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The antimicrobial interventions of cilantro (Coriandrum sativum) in mitigating cross-contamination of foodborne pathogens during the retail soaking process

The antimicrobial interventions of cilantro (Coriandrum sativum) in mitigating... The efficacy of commercially available antimicrobials for fresh produce—electrolyzed water (EW; around 60  mg/L of free chlorine), a com - bination of lactic acid and phosphoric acid-based (LPA) and citric acid-based (CA)—was compared with tap water (TW) in preventing cross- contamination during the soaking step of crisping at a retail setting. A bunch of cilantro ((103.7±14.9) g/bunch) was inoculated with a three-strain cocktail of nalidixic acid-resistant Salmonella enterica (S. enterica) and Escherichia coli (E. coli) O157:H7, S. enterica and Listeria monocytogenes (L. monocytogenes), or E. coli O157:H7 and L. monocytogenes (around 5.0 log CFU/g). One inoculated and seven non-inoculated cilantro bunches were soaked in 76 L of TW, EW, LPA, and CA for 5 min. Two additional soakings, each with eight bunches of non-inoculated cilantro, were performed in the same soaking water. To determine the cross-contamination of inoculated foodborne pathogens via soaking water, the cilantro samples and soaking water following each soaking step were subjected to microbiological analyses using selective media supplemented with nalidixic acid (100 μg/mL). During the first soaking, significantly greater reductions in Salmonella ((2.9±0.5) log CFU/g), E. coli O157:H7 ((3.0±0.1) log CFU/g), and L. monocytogenes ((2.7±0.3) log CFU/g) on cilantro were achieved with EW compared to soaking with TW, LPA, and CA (P<0.05). Cross-contamination of foodborne pathogens from inoculated cilantro to non-inoculated cilantro was completely mitigated by EW during three subsequent soaking events. With the exception of TW soaking water, no inoculated foodborne pathogens were detected in the 100 mL soaking water of EW, CA, and LPA collected. Including an appropriate concentration of chemical antimicrobial in water during the soaking step of crisping aids in mitigating cross-contamination of foodborne pathogen(s) in cilantro bunches. Keywords: Cross-contamination; retail settings; cilantro; antimicrobials; post-harvest washing; Escherichia coli O157:H7; Salmonella; Listeria monocytogenes. Introduction 1.60% of the overall contamination (4.40%) by Salmonella and Shigella in the U.S. Food and Drug Administration (FDA) Fresh cilantro, also referred to as green coriander and Mexican survey of imported fresh produce (FDA, 2001). Su et al. (2021) or Chinese parsley, is a popular culinary herb used in specialty evaluated the microbiological quality of fresh cilantro, green foods of Mexico and some Asian countries such as India (Luo onions, jalapeños, and serrano peppers in three U.S. States and et al., 2004; Wang et al., 2004). Fresh herbs, including cilantro reported that cilantro was the produce that had the highest aer- and basil, are likely to be added to the dishes immediately prior obic plate count (APC; (7.30±0.94) log colony-forming units to consumption without going through a thermal process. (CFU)/g) than the other three produce (P<0.05), indicating Fresh herbs have been sporadically associated with foodborne poor hygienic quality of cilantro. illness outbreaks (Campbell et al., 2001; Maistro et al., 2012; As the demand for fresh herbs continues to grow, fresh Pan et al., 2015; Chicago Department of Public Health, 2016; cilantro is available year-round at grocery stores, including Korir et al., 2016). At least 13 cilantro-linked outbreaks were supermarkets and farmer’s markets. In those settings, cil- reported to CDC’s National Outbreak Reporting System be- antro bunches with or without roots are likely to be crisped tween 2011 and 2020 (CDC, 2020). According to the Food (soaked) to remove soil/debris and to rehydrate prior to and Agriculture Organization of the United Nations (FAO) display in a refrigerated cabinet. Crisping is a general prac- and World Health Organization (WHO), cilantro was ranked tice performed in retail settings and entails the soaking of as a level 1 priority group, which indicates the highest micro- a commodity (typically leafy greens and fresh herbs) for biological hazard (FAO/WHO, 2008). Moreover, Reddy et al. up to 10 min in water to rehydrate and improve consumer (2016) reported that the prevalence of Salmonella in cil- acceptance. Crisping improves the freshness, but may me- antro was 0.34% (n=9245), which was the highest among 15 diate cross-contamination of foodborne pathogens among commodities evaluated between 2004 and 2012 in the USA. batches/commodities during the soaking process, which is Cilantro was one of three produce items with the greatest inci- generally performed in a food preparation sink with up to dence of pathogenic microbial contamination, accounting for Received 12 November 2021; Revised 8 March 2022; Editorial decision 11 March 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Y. Jung et al. 24 bunches/heads of a commodity. For example, crisping was use (≤24  h). The roots of cilantro were removed prior to suspected as a cross-contamination point in an Escherichia processing; a rubber band was used to secure each bunch. coli (E.  coli) O157:H7 outbreak that occurred in Montana After removing roots ((103.7±14.9) g/bunch), two bunches (USA) in 1995 (Ackers et al., 1998). Brochures and training from each wooden crate were randomly selected and were materials from industry and commodity groups, such as chopped in a separate sterile tray. Then, two samples (25  g ‘Crisping fresh produce procedures’ (IGA Institute, 2005), each) of the chopped cilantro were separately placed into sam- ‘The Network for a Healthy California—Retail program: pling bags containing 100 mL of sterile 0.10% peptone water, Retail fruit and vegetable marketing guide’ (California and its aliquots (100 µL) were spread out onto tryptic soy Department of Public Health, 2011), ‘DeCA manual 40-4.1: agar (TSA; Difco, Becton Dickinson (BD), Sparks, MD, USA) Produce department operations’ (Defense Commissary to determine the total APC of cilantro. Also, two aliquots Agency, 2018), ‘Guidance for processing fresh-cut produce (100 µL) were plated out onto TSA containing