Abstract
Salmonella spp. are one of the most common causes of foodborne disease outbreaks worldwide. Disinfectants are widely used in the food industry to reduce pathogen contamination, but the increase in antimicrobial resistance has reinforced the global need for effective and environmentally friendly alternatives. In addition, Salmonella Heidelberg, an emergent serotype, has been described as highly persistent in facilities of poultry production chain. In this context, the present study aimed to evaluate the action of electrochemically activated water (ECAW), a biocide produced from water, salt, and electricity, against aerobic bacteria, including S. Heidelberg, experimentally inoculated in chicken breasts. Chicken breasts fragments (2 g) were inoculated by immersion in the bacterial inoculum solution and then were left in a petri dish for 10 min to allow microbial attachment. Fragments were treated by immersion in ECAW (treated group) or 0.1% sterile peptone water solution (control group) at 25 °C for 10 min. After, chicken breasts slices were transferred to sterile tubes and were incubated at 7 °C and at three contact times of 5, 30, and 60 min to simulate chiller environment. The average reduction was 1.07 log10 CFU/g after treatment, and the bacterial counts decreased significantly (p < 0.05) with increasing contact time. These results demonstrate the potential use of this technology in chicken slaughter plants.
Introduction
Salmonella spp. are common causes of foodborne disease outbreaks. Salmonellosis is the most common zoonotic disease in the United States, and the second most common the European Union (EU). In the EU, over 91,000 salmonellosis cases are reported annually, and the overall annual economic burden is approximately €3 billion. In the United States, 1.35 million infections occur each year, resulting in medical costs exceeding $3.7 billion (CDC, 2023; EFSA, 2023; USDA, 2023). Poultry is an important source of Salmonella; therefore, the control of this pathogen is essential during the entire production chain, especially during slaughter, due to cross-contamination (Wessels et al., 2021).
Companies routinely adopt several control programs. However, the identification of a variety of Salmonella serotypes is challenging. The emergence of Salmonella Heidelberg in Canada, the United States, and Brazil in the last decade has demonstrated the potential for the persistence of this serotype in poultry farms and slaughterhouses (Souza et al., 2019). S. Heidelberg establishes a condition similar to the commensal microbiota of birds but is one of the main serotypes responsible for outbreaks related to the consumption of poultry products (Dewi et al., 2021; Gieraltowski et al., 2016).
Disinfectants are widely used in the food industry to reduce pathogen contamination and eliminate spoilage bacteria (Maillard and Pascoe, 2024). However, antimicrobial resistance is a global concern, and in recent years, there has been a need for effective and environmentally friendly alternatives. Electrochemically activated water (ECAW) is a biocidal, nontoxic, and biodegradable compound produced from water, salt, and electricity. Hypochlorous acid (HClO), its main component, acts by increasing cell membrane permeability, leading to the leakage of intracellular contents and decreased dehydrogenase and nitrate reductase activities (Zeng et al., 2010). Oxidation damages the cell membranes of microorganisms and affects their metabolic processes, resulting in cell death (Liao et al., 2007). In addition, HClO exhibits sanitizing power 80-fold greater than that of the hypochlorite ion (ClO–) at pH 5.0–6.5 (Rahman et al., 2016).
The antimicrobial and antibiofilm effect of ECAW against S. Heidelberg has been previously demonstrated in in vitro studies (Wilsmann et al., 2020, 2023). However, to date, there has been no information regarding the antimicrobial effect of ECAW against S. Heidelberg, a multidrug-resistant and biofilm-producing serotype (Borges et al., 2018; Gieraltowski et al., 2016), in chicken carcasses. Thus, the present study aimed to evaluate the antimicrobial activity of ECAW against aerobic bacteria, including S. Heidelberg, in experimentally inoculated into chicken breasts and to provide data for future regulation of the use of ECAW in Brazil.
Materials and methods
Slices of chicken breast
Chicken breasts were purchased from a local market in Rio Grande do Sul (RS, Brazil) and belonged to a single company under the authority of the Federal Inspection System (SIF). Chicken breasts were cut into 1 × 1 cm fragments (2 g each). Slices were placed in sterile Petri dishes and exposed to UV light for 15 min on each side to eliminate bacterial contamination (Morales-Partera et al., 2017). Control fragments were used to confirm the efficacy of sterilization by ultraviolet (UV) light.
Production of ECAW
ECAW was produced in a generator (Centrego, Frome, UK) with a production capacity of 200 L/h using supply water and a 0.1% sodium chloride (NaCl) solution. The free chlorine and oxidation-reduction potential (ORP) of the solution were measured using a Micro 7 Plus meter (Akso, São Leopoldo, Brazil) after ECAW production. The ORP values varied from 800 to 900 mV, and the average concentration of free chlorine obtained in the initial ECAW solution ranged from 350 to 400 ppm. The ECAW fresh solution was diluted with distilled water to a concentration of 50 ppm, which is the limit of free chlorine allowed in washing chicken carcasses, according to United States legislation (USDA, 2021). In Brazil, there is still no regulation regarding ECAW.
