 6.35 log10 CFU/g for S.enteritidis, E.coli O157:H7 and S.aureus, respectively. Fig.1a showed that spraying with SAEW, AlEW + SAEW, CD, AEW and NaClO solution at ACCs of 60, 80 and 100 mg/L resulted in a reduction of 2.14 - 2.40, 2.36 - 2.82, 1.89 - 2.16, 2.07 - 2.40 and 1.98 - 2.22 log10 CFU/g for S.enteritidis on the shell eggs, respectively. The highest population reduction of S.enteritidis was observed in shell eggs treated with AlEW + SAEW100 with values of 2.82 log10 CFU/g. As compared to deionized water (DW), all treatment solutions reduced the population of S.enteritidis significantly (P < 0.05). The bactericidal activity of SAEW and AlEW + SAEW were significantly higher than that of CD and NaClO solution at ACCs of 80 and 100 mg/L(P < 0.05), however, no significant differences were observed between SAEW and NaClO solution at an ACC of 60 mg/L, and SAEW and AEW showed no significant sanitization difference at the same ACC. Moreover, CD and NaClO solution had no significant bactericidal difference (P > 0.05).
Reduction of S.enteritidis (a), E.coli O157:H7 (b) and S.aureus (c) after spraying or immersing shell eggs with slightly acidic electrolyzed water (SAEW), alkaline electrolyzed water followed by slightly acidic electrolyzed water (AlEW+SAEW), chlorine dioxide (CD), acidic electrolyzed water (AEW) and sodium hypochlorite (NaClO) solution at available chlorine concentrations of 60, 80 and 100 mg/L, and with deionized water (DW) and alkaline electrolyzed water (AlEW). Vertical bars represent mean ± standard deviations of mean. Bars labeled with different letters within the same application method between treatments indicate significant differences (P < 0.05).
Spray treatment of SAEW, AlEW + SAEW, CD, AEW and NaClO solution decreased the viable cells of E.coli O157:H7 on shell eggs by 1.50 - 2.71, 2.16 - 3.48, 1.49 - 2.17, 1.89 - 2.76 and 1.52 - 2.11 log10 CFU/g, respectively (Fig.1b). Combined treatment of AlEW+SAEW100 significantly (P < 0.05) decreased populations of E.coli O157:H7 by 3.48 log10 CFU/g compared with each treatment alone. In comparison with DW, AlEW, SAEW, AlEW+SAEW, CD, AEW and NaClO solution reduced the population of E.coli O157:H7 significantly (P < 0.05). AlEW+SAEW treatment showed markedly higher efficacy than SAEW, CD, AEW and NaClO treatment at ACCs of 60, 80 and 100 mg/L, and SAEW was more effective than CD and NaClO solution at an ACC of 100 mg/L, while no significant differences were found among SAEW, CD and NaClO solution at ACCs of 60 and 80 mg/L (P > 0.05). Furthermore, the differences of disinfection effect between SAEW and AEW, and between CD and NaClO were not significant (P > 0.05).
The reduction in S.aureus population on shell eggs after spraying with SAEW, AlEW+SAEW, CD, AEW and NaClO solution was 2.16 - 2.78, 2.17 - 2.81, 2.20 - 2.82, 2.20 - 3.01 and 2.17 - 2.75 log10 CFU/g, respectively (Fig.1c). A significant reduction in the population of S.aureus was observed when comparing SAEW, AlEW+SAEW, CD, AEW and NaClO to DW (P < 0.05), while no significant differences were found between SAEW and AlEW+SAEW at the same concentration, nor were there any significant differences between SAEW and AEW. Moreover, no significant differences were found among SAEW, CD and NaClO solution at ACCs of 60 and 100 mg/L, and CD and NaClO showed no significant bactericidal difference (P > 0.05). However, the population of S.enteritidis, E.coli O157:H7 and S.aureus on the surface of shell eggs was only reduced by 0.80 - 1.14 log10 CFU/g for DW and 0.86 - 1.75 log10 CFU/g for AlEW treatment. When eggs were immersed with treatment solutions, although higher reductions were obtained for all treatments, the best results were also achieved with AlEW+SAEW100 for S.enteritidis and S.aureus and AlEW+SAEW80 for E.coli O157:H7 (Fig.1). SAEW and AEW had similar bactericidal activities for each pathogen, and there were no significant differences among SAEW, CD and NaClO solution at an ACC of 60 mg/L (P > 0.05), but SAEW100 was statistically more effective at reducing S.enteritidis population than CD100 and NaClO100 (P < 0.05).