nalidixic acid in retail operations’ (Schneider et al., 2004), and ‘Produce (TSA-N, 100 µg/mL; Alfa Aesar, Heysham, UK) to screen nat- safety best practices guide for retailers’ (FMI, 2014), pro- urally occurring nalidixic acid-resistant background micro- mote crisping as a strategy to extend the shelf life of fresh organisms. Moreover, another two 25  g chopped cilantro produce, but a significant knowledge gap exists concerning samples were enriched in a 100  mL of tryptic soy broth the retail practices of crisping from a microbial food safety (TSB) at 37 °C for 18–20 h, and then a loopful (10 µL) of en- perspective. riched samples was streaked onto selective media containing The FDA Food Code 2017 (section 3-302.15) stipulates 100 µg/mL nalidixic acid to recover any naturally existing that ‘fruits and vegetables may be washed by using chem- nalidixic acid-resistant Salmonella enterica (S. enterica), E. coli icals as specified under section 7-204.12’ ( FDA, 2017). This O157:H7, and Listeria monocytogenes (L. monocytogenes); directs that using antimicrobial chemicals is not mandated for the selective media used were Xylose Lactose Tergitol™ washing fresh produce. Potable water is primarily used to re- 4 (XLT4; Difco, BD) containing XLT4 supplement and move soil and debris on commodities, but in soaking scenarios 100 µg/mL nalidixic acid (XLT4-N) for S. enterica, Sorbitol water without or with an insufficient level of antimicrobials MacConkey (SMAC; Difco, BD) agar containing 100 µg/mL can serve as a vehicle of microbial cross-contamination when nalidixic acid (SMAC-N) for E. coli O157:H7, and Palcam the contamination exists on the fresh commodity (Gombas et base agar (Palcam; Difco, BD) containing Palcam sup- al., 2017). Not surprisingly, a wealth of research has been con- plement and 100 µg/mL nalidixic acid (Palcam-N) for ducted to examine the immediate efficacy of antimicrobials L. monocytogenes. (e.g. sodium and calcium hypochlorite, chlorine dioxide, ozone, organic acids, electrolyzed water (EW), and so on) and Bacterial strains and inoculation factors affecting their efficacy in controlling microbial popu - Three nalidixic acid-resistant strains of E. coli O157:H7, lations on fresh produce under various conditions (Gil et al., S. enterica, and L. monocytogenes were obtained from Dr. 2009; Tomás-Callejas et al., 2012; Goodburn and Wallace, Joshua Gurtler (Eastern Regional Research Center, United 2013; Gombas et al., 2017). However, studies related to the States Department of Agriculture, Wyndmoor, PA, USA), retail handling of fresh produce are limited. In our previous including E. coli O157:H7 06F00475, 6535, 7386; Salmonella research on the efficacy of commercially available sanitizers in Newport H1275, Salmonella Stanley H0558, Salmonella mitigating the cross-contamination of foodborne pathogens Montevideo G4639; L. monocytogenes L008 (serotype 4b), during the soaking process of heads of lettuce, EW showed sig- L2624 (1/2b), and L2625 (1/2a). All strains were associated nificant effects to prevent cross-contamination among lettuce with fresh produce outbreaks. Stock cultures were maintained heads (Jung et al., 2017). Cilantro is a low-growing vegetable with glycerol at –80 °C, and their working cultures on TSA crop, and its leaf morphology and the packing process means (Difco, BD) plates stored at 4 °C were replaced every month. it contains high natural microflora and organic matters, such For inoculum, one single colony was suspended in 30 mL of as soil and debris (Luo et al., 2004; Su et al., 2021). Therefore, TSB (Difco, BD) containing nalidixic acid (100 µg/mL) for the first objective of this study was to evaluate the natural E. coli O157:H7 and S. enterica or brain heart infusion broth microbial load of a cilantro bunch before the soaking pro- (BHI broth; Difco, BD) containing nalidixic acid (100 µg/mL) cess and the change in free chlorine concentration after each for L. monocytogenes, and then incubated for 18–20  h at soaking process. Next, the immediate efficacy of the soaking 37 °C with shaking. process with or without antimicrobials on the reduction of For inoculation of cilantro bunches, the overnight cul- foodborne pathogens on cilantro was determined. Lastly, the ture of each strain was centrifuged at 4500 r/min for 15  min potential cross-contamination of foodborne pathogens among (Allegra TM 21R, Beckman Coulter, Palo Alto, CA, USA), cilantro bunches and the presence of foodborne pathogens in the supernatant decanted, and the cell pellet washed with the soaking water were assessed. The results would contribute 0.10% peptone (Difco, BD). Cells of each strain were pooled to filling the knowledge gap to use antimicrobials during the in 30  mL of sterile tap water (to achieve approximately soaking process of retail crisping practice. 9 log CFU/mL) to prepare the inoculum. A cilantro bunch was spot-inoculated at 30 different locations (5 µL inoculum per spot) with S. enterica and E. coli O157:H7, E. coli O157:H7 and L. Materials and Methods monocytogenes, or S. enterica and L. monocytogenes (double Preparation of cilantro bacterial inoculation, each inoculum for a given pathogen was Wooden crates of fresh cilantro with roots (24 bunches per applied independently), respectively. The initial concentration crate) were purchased from a local retail market (Institute, of each inoculated pathogen onto cilantro was approximately WV, USA). The cilantro was not subjected to any treatment 5.0 log CFU/g of cilantro. Inoculated cilantro bunches were dried at the market. Crates of cilantro were stored at 4 °C until on a stainless steel screen in a biosafety cabinet for at least 60 min. Antimicrobial interventions of cilantro 3 Preparation of soaking water (LPA; Suma Eden antimicrobial fruit & vegetable wash, Diversey, Charlotte, NC, USA) were prepared based on the Before filling the sink with tap water or tap water with manufacturer’s instruction; the concentrated antimicrobial antimicrobials, sinks were cleaned with a quaternary sani- solution was added into 76 L of tap water in a sink according tizer (Steramine, Edwards-Councilor Co., Virginia Beach, VA, to the recommended dilution rate (pH of ≤3.0 of CA and LPA USA) and then rinsed with tap water. Sinks were permitted should be maintained). to dry, and the presence of E. coli O157:H7, S. enterica, and Before and after each soaking event, a 50-mL aliquot of L. monocytogenes was tested by swabbing two surface areas 2 2 water was collected from each sink to measure temperature, (one side and one bottom, 10 cm ×10 cm ) of each sink. The pH, and concentrations of free chlorine in soaking water swabs were separately transferred to 10  mL of Dey/Engley (Figure 1A). Free residual chlorine was measured using a neutralizing broth (D/E broth; Hardy Diagnostics, Santa Palintest 1000 chlorometer (measurement range: 0–200 mg/L; Maria, CA, USA) and incubated at 37 °C for 18–20  h. The Palintest Ltd., Gateshead, UK). Photometer tablet reagents aliquots of enriched broth were streaked onto XLT4-N for (Palintest Ltd., Gateshead, UK) were added, crushed, and S. enterica, SMAC-N for E. coli O157:H7, and Palcam-N for mixed with 10  mL of water sample in a test tube, and a L. monocytogenes. 