S. Heidelberg strains and inoculum preparation
Eight S. Heidelberg strains isolated from poultry sources between 2018 and 2019 were selected for the present study and analyzed in pools. Strains were serotyped by the Oswaldo Cruz Institute Foundation (Fiocruz, Brazil). The bacterial isolates were stored at ‒80 °C in brain heart infusion broth (BHI; Oxoid, Basingstoke, UK) supplemented with 15% glycerin (Synth, Diadema, Brazil). Strains were reactivated in BHI for 18–24 h at 37 °C and then in xylose lysine deoxycholate (XLD) agar (Oxoid) for 18–24 h at 37 °C.
The strains were cultured on 200 mL 0.1% sterile buffered peptone water solution (BPW; Merck; Darmstadt, Germany) for 24 h at 37 °C. The turbidity of the bacterial suspension was adjusted using a spectrophotometer SP-22 (Biospectro, Brazil) at a wavelength of 620 nm and ranged from 0.224 to 0.300, corresponding to 107 CFU/mL.
Inoculation of chicken breast slices
Chicken breast slices were inoculated following the methodology described by Morales-Partera et al. (2017). Briefly, fragments were divided in two groups: (1) fragments inoculated with S. Heidelberg and treated with ECAW (treated group); (2) fragments inoculated with S. Heidelberg and nontreated (control group).
All fragments were inoculated by immersion for 2 min in 18 mL of bacterial inoculum (ratio 1:10) to obtain an initial bacterial load of approximately 105 CFU/mL. After immersion, chicken breasts slices were placed on Petri dishes at room temperature (25 °C) for 10 min to allow microbial attachment. Fragments were then treated by immersion in 18 mL of ECAW (ratio 1:10) at room temperature (25 °C) for 10 min. After immersion, chicken breasts slices were transferred to sterile tubes and were incubated at 7 °C, which is the maximum temperature allowed for carcasses after the cooling stage (MAPA, 1998), for 5, 30, or 60 min. Nontreated fragments (control) were immersed in 0.1% sterile peptone water solution (APT; Merck, Darmstadt, Germany) instead of ECAW. A total of three chicken breasts fragments were used per treatment. The experiment was repeated three times.
Bacterial recovery
The number of bacteria remaining in the chicken breast slices was determined by counting. Fragments were immersed in 18 mL of 0.1% sterile BPW and homogenized by stomaching for 2 min. For bacterial counts in CFU/mL, serial dilutions (10−1 to 10−4) were performed in 0.85% sterile saline solution and then seeded on standard agar for counting (PCA; Merck; Darmstadt, Germany) for quantification using the drop plate method (Milles and Misra, 1938), followed by incubation at 37 °C for 24 h. The bacterial count (CFU/g) was determined as described by the International Organization for Standardization (ISO) guideline 7218 (2007).
Statistical analyses
All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). Descriptive statistics were used to determine the mean bacterial count for each treatment. Bacterial counts were transformed into log10. One-way analysis of variance (ANOVA), followed by Tukey's honest significant difference (HSD) test, was used to detect differences in bacterial counts among time of contacts and treatments and control. Statistical significance was set at p < 0.05.
Results and discussion
ECAW is an ecologically safe technology that promotes the rational use of water resources and reduces the volume of harmful substances discharged into the environment. In addition, the use of ECAW only requires basic occupational safety precautions. It is biodegradable and exhibits low cytotoxicity (Wang et al., 2019). Studies on electrolyzed water have been conducted in medicine, dentistry, agriculture, and the food industry. In food-processing plants, they have been used as antimicrobial agents in cutting tools, eggs, chicken carcasses, fruits, and vegetables (Afari and Hung, 2018; Huang et al., 2008; Wang et al., 2018; Yan and Xie, 2021).
The application of the ECAW laboratory environment or poultry slaughterhouses may not only reduce the initial bacterial load, but also extend the shelf life of carcasses. Duan et al. (2017) demonstrated that poultry carcasses sprayed with ECAW for 15 s had an extended shelf life when compared to other treatments with sodium hypochlorite (NaClO), dioxide, and lactic acid. The antibiofilm activity of ECAW against S. Heidelberg was previously demonstrated by our research group (Wilsmann et al., 2023). However, the antimicrobial activity of ECAW on chicken meat against this pathogen has not been studied.