Effect of SAEW on the natural microflora of shell eggs The microbial counts naturally present on shell eggs treated with SAEW, AlEW+SAEW, CD, AEW and NaClO solution with ACC of 60, 80 and 100 mg/L are given in Table 2. The initial microbial populations of total aerobic bacteria, coliforms, staphylococci, and moulds and yeasts in control were 6.52, 4.37, 4.78, and 4.27 log10 CFU/g, respectively. When spray disinfection was used, the reductions of indigenous microbiota on shell eggs were 1.30 - 2.97, 1.38 - 3.40, 1.25 - 2.67, 1.79 - 2.72 and 1.38 - 2.65 log10 CFU/g of total aerobic bacteria for SAEW, AlEW+SAEW, CD, AEW and NaClO solution, respectively. SAEW, AlEW+SAEW, CD, AEW and NaClO solution reduced the populations of total aerobic bacteria significantly when compared with DW (P < 0.05). At an ACC of 80 mg/L, the reduction obtained after spraying with SAEW was significantly higher than that obtained with CD and NaClO solution (P < 0.05), whereas no significant differences were found among SAEW, CD and NaClO solution at ACCs of 60 and 100 mg/L (P > 0.05). Additionally, the differences between SAEW and AEW at ACCs of 80 and 100 mg/L, and between CD and NaClO solution were not statistically significant (P > 0.05), and AlEW+SAEW at an ACC of 100 mg/L yielded the lowest bacterial counts than any other treatment.
| Treatment | Surviving population (log10 CFU/g) | |||||||
|---|---|---|---|---|---|---|---|---|
| Total aerobic bacteria | Coliforms | Staphylococci | Moulds and yeasts | |||||
| Spraying | Immersion | Spraying | Immersion | Spraying | Immersion | Spraying | Immersion | |
| Control | 6.52 ± 0.16a | 6.52 ± 0.16a | 4.37 ± 0.09a | 4.37 ± 0.09a | 4.78 ± 0.15a | 4.78 ± 0.15a | 4.27 ± 0.06a | 4.27 ± 0.06a |
| DW | 5.67 ± 0.05b | 5.64 ± 0.17b | 4.20 ± 0.23ab | 3.42 ± 0.40b | 4.49 ± 0.29a | 4.40 ± 0.39ab | 3.47 ± 0.20b | 2.81 ± 0.51b |
| AlEW | 5.44 ± 0.22bc | 5.33 ± 0.07b | 4.19 ± 0.16ab | 3.48 ± 0.11b | 4.45 ± 0.12a | 3.54 ± 0.24bc | 3.28 ± 0.31bc | 2.58 ± 0.34bc |
| SAEW60 | 5.22 ± 0.05cde | 3.69 ± 0.54cd | 4.02 ± 0.21abc | 2.65 ± 0.68cd | 3.79 ± 0.22b | 3.25 ± 0.30cd | 2.95 ± 0.16cd | 2.61 ± 0.57bc |
| SAEW80 | 4.20 ± 0.06g | 3.10 ± 0.15efg | 3.65 ± 0.64cdef | ND | 3.61 ± 0.37bcd | 2.16 ± 0.24e | 2.16 ± 0.24fgh | ND |
| SAEW100 | 3.55 ± 0.34i | 3.44 ± 0.19cdef | 2.95 ± 0.60ghi | ND | 3.66 ± 0.07bc | ND | 1.74 ± 0.17hi | ND |
| AlEW+SAEW60 | 5.14 ± 0.08cde | 2.97 ± 0.63fgh | 3.45 ± 0.17defg | 1.84 ± 0.17f | 3.71 ± 0.29b | 2.27 ± 0.57e | 2.72 ± 0.26de | 1.84 ± 0.17d |
| AlEW+SAEW80 | 4.15 ± 0.18gh | 2.51 ± 0.51h | 3.38 ± 0.38efgh | ND | 3.31 ± 0.11d | ND | ND | ND |
| AlEW+SAEW100 | 3.12 ± 0.16j | ND* | 3.18 ± 0.12fghi | ND | 2.70 ± 0.18e | ND | ND | ND |
| CD60 | 5.27 ± 0.04cd | 3.81 ± 0.51c | 4.06 ± 0.31abc | 3.18 ± 0.22bc | 3.81 ± 0.19b | 2.86 ± 0.18cd | 2.80 ± 0.65de | 2.63 ± 0.33bc |
| CD80 | 5.09 ± 0.15de | 3.38 ± 0.11cdefg | 3.87 ± 0.20abcde | 2.17 ± 0.40de | 3.62 ± 0.39bcd | 2.27 ± 0.57e | 2.88 ± 0.05cde | ND |
| CD100 | 3.85 ± 0.34hi | 3.61 ± 0.18cde | 3.19 ± 0.23fghi | ND | 3.36 ± 0.26cd | ND | ND | ND |
| AEW60 | 4.73 ± 0.09f | 3.64 ± 0.05cde | 3.73 ± 0.04bcde | 2.20 ± 0.57de | 3.69 ± 0.14bc | 3.19 ± 0.20cd | 2.73 ± 0.52de | 2.38 ± 0.50bc |
| AEW80 | 4.20 ± 0.30g | 3.13 ± 0.50defg | 3.43 ± 0.31defg | 1.97 ± 0.17e | 3.64 ± 0.16bcd | 2.11 ± 0.06e | 2.12 ± 0.23fgh | 1.17 ± 0.27e |
| AEW100 | 3.80 ± 0.15i | 2.84 ± 0.34gh | 2.74 ± 0.51i | ND | 3.35 ± 0.04cd | ND | 1.59 ± 0.22i | ND |
| NaClO60 | 5.14 ± 0.06cde | 3.84 ± 0.17c | 4.01 ± 0.06abc | 2.58 ± 0.62cde | 3.78 ± 0.06b | 2.57 ± 0.50d | 2.27 ± 0.10fg | 2.29 ± 0.75cd |