3 3 3 chlorometer reading was taken. A pH meter (Accumet AB15, Each sink (inside size: 60  cm ×60  cm ×35  cm ) was filled Fisher Scientific, Pittsburgh, PA, USA) and an infrared therm - with 76 L of tap water alone (TW) or tap water with commer- TM ometer (Traceable , Fisher Scientific, Pittsburgh, PA, USA) cially available antimicrobials. Antimicrobials were prepared were used to measure the pH and temperature of the water, followed the manufacturer’s instructions. A commercial respectively. All soaking water were prepared within 1 h prior concentrated EW (ProduceFresh Concentration, Sterilox™, to treatment. Lithia Springs, GA, USA) was electrochemically generated hypochlorous acid solution at a manufacturer site, and a dis- Soaking procedures penser provided by the manufacturer was set up on the wall of the laboratory and was connected to tap water to dilute the Figure 1 presents the process flow of the three consecutive concentrated EW. Citric acid-based antimicrobial (CA; CA li- soaking procedures. For the first soaking, one inoculated quid fruit & vegetable wash, Chemstar, Lithia Springs, GA, cilantro bunch (tied with a red rubber band) and seven non- USA) and lactic acid and phosphoric acid-based antimicrobial inoculated bunches (tied with a blue rubber band) were Figure 1. Process flow of the three consecutive soaking procedures. Cilantro was inoculated with Salmonella enterica, Escherichia coli O157:H7, and/ or Listeria monocytogenes. A sink was filled with 76 L of tap water alone (TW), electrolyzed water (EW), citric acid-based antimicrobial (CA), or lactic acid and phosphoric acid-based antimicrobial (LPA). (A) Before and after soaking processes, pH, free chlorine, and temperature of the soaking water measured, (B) after each soaking process, all cilantro bunches were removed for microbiological analysis, and (C) after each soaking process, water samples (100 mL and 100 µL aliquots) were collected for microbiological analysis. 4 Y. Jung et al. submerged together in the 76 L of TW, EW, CA, or LPA for in case too many colonies were recovered on the filter. All 5 min; floating bunches were pushed down into the soaking plates were incubated at 37 °C for 18–36 h. water with sterile tongs. After the 5-min soaking, cilantro bunches were removed from the water using a tong and Statistical analysis placed in perforated crisping trays to allow excess water to All measurable data (pH values, free chlorine concentration, drain; inoculated or non-inoculated cilantro were placed onto temperature of the soaking water, and APC) were recorded separate trays. Then, two subsequent soaking processes with ® in a spreadsheet (Microsoft Excel, Redmond, WA, USA). eight bunches of non-inoculated cilantro were performed in The mean values of the three trials and their standard devi- the same soaking water used for the first soaking. After the ation were calculated in Microsoft Excel. The reduction of second or third soakings, the eight bunches of non-inoculated foodborne pathogen levels on inoculated cilantro after the cilantro were placed in separate perforated trays for micro- first soaking process was determined as the mean of two sam - biological analysis. ples at each sampling for each of the three trials. The mean of log CFU data among different soaking treatments (TW, Microbiological analysis of cilantro EW, CA, and LPA) were compared by analysis of variance (ANOVA)–Duncan’s posthoc analysis using a SAS Education After the first soaking, as shown in Figure 1B, cilantro Analytical Suite 9.4 package (SAS Institute, Cary, NC, USA). bunches were chopped into 3-cm long pieces using a sterile The P-value was set at 0.05. knife and mixed well in a sterile tray. Two samples (25 g each) were collected from the inoculated bunch, and four samples (25  g each) were collected from the seven non-inoculated Results and Discussion cilantro bunches. Then, after the second or third soakings, four samples (25 g each) were obtained from the eight non- APC of untreated cilantro and changes in free inoculated cilantro bunches for microbiological analysis. chorine and pH during soaking processes Each 25  g sample treated with TW or EW was then trans- Because cilantro is often harvested with roots intact and field- ferred to a sampling bag containing 100  mL of D/E broth packed, it typically carries a high population of bacteria com- to neutralize the antimicrobials; double-strength D/E broth pared to other leafy greens. In the present study, the APC of was prepared for the samples treated with CA or LPA to neu- cilantro was (7.4±0.8) log CFU/g before the soaking/crisping tralize acid-based antimicrobials effectively (Park and Chen, process. This result is in line with other studies with cilantro 2011). Then, the sample in the D/E broth was homogenized having an APC of 6.7 log CFU/g for unwashed cilantro leaves for 1 min (easyMIX™, BioMerieux, Marcy-l’Etoile, France). and aerobic bacterial count of (7.0±0.1) log CFU/g for fresh- The undiluted and diluted (1:10) homogenized samples in cut cilantro (Wang et al., 2004; Allende et al., 2009). Stefano 0.10% sterile peptone water were surface plated in duplicate and colleagues (2016) reported fresh cilantro purchased from onto selective agar plates containing 100 µg/mL of nalidixic farmer’s markets in Virginia and North Carolina (USA) had acid to recover inoculated foodborne pathogens; XLT4-N APCs of 7.7 log