According to the US legislation, electrochemically generated HClO can be used in various processes in the food industry (USDA, 2021). In the United States, a concentration of up to 50 ppm of free chlorine is allowed for washing poultry carcasses, chiller water, and offal (FDA, 2012). Previous studies have demonstrated that at this concentration, ECAW reduced the bacterial counts of S. Typhimurium, Campylobacter spp., and Listeria monocytogenes in broiler chicken carcasses (Hernández-Pimentel et al., 2020; Rahman et al., 2012a; Rasschaert et al., 2013). In Brazil, no legislation determines or regulates the use of ECAW to wash poultry carcasses. Initial tests carried out by our research group demonstrated that ECAW is capable of reducing S. Heidelberg in vitro at 50 ppm, but not at 5 ppm (Wilsmann et al., 2020). Therefore, 50 ppm was selected for subsequent concentration tests. The aerobic bacterial counts (CFU/g) after treatment ECAW on chicken breast experimentally inoculated with S. Heidelberg are shown in Table 1.
Mean bacterial counts (log10 CFU/g) after treatment with electrochemically activated water (ECAW) of chicken breasts experimentally inoculated with Salmonella Heidelberg, according to contact time.
Different lowercase letters on the same line indicate statistically significant differences (p < 0.05) among the contact times for the same treatment.
Different capital letters in the same column indicate statistically significant differences (p < 0.05) between treatment and control at the same contact time.
The aerobic bacterial count after treatment with 50 ppm ECAW was significantly lower (p < 0.0001) than that in the control group, regardless of contact time. The bacterial counts significantly decreased (p < 0.0001) with increasing contact time during ECAW treatment. The antimicrobial activity of ECAW has been previously demonstrated for bacterial contamination of spinach, lettuce, surfaces in food services, and fresh chicken breasts (Guentzel et al., 2008; Rahman et al., 2012b). This study demonstrates the antimicrobial effect of ECAW against aerobic bacteria, including S. Heidelberg, on chicken breasts fragments.
No treatment completely eliminated the bacteria from chicken breasts. In addition to ORP and pH, the presence of organic matter may also influence ECAW activity (Rahman et al., 2012b). The antimicrobial activity of ECAW may be reduced in the presence of organic matter, particularly when lower concentrations of the product are used (Al-Holy and Rasco, 2015). However, it is noteworthy that the effect of traditional disinfectants is reduced in the presence of organic matter (Pilotto et al., 2007). In addition, some authors agree that the reduction in foodborne pathogens, such as Escherichia coli O157:H7, Salmonella spp., and L. monocytogenes, by ECAW may be more significant for smooth-surfaced foods, such as eggs and tomatoes, whereas it may be lower in meat products (Afari and Hung, 2018).
The presence of organic matter in water undergoing chlorination can result in the formation of trihalomethanes (THM), which are chlorinated organic compounds, and the main by-product of the water treatment process. They are also indicators of the presence of other organic compounds in water (Araujo et al., 2020). In the presence of organic matter, the addition of NaClO to water led to the formation of THM via oxidation. This reaction reduces the action of NaClO. Chloroform is considered an indicator of the presence of these toxic compounds and was detected in broiler chicken carcasses after treatment with NaClO. This represents a chemical hazard owing to the carcinogenic properties of chloroform (Vizzier-Thaxton et al., 2010). To date, no study has demonstrated the formation of these toxic compounds after the use of ECAW in water-containing organic matter (Hernández-Pimentel et al., 2020).
ECAW has several advantages, including on-the-spot production, low cost, environmental friendliness, and safe production. It does not irritate hands, skin, or mucous membranes. In addition, ECAW presents a similar potential corrosion of stainless steel to that of commercially available disinfectants, including NaClO-based disinfectants, even after long exposure (>30 days) (Tanaka et al., 1999; Thorn et al., 2012; Wang et al., 2019; Wilsmann et al., 2023). Another important feature of ECAW is the lack of evidence of bacterial resistance (Al-Holy and Rasco, 2015; Huang et al., 2008). ECAW is not a stable solution and can transform into salt and water, depending on the production conditions. Thus, it is possible that ECAW does not cause microbial resistance, making it a potential alternative to currently used disinfectants (Thorn et al., 2012). The use of ECAW is seen as an opportunity to reduce the use and costs of chemical compounds because the inputs used for its production are low-cost and abundant. In addition, the need to stock products or reagents and ample storage facilities is reduced because ECAW can be produced on-site using generating equipment purchased by the company (Huang et al., 2008; Khalid et al., 2018; Liao et al., 2007; Wang et al., 2019).
The current study demonstrates that ECAW can significantly reduce the bacterial load in experimentally inoculated chicken breasts. These results demonstrated the potential of this technology to reduce biological risks in chicken meat.
Footnotes
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This research was supported by the National Council for Scientific and Technological Development (CNPq) through the concession of a scholarship to Daiane Elisa Wilsmann.