| NaClO80 | 4.93 ± 0.24ef | 3.67 ± 0.26cde | 3.91 ± 0.13abcd | 2.26 ± 0.35de | 3.60 ± 0.13bcd | 2.20 ± 0.15e | 2.50 ± 0.30ef | 1.84 ± 0.13d |
| NaClO100 | 3.87 ± 0.24hi | 3.19 ± 0.49defg | 2.92 ± 0.09hi | ND | 3.32 ± 0.05d | ND | 1.98 ± 0.23ghi | ND |
Control (not treated), DW (deionized water), AlEW (Alkaline electrolyzed water), SAEW (slightly acidic electrolyzed water), AlEW + SAEW (alkaline electrolyzed water followed by slightly acidic electrolyzed water), CD (chlorine dioxide), AEW(acidic electrolyzed water), and NaClO (sodium hypochlorite) solution at available chlorine concentrations of 60, 80 and 100 mg/L, Values reported as the means of triplicate measurements ± standard deviation. Means within the same column with different lowercase letters were significantly different (P < 0.05).
*Not detectable on direct plate count, and negative on enrichment media.
The population of coliforms on shell eggs was reduced by 0.35 - 1.42, 0.92 - 1.19, 0.31 - 1.18, 0.64 - 1.63 and 0.36 - 1.45 log10 CFU/g for treatment of SAEW, AlEW+SAEW, CD, AEW and NaClO solution when compared with the control group. In comparison with DW, SAEW80, SAEW100, AlEW+SAEW, CD100, AEW80, AEW100 and NaClO100 reduced the population of coliforms significantly (P < 0.05), while SAEW60, CD60, CD80, AEW60, NaClO60 and NaClO80 were no more effective than DW, and there were no significant difference among SAEW, CD, AEW and NaClO solution at the same ACC (P > 0.05). Similar results were also obtained in the disinfection efficacy to shell eggs on the population of staphylococci. When compared to untreated control, SAEW, AlEW+SAEW, CD, AEW and NaClO solution decreased the population of staphylococci by 0.99 - 1.17, 1.07 - 2.08, 0.97 - 1.43, 1.00 - 2.08 and 0.97 - 1.46 log10 CFU/g, respectively. With regard to DW treatment, SAEW, AlEW+SAEW, CD, AEW and NaClO solution reduced the population of staphylococci significantly (P < 0.05), while no significant differences were observed among SAEW, CD and AEW, and SAEW was not significantly different from NaClO at ACCs of 60 and 80 mg/L (P > 0.05). AlEW+SAEW at ACC of 100 mg/ L was the most effective treatment and achieved 2.08 log10 CFU/g reduction for staphylococci.
It was also found that SAEW, AlEW+SAEW, CD, AEW and NaClO treatment significantly reduced the populations of moulds and yeasts relative to DW treatment group (P < 0.05). Treatment of AlEW+SAEW at ACCs of 80 and 100 mg/L and CD at 100 mg/L decreased the population to undetectable levels, achieving reduction of 4.27 log10 CFU/g for moulds and yeasts. No significant differences in population of moulds and yeasts were found among SAEW60, CD60 and CD80, and the differences of disinfection effect between SAEW and AEW, and between SAEW and NaClO solution at ACCs of 80 and 100 mg/L were not significant (P > 0.05). However, DW treatment reduced the populations by 0.17 - 0.85 log10 CFU/g, while AlEW treatment showed slightly greater reduction of 0.18 - 1.08 log10 CFU/g for total aerobic bacteria, coliforms, staphylococci, and moulds and yeasts, respectively compared to the untreated control. For the immersion treatment, SAEW, AlEW + SAEW, CD, AEW and NaClO solution exhibited significant decreases in indigenous microbiota on shell eggs when compared with DW, ranging from 1.53 - 6.52 log10 CFU/g, and reductions were always higher than spraying treatment. The population of total aerobic bacteria was reduced to undetectable levels with AlEW+SAEW at ACC of 100 mg/L, similar results were also observed with SAEW100, AlEW+SAEW80, AlEW+SAEW100, CD100, AEW100, and NaClO100, achieving reductions of 4.37, 4.78 and 4.27 log10 CFU/g for coliforms, staphylococci and moulds and yeasts, respectively.
The results obtained in this study showed that SAEW with an ACC of 60 - 100 mg/L had the comparative or higher powerful bactericidal activity to reduce foodborne pathogens and indigenous microbiota present on shell eggs compared to chlorine dioxide solution at the same available chlorine concentration. Several studies have investigated the efficacy of AEW on shell eggs. Bialka et al. (2004) evaluated the efficacy of AEW (pH of 2.7, free chlorine level of 70 - 80 mg/L) for inactivating S.enteritidis and Escherichia coli K12 on artificially inoculated shell eggs and found reductions of more than 2.1 and 2.3 log10 CFU/g for S.enteritidis and E.coli K12 after a 3 min immersion treatment at 45°C, respectively. Our results showed that spraying eggs with SAEW and AEW at an ACC of 80 mg/L reduced about by 2.30 - 2.33 log10 CFU/g for Salmonella enteritidis, and 1.90 - 2.00 for E.coli O157:H7, respectively, and a reduction of 2.35 - 2.43 log10 CFU/g for S.enteritidis and 2.67 - 2.68 log10 CFU/g for E.coli O157:H7 were obtained when using immersion method. This may be in part due to differences in the way the SAEW and AEW was applied to the eggs and the treatment temperature and time. It has been reported that mild heat treatment improved the efficacy of a sanitizer at killing or removing microorganisms on produce (Rahman et al., 2011).
Our results also revealed that combined AlEW and SAEW treatment decreased more foodborne pathogens or indigenous microbiota on shell eggs when compared to each single treatment alone, which demonstrate a synergistic effect of AlEW and enhancing bactericidal activity of SAEW on shell eggs. The reductions of foodborne pathogens on shell eggs by the combination treatment of AlEW and SAEW at an ACC of 60 mg/L are similar to chlorine dioxide and NaClO solution with ACCs of 80 or 100mg/L, indicating a potential for combining SAEW and AlEW to replace high concentration of chlorinated water for shell egg washing. The results of our study indicated that spray application was less effective than immersion. However, spraying is more easily accomplished in most situations, particularly on a farm, and is more practical than immersion (Davies et al., 1997; Cox et al., 1997). Moreover, most eggs purchased in the shell are required to be washed and sanitized in the United States, but immersion is disallowed during washing or sanitization (Musgrove et al., 2010).
In addition, our study indicated that the differences of disinfection effect between SAEW and AEW were not statistically significant, and SAEW had an equivalent or high disinfectant efficacy for shell eggs compared to NaClO solution, which agree with the findings of Cao et al. (2009), who demonstrated that SAEW had similar bactericidal activities with AEW and NaClO solution at the same ACC. The results of a study by Zhang et al. (2011) showing that SAEW is an effective method to reduce foodborne pathogens on seeds and sprouts with less effects on the viability of seeds. Pangloli et al. (2011) suggested that application of SAEW to wash and chill lettuce and tomatoes in food service kitchens could minimize cross-contamination and reduce the risk of E.coli O157:H7 present on the produce. The results obtained by Issa-Zacharia et al. (2011) and Hao et al. (2011) showed that SAEW and NaClO solution had no significant sanitization difference and SAEW could be an alternative of AEW and NaClO solution. These results indicate that SAEW with a near neutral pH may be a potential sanitizer represent an alternative to chlorine dioxide and NaClO solution used in food industry.
In conclusion, this present study demonstrated that SAEW has an equivalent or higher efficiency to reduce S.enteritidis, E.coli O157:H7, S.aureus and indigenous microbiota present on shell eggs compared to chlorine dioxide and NaClO solution, and has similar bactericidal activities with AEW at the same available chlorine concentration. The combination treatment of SAEW with AlEW had better reduction than SAEW or AlEW alone. Therefore, SAEW shows the potential to be used for sanitization of egg shells as an environmentally friendly disinfection agent. Further studies should be elucidated to determine the levels of bacterial inoculation and the synergistic effects of combining technologies.
Acknowledgments This study was supported by the China Agricultural Research System (Project No: CARS- 41), the Chinese Universities Scientific Fund (Grant No: 2012YJ015), and by the Program for Key Laboratory of Animal Disease Prevention and Food Safety of Sichuan Province. The authors are thankful to Beijing Zhouji Ziyuan Huanbao Co Ltd., China, for providing the electrolyzed water generators.