CFU/g, which was the highest counts among for S. enterica, SMAC-N for E. coli O157:H7, and Palcam-N 14 different commodities of leafy greens and herbs evaluated. for L. monocytogenes. The selective plates were incubated Korir et al. (2016) indicated that cilantro sold in the retail at 37 °C for 24 h to enumerate the typical colonies of each markets in Maryland (USA) had an APC of 9.0 log CFU/g. pathogen. The detection limit of this direct plating method Parameters of soaking water such as temperature, pH, was 1.7 log CFU/g. The presence of inoculated foodborne contact time, the ratio of produce and water, residual concen- pathogens below the detection limit was determined by en- tration of active compounds (i.e. free chlorine), and amount richment; all homogenized samples in D/E broth were further of organic matter from produce are important factors for the incubated at 37 °C for 18–20 h, and aliquots were streaked efficacy of antimicrobials in washing fresh produce ( Gil et al., onto XLT4-N, SMAC-N, or Palcam-N plates. The number of 2009; Banach et al., 2015). In this study, the temperature of positive samples after enrichment of non-inoculated cilantro the water was not adjusted to simulate standard retail prac- samples was recorded to determine the cross-contamination tice where water for crisping/soaking is used directly from of inoculated foodborne pathogens (present below the de- the tap without temperature adjustment. The temperature tection limit) from the inoculated cilantro to non-inoculated of tap water was (17.7±2.9) °C in this study. Fluctuation in cilantro. water temperature was minimal even though experiments were conducted during the summer and winter seasons. Water quality during soaking processes Hsu (2005) reported that the impact of water temperature Four water samples (100 mL each) were collected from each in the range of 10–40 °C was minor on the total chlorine batch of soaking water (TW, EW, CA, or LPA) after every concentration in EW. Table 1 shows measurements of pH soaking process (Figure 1C). The collected water sample was and concentration of free chlorine in the soaking water be- passed through a filtering apparatus with a 0.2- μm pore size fore and after soaking processes. Before soaking the cilantro filter (Analytical Filter Units, Nalgene™, Rochester, NY, USA) bunches, the pH of TW, EW, CA, and LPA were 7.3±0.3, followed by rinsing the filter with 100 mL of sterile distilled 6.7±0.1, 2.7±0.1, and 2.9±0.1, respectively. The pH of acid- water to aid in the removal of residual sanitizing agents. based chemical antimicrobials (CA and LPA) was adjusted Then, the filters were aseptically placed onto D/E neutralizing to below pH 3.0 based on the manufacturer’s instruction agar (Difco, BD) and selective media (XLT4-N, SMAC-N, or to achieve optimal efficacy. Antimicrobials with low pH Palcam-N) to determine total APC and the presence of inocu- can cause some quality issues (e.g. discoloration) of fresh lated foodborne pathogens in the soaking water, respectively. produce, but no visible quality change was observed during Additionally, two aliquots (100 μL) of each soaking water soaking processes with CA and LPA. As shown in Table 1, were spread out onto D/E agar to enumerate viable total APC the concentration of free chlorine in the tap water was at Antimicrobial interventions of cilantro 5 0. The initial concentration of free chlorine in EW was in chlorine due to a lower oxidation strength (Gombas et al., the range of 61–64 (62±2) mg/L, and this dropped to 38–56 2017). The contribution of organic matter to processing (47±13) mg/L after three soaking events. Our previous re- waters can also be the result of cellular exudate leakage from search (Jung et al., 2017) showed less than 5 mg/L decreases the cut surfaces of vegetables and fruits. in free chlorine concentration after three soakings of lettuce heads (8 heads/soaking). The variability in the concentration APCs of the soaking water of free chlorine is likely associated with the levels of organic matter from the cilantro during soaking. The cilantro used in Table 2 shows the APCs of the soaking water following cil- this study carried a considerable amount of soil and organic antro processing. After the first soaking of cilantro bunches matter debris because the roots were intact when purchased. (one inoculated bunch and seven non-inoculated bunches), This suggests that the concentration of antimicrobials should the level of aerobic bacteria exceeded 80 CFU/100  mL of be monitored to maximize the efficacy of antimicrobials de- TW by a filter method. Thus, for better quantification, we pending on the commodities. In a study conducted by Shen plated out the aliquots of 100 µL soaking water. No signifi - (2014), the concentration of free chlorine decreased from 40 cant difference was observed on the APCs of the acid-based to 15 mg/L after the third cycle among seven washing cycles antimicrobials (LPA and CA) soaking water compared to TW of spinach and lettuce and further declined to 0.5  mg/L (P>0.05). In contrast, the APC levels of EW soaking water after the completion of seven cycles of washing. Ampiaw was significantly lower than TW, CA, and LPA ( P<0.05), and et al. (2021) and Luo et al. (2012) also reported that the ef- there was no significant difference among the three consecu - ficacy of antimicrobials (e.g. hypochlorous acid) in soaking tive cycles of the process (P>0.05). This indicates that the EW water is highly affected by the organic and inorganic sub- was not able to inactivate microorganisms completely, but stances during the processing of fresh and fresh-cut produce mitigated the population of the aerobic bacteria from cilantro (Gil et  al., 2009; Luo et al., 2012). Peracetic acid, ozone, during three soaking cycles compared to TW, LPA, and CA. A and chlorine dioxide are less sensitive to organic load than high APC in food/water samples does not necessarily indicate Table 1. The pH and concentrations of free chlorine in soaking water Parameter Soaking event Soaking water TW EW CA LPA pH Before soaking 7.3±0.3 6.7±0.1 2.7±0.1 2.9±0.1 After 1st soaking 7.4±0.2 6.7±0.2 2.7±0.1 2.9±0.1 After 2nd soaking 7.4±0.3 6.7±0.1 2.8±0.1 2.9±0.1 After 3rd soaking 7.4±0.3 6.7±0.1 2.8±0.1 3.0±0.1 Free chlorine (mg/L) Before soaking ND 62±2.1 ND ND After 1st soaking ND 56±1.5 ND ND AB After 2nd soaking ND 49±7.6 ND ND After 3rd soaking ND 47±12.5 ND ND Soaking event: before and after the soaking event, each soaking water in a sink was vigorously stirred and then collected to measure pHs and the concentrations of free chlorine. Values are the mean of at least three replicates±standard deviation. Different capital letters (subscript) in free chlorine level indicate statistical difference (P<0.05). TW, tap water alone; EW, electrolyzed water; CA, citric acid-based antimicrobial; LPA, lactic acid and phosphoric acid-based antimicrobial; ND, not detected. Table 2. Microbial quality of soaking water Soaking cycle Aerobic plate count Soaking water TW EW CA LPA 1st soaking Filter (CFU/100 mL) >80 1.8±1.7 >80 >80 Direct plating (log CFU/mL) 2.4±1.3 BD 2.5±1.4 2.4±1.2 2nd soaking Filter (CFU/100 mL) > 80 2.0±2.8 >80 >80 Direct plating (log CFU/mL) 2.9±1.3 BD 2.8±1.6 2.9±1.0 3rd soaking Filter (CFU/100 mL) >80 3.2±3.9 >80 >80 Direct plating (log CFU/mL) >3.4 BD 2.9±1.4 2.9±1.5 Aerobic plate count: filter method (average of total colony number on the filter after 100 mL soaking water passed through); direct plating method (average of total colony number of 100 µL soaking water); values are the mean of at least three replicates±standard deviation. Aerobic plate count of EW was significant lower than in TW, CA, and LPA (P<0.05). No significant difference was observed among three consecutive soaking steps (P>0.05). TW, tap water alone; EW, electrolyzed water; CA, citric acid-based antimicrobial; LPA, lactic acid and phosphoric acid-based antimicrobial; BD, below detection limit (<1 log CFU/mL). 6 Y. Jung et al. fecal contamination or the presence of foodborne pathogens; treated with 200  mL of 200  mg/L chlorine, achieving a however, the APC level may be used for the quality control 2.6 log CFU/g S. enterica reduction, while a 1.0 log CFU/g re- of fresh produce (Mendonca et al., 2020; Su et al., 2021). duction was achieved with water. Other researchers reported Compared to our previous study (Jung et al., 2017), the APC that immersion of 100  g of fresh-cut cilantro in 300  mL of the EW soaking water for soaking cilantro bunches was of slightly acidic electrolyzed water (pH (5.85±0.05)) and higher than that for soaking lettuce heads, indicating the ini- acidic electrolyzed water (pH (2.48±0.07)) for 5  min re- tial microbial load of cilantro affects the number of aerobic sulted in reductions of 1.5 log CFU/g and 2.5 log CFU/g bacteria in water. of E. coli O78, respectively (Hao et al., 2011). The anti- microbial effect of acid-based sanitizers was limited; the reductions of foodborne pathogens achieved by TW, CA, Pathogen reduction on cilantro following 5-min and LPA were in the range of 1.0–1.6 log CFU/g. No sig- soaking nificant difference between TW and acid-based chemical Spot-inoculation on cilantro achieved populations of antimicrobials (CA and LPA) was noted (P>0.05; Figure 2). S. enterica, E. coli O157:H7, and L. monocytogenes López-Gálvez et al. (2009) also demonstrated that commer- of (4.9±0.3), (5.1±0.5), and (5.4±0.2) log CFU/g, re- cial acid-based chemical antimicrobials in 5  L wash water spectively. Prior to inoculation, we screened for natur- ® ® (Citrox , London, UK; Purac , Lund, Sweden) were not ef- ally occurring foodborne pathogens resistant to nalidixic fective in reducing the E. coli population on fresh-cut let- acid (100 µg/mL) in non-inoculated cilantro and found tuce or in preventing cross-contamination of E. coli from no viable colonies in the representative samples (2 of 24 inoculated fresh-cut lettuce (100 g) to non-inoculated lettuce bunches). However, it should be noted that we may over- (900  g). Meta-analysis on the efficacy of different chemical estimate the number of foodborne pathogens if the cil- antimicrobials to reduce foodborne pathogens on a com- antro sample harbored any naturally occurring nalidixic modity suggests slightly acidic electrolyzed water possessed acid-resistant foodborne pathogens (viable at 100 µg/mL greater efficacy to inactivate foodborne pathogens than acid- nalidixic acid). Also, we inoculated foodborne pathogens based chemical antimicrobials (Prado-Silva et al., 2015). in pairs (S. enterica and E. coli O157:H7, S. enterica and L. monocytogenes, E. coli O157:H7 and L. monocytogenes) to mimic the situation of co-occurrence of foodborne patho- gens in the samples. We utilized selective media containing Cross-contamination during processing nalidixic acid and observed colonies characteristic of species Figure 3 presents the cross-contamination of E. coli when enumerating colonies. O157:H7, S. enterica, and L. monocytogenes from inocu- Figure 2 shows the reduction in the population of each lated cilantro bunches introduced in the first soaking event pathogen following five minutes of soaking in soaking water to non-inoculated cilantro bunches throughout the three (TW, EW, CA, and LPA). EW was significantly effective in soaking events. The results reported in Figure 3 are associated reducing the population of Salmonella ((2.9±0.5) log CFU/g with enrichment because no colonies were detected through reduction), E. coli O157:H7 ((3.0±0.1) log CFU/g reduc- direct plating (detection limit: 1.7 log CFU/g) of samples. tion), and L. monocytogenes ((2.7±0.3) log CFU/g reduc- These results indicate that cross-contamination does occur tion) on cilantro compared to TW, CA, and LPA (P<0.05). during the soaking/crisping of cilantro. Because enrichment Villagómez et al. (2010) investigated the efficacy of decon - was required for the detection of each foodborne pathogen, tamination treatments to reduce S. enterica on cilantro. the number of cells transferred was likely low, although a Whole leaf cilantro (10  g) inoculated with S. enterica was human health concern remains and could be exacerbated should the cilantro be temperature-abused. As shown in Figure 3, only EW completely inhibited the cross-contamin- ation of E. coli O157:H7, S. enterica, and L. monocytogenes on cilantro during three consecutive soaking processes. TW, CA, and LPA were ineffective at preventing cross-contam- ination of cilantro with E. coli O157:H7, S. enterica, and L. monocytogenes. Interestingly, no inoculated foodborne pathogens were detected in the 100  mL soaking water of EW, CA, and LPA (Figure 1). Shen (2014) evaluated the ef- ficacy of chlorine to prevent cross-contamination from in - oculated spinach (20  g) to non-inoculated shredded lettuce (1000 g) in 20 L of chlorinated water (40 mg/L of initial free chlorine) during seven consecutive cycles of 1-min washing. In Shen’s research, E. coli O157:H7 cross-contamination of non-inoculated lettuce occurred when residual free chlorine Figure 2. Reduction of Salmonella enterica, Escherichia coli O157:H7, was less than 4 mg/L. Luo et al. (2012) reported that E. coli and/or Listeria monocytogenes following a 5-min soaking of inoculated cilantro bunches during the soaking step of crisping: tap water alone O157:H7 was transferred from inoculated lettuce to non- (TW), electrolyzed water (EW; around 60 mg/L free chlorine), citric inoculated lettuce when the wash solution contained 5 mg/L acid-based antimicrobial (CA), and lactic acid and phosphoric acid- free chlorine; E. coli O157:H7 did not survive in the wash based antimicrobial (LPA). Bars represent the mean of three replicate water. These findings suggest that an active concentration of experiments (2 samples per experiment), and error bars indicate positive a chemical antimicrobial added to water is important in con- standard deviation. Different capital letters above the bars in the same trolling microbial populations in the water and minimizing foodborne pathogen represent significant differences among different cross-contamination. soaking/crisping water (P<0.05). Antimicrobial interventions of cilantro 7 Figure 3. Cross-contamination of cilantro during initial and subsequent soaking cycles. Bars indicate the averaged positive cilantro sample numbers after the sample was subjected to an enrichment process to detect the presence of inoculated foodborne pathogens. Black down-pointing triangle indicates the presence of foodborne pathogens in the 100 mL of soaking water after soaking of cilantro bunches. The x-axis indicates different soaking water: tap water alone (TW), electrolyzed water (EW), citric acid-based antimicrobial (CA), and lactic acid and phosphoric acid-based antimicrobial (LPA). Conclusions References Ackers, M., Mahon, B., Leahy, E., et al. (1998). An outbreak of Research conducted to evaluate the efficacy of antimicrobial Escherichia coli O157: H7 infections associated with leaf lettuce agents added to water typically focuses on the reduction of consumption. 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The results of the present study suggest eering, 43: 102228. Banach, J., Sampers, I., Van Haute, S., et al. (2015). Effect of disinfect- that the addition of an antimicrobial to processing water aids ants on preventing the cross-contamination of pathogens in fresh in controlling microbial populations in the water, limits/pre- produce washing water. International Journal of Environmental vents cross-contamination, and reduces the microbial popu- Research and Public Health, 12(8): 8658–8677. lation on cilantro. Moreover, monitoring the soaking water California Department of Public Health. (2011). Retail Fruit and Vege- quality both in microbiological and physicochemical param- table Marketing Guide [Online]. https://ucanr.edu/sites/Postharvest_ eters is critical to maximizing the efficacy of antimicrobials. Technology_Center_/files/231893.pdf. Accessed on March 14, 2022. The efficacy of antimicrobials evaluated in this study varied Campbell, J., Mohle-Boetani, J., Reporter, R., et al. 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Food Control, 28(2): 258–264. 949–956. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food Quality and Safety Oxford University Press

The antimicrobial interventions of cilantro (Coriandrum sativum) in mitigating cross-contamination of foodborne pathogens during the retail soaking process

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Abstract

The efficacy of commercially available antimicrobials for fresh produce—electrolyzed water (EW; around 60  mg/L of free chlorine), a com - bination of lactic acid and phosphoric acid-based (LPA) and citric acid-based (CA)—was compared with tap water (TW) in preventing cross- contamination during the soaking step of crisping at a retail setting. A bunch of cilantro ((103.7±14.9) g/bunch) was inoculated with a three-strain cocktail of nalidixic acid-resistant Salmonella enterica (S. enterica) and Escherichia coli (E. coli) O157:H7, S. enterica and Listeria monocytogenes (L. monocytogenes), or E. coli O157:H7 and L. monocytogenes (around 5.0 log CFU/g). One inoculated and seven non-inoculated cilantro bunches were soaked in 76 L of TW, EW, LPA, and CA for 5 min. Two additional soakings, each with eight bunches of non-inoculated cilantro, were performed in the same soaking water. To determine the cross-contamination of inoculated foodborne pathogens via soaking water, the cilantro samples and soaking water following each soaking step were subjected to microbiological analyses using selective media supplemented with nalidixic acid (100 μg/mL). During the first soaking, significantly greater reductions in Salmonella ((2.9±0.5) log CFU/g), E. coli O157:H7 ((3.0±0.1) log CFU/g), and L. monocytogenes ((2.7±0.3) log CFU/g) on cilantro were achieved with EW compared to soaking with TW, LPA, and CA (P<0.05). Cross-contamination of foodborne pathogens from inoculated cilantro to non-inoculated cilantro was completely mitigated by EW during three subsequent soaking events. With the exception of TW soaking water, no inoculated foodborne pathogens were detected in the 100 mL soaking water of EW, CA, and LPA collected. Including an appropriate concentration of chemical antimicrobial in water during the soaking step of crisping aids in mitigating cross-contamination of foodborne pathogen(s) in cilantro bunches. Keywords: Cross-contamination; retail settings; cilantro; antimicrobials; post-harvest washing; Escherichia coli O157:H7; Salmonella; Listeria monocytogenes. Introduction 1.60% of the overall contamination (4.40%) by Salmonella and Shigella in the U.S. Food and Drug Administration (FDA) Fresh cilantro, also referred to as green coriander and Mexican survey of imported fresh produce (FDA, 2001). Su et al. (2021) or Chinese parsley, is a popular culinary herb used in specialty evaluated the microbiological quality of fresh cilantro, green foods of Mexico and some Asian countries such as India (Luo onions, jalapeños, and serrano peppers in three U.S. States and et al., 2004; Wang et al., 2004). Fresh herbs, including cilantro reported that cilantro was the produce that had the highest aer- and basil, are likely to be added to the dishes immediately prior obic plate count (APC; (7.30±0.94) log colony-forming units to consumption without going through a thermal process. (CFU)/g) than the other three produce (P<0.05), indicating Fresh herbs have been sporadically associated with foodborne poor hygienic quality of cilantro. illness outbreaks (Campbell et al., 2001; Maistro et al., 2012; As the demand for fresh herbs continues to grow, fresh Pan et al., 2015; Chicago Department of Public Health, 2016; cilantro is available year-round at grocery stores, including Korir et al., 2016). At least 13 cilantro-linked outbreaks were supermarkets and farmer’s markets. In those settings, cil- reported to CDC’s National Outbreak Reporting System be- antro bunches with or without roots are likely to be crisped tween 2011 and 2020 (CDC, 2020). According to the Food (soaked) to remove soil/debris and to rehydrate prior to and Agriculture Organization of the United Nations (FAO) display in a refrigerated cabinet. Crisping is a general prac- and World Health Organization (WHO), cilantro was ranked tice performed in retail settings and entails the soaking of as a level 1 priority group, which indicates the highest micro- a commodity (typically leafy greens and fresh herbs) for biological hazard (FAO/WHO, 2008). Moreover, Reddy et al. up to 10 min in water to rehydrate and improve consumer (2016) reported that the prevalence of Salmonella in cil- acceptance. Crisping improves the freshness, but may me- antro was 0.34% (n=9245), which was the highest among 15 diate cross-contamination of foodborne pathogens among commodities evaluated between 2004 and 2012 in the USA. batches/commodities during the soaking process, which is Cilantro was one of three produce items with the greatest inci- generally performed in a food preparation sink with up to dence of pathogenic microbial contamination, accounting for Received 12 November 2021; Revised 8 March 2022; Editorial decision 11 March 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Y. Jung et al. 24 bunches/heads of a commodity. For example, crisping was use (≤24  h). The roots of cilantro were removed prior to suspected as a cross-contamination point in an Escherichia processing; a rubber band was used to secure each bunch. coli (E.  coli) O157:H7 outbreak that occurred in Montana After removing roots ((103.7±14.9) g/bunch), two bunches (USA) in 1995 (Ackers et al., 1998). Brochures and training from each wooden crate were randomly selected and were materials from industry and commodity groups, such as chopped in a separate sterile tray. Then, two samples (25  g ‘Crisping fresh produce procedures’ (IGA Institute, 2005), each) of the chopped cilantro were separately placed into sam- ‘The Network for a Healthy California—Retail program: pling bags containing 100 mL of sterile 0.10% peptone water, Retail fruit and vegetable marketing guide’ (California and its aliquots (100 µL) were spread out onto tryptic soy Department of Public Health, 2011), ‘DeCA manual 40-4.1: agar (TSA; Difco, Becton Dickinson (BD), Sparks, MD, USA) Produce department operations’ (Defense Commissary to determine the total APC of cilantro. Also, two aliquots Agency, 2018), ‘Guidance for processing fresh-cut produce (100 µL) were plated out onto TSA containing nalidixic acid in retail operations’ (Schneider et al., 2004), and ‘Produce (TSA-N, 100 µg/mL; Alfa Aesar, Heysham, UK) to screen nat- safety best practices guide for retailers’ (FMI, 2014), pro- urally occurring nalidixic acid-resistant background micro- mote crisping as a strategy to extend the shelf life of fresh organisms. Moreover, another two 25  g chopped cilantro produce, but a significant knowledge gap exists concerning samples were enriched in a 100  mL of tryptic soy broth the retail practices of crisping from a microbial food safety (TSB) at 37 °C for 18–20 h, and then a loopful (10 µL) of en- perspective. riched samples was streaked onto selective media containing The FDA Food Code 2017 (section 3-302.15) stipulates 100 µg/mL nalidixic acid to recover any naturally existing that ‘fruits and vegetables may be washed by using chem- nalidixic acid-resistant Salmonella enterica (S. enterica), E. coli icals as specified under section 7-204.12’ ( FDA, 2017). This O157:H7, and Listeria monocytogenes (L. monocytogenes); directs that using antimicrobial chemicals is not mandated for the selective media used were Xylose Lactose Tergitol™ washing fresh produce. Potable water is primarily used to re- 4 (XLT4; Difco, BD) containing XLT4 supplement and move soil and debris on commodities, but in soaking scenarios 100 µg/mL nalidixic acid (XLT4-N) for S. enterica, Sorbitol water without or with an insufficient level of antimicrobials MacConkey (SMAC; Difco, BD) agar containing 100 µg/mL can serve as a vehicle of microbial cross-contamination when nalidixic acid (SMAC-N) for E. coli O157:H7, and Palcam the contamination exists on the fresh commodity (Gombas et base agar (Palcam; Difco, BD) containing Palcam sup- al., 2017). Not surprisingly, a wealth of research has been con- plement and 100 µg/mL nalidixic acid (Palcam-N) for ducted to examine the immediate efficacy of antimicrobials L. monocytogenes. (e.g. sodium and calcium hypochlorite, chlorine dioxide, ozone, organic acids, electrolyzed water (EW), and so on) and Bacterial strains and inoculation factors affecting their efficacy in controlling microbial popu - Three nalidixic acid-resistant strains of E. coli O157:H7, lations on fresh produce under various conditions (Gil et al., S. enterica, and L. monocytogenes were obtained from Dr. 2009; Tomás-Callejas et al., 2012; Goodburn and Wallace, Joshua Gurtler (Eastern Regional Research Center, United 2013; Gombas et al., 2017). However, studies related to the States Department of Agriculture, Wyndmoor, PA, USA), retail handling of fresh produce are limited. In our previous including E. coli O157:H7 06F00475, 6535, 7386; Salmonella research on the efficacy of commercially available sanitizers in Newport H1275, Salmonella Stanley H0558, Salmonella mitigating the cross-contamination of foodborne pathogens Montevideo G4639; L. monocytogenes L008 (serotype 4b), during the soaking process of heads of lettuce, EW showed sig- L2624 (1/2b), and L2625 (1/2a). All strains were associated nificant effects to prevent cross-contamination among lettuce with fresh produce outbreaks. Stock cultures were maintained heads (Jung et al., 2017). Cilantro is a low-growing vegetable with glycerol at –80 °C, and their working cultures on TSA crop, and its leaf morphology and the packing process means (Difco, BD) plates stored at 4 °C were replaced every month. it contains high natural microflora and organic matters, such For inoculum, one single colony was suspended in 30 mL of as soil and debris (Luo et al., 2004; Su et al., 2021). Therefore, TSB (Difco, BD) containing nalidixic acid (100 µg/mL) for the first objective of this study was to evaluate the natural E. coli O157:H7 and S. enterica or brain heart infusion broth microbial load of a cilantro bunch before the soaking pro- (BHI broth; Difco, BD) containing nalidixic acid (100 µg/mL) cess and the change in free chlorine concentration after each for L. monocytogenes, and then incubated for 18–20  h at soaking process. Next, the immediate efficacy of the soaking 37 °C with shaking. process with or without antimicrobials on the reduction of For inoculation of cilantro bunches, the overnight cul- foodborne pathogens on cilantro was determined. Lastly, the ture of each strain was centrifuged at 4500 r/min for 15  min potential cross-contamination of foodborne pathogens among (Allegra TM 21R, Beckman Coulter, Palo Alto, CA, USA), cilantro bunches and the presence of foodborne pathogens in the supernatant decanted, and the cell pellet washed with the soaking water were assessed. The results would contribute 0.10% peptone (Difco, BD). Cells of each strain were pooled to filling the knowledge gap to use antimicrobials during the in 30  mL of sterile tap water (to achieve approximately soaking process of retail crisping practice. 9 log CFU/mL) to prepare the inoculum. A cilantro bunch was spot-inoculated at 30 different locations (5 µL inoculum per spot) with S. enterica and E. coli O157:H7, E. coli O157:H7 and L. Materials and Methods monocytogenes, or S. enterica and L. monocytogenes (double Preparation of cilantro bacterial inoculation, each inoculum for a given pathogen was Wooden crates of fresh cilantro with roots (24 bunches per applied independently), respectively. The initial concentration crate) were purchased from a local retail market (Institute, of each inoculated pathogen onto cilantro was approximately WV, USA). The cilantro was not subjected to any treatment 5.0 log CFU/g of cilantro. Inoculated cilantro bunches were dried at the market. Crates of cilantro were stored at 4 °C until on a stainless steel screen in a biosafety cabinet for at least 60 min. Antimicrobial interventions of cilantro 3 Preparation of soaking water (LPA; Suma Eden antimicrobial fruit & vegetable wash, Diversey, Charlotte, NC, USA) were prepared based on the Before filling the sink with tap water or tap water with manufacturer’s instruction; the concentrated antimicrobial antimicrobials, sinks were cleaned with a quaternary sani- solution was added into 76 L of tap water in a sink according tizer (Steramine, Edwards-Councilor Co., Virginia Beach, VA, to the recommended dilution rate (pH of ≤3.0 of CA and LPA USA) and then rinsed with tap water. Sinks were permitted should be maintained). to dry, and the presence of E. coli O157:H7, S. enterica, and Before and after each soaking event, a 50-mL aliquot of L. monocytogenes was tested by swabbing two surface areas 2 2 water was collected from each sink to measure temperature, (one side and one bottom, 10 cm ×10 cm ) of each sink. The pH, and concentrations of free chlorine in soaking water swabs were separately transferred to 10  mL of Dey/Engley (Figure 1A). Free residual chlorine was measured using a neutralizing broth (D/E broth; Hardy Diagnostics, Santa Palintest 1000 chlorometer (measurement range: 0–200 mg/L; Maria, CA, USA) and incubated at 37 °C for 18–20  h. The Palintest Ltd., Gateshead, UK). Photometer tablet reagents aliquots of enriched broth were streaked onto XLT4-N for (Palintest Ltd., Gateshead, UK) were added, crushed, and S. enterica, SMAC-N for E. coli O157:H7, and Palcam-N for mixed with 10  mL of water sample in a test tube, and a L. monocytogenes. 3 3 3 chlorometer reading was taken. A pH meter (Accumet AB15, Each sink (inside size: 60  cm ×60  cm ×35  cm ) was filled Fisher Scientific, Pittsburgh, PA, USA) and an infrared therm - with 76 L of tap water alone (TW) or tap water with commer- TM ometer (Traceable , Fisher Scientific, Pittsburgh, PA, USA) cially available antimicrobials. Antimicrobials were prepared were used to measure the pH and temperature of the water, followed the manufacturer’s instructions. A commercial respectively. All soaking water were prepared within 1 h prior concentrated EW (ProduceFresh Concentration, Sterilox™, to treatment. Lithia Springs, GA, USA) was electrochemically generated hypochlorous acid solution at a manufacturer site, and a dis- Soaking procedures penser provided by the manufacturer was set up on the wall of the laboratory and was connected to tap water to dilute the Figure 1 presents the process flow of the three consecutive concentrated EW. Citric acid-based antimicrobial (CA; CA li- soaking procedures. For the first soaking, one inoculated quid fruit & vegetable wash, Chemstar, Lithia Springs, GA, cilantro bunch (tied with a red rubber band) and seven non- USA) and lactic acid and phosphoric acid-based antimicrobial inoculated bunches (tied with a blue rubber band) were Figure 1. Process flow of the three consecutive soaking procedures. Cilantro was inoculated with Salmonella enterica, Escherichia coli O157:H7, and/ or Listeria monocytogenes. A sink was filled with 76 L of tap water alone (TW), electrolyzed water (EW), citric acid-based antimicrobial (CA), or lactic acid and phosphoric acid-based antimicrobial (LPA). (A) Before and after soaking processes, pH, free chlorine, and temperature of the soaking water measured, (B) after each soaking process, all cilantro bunches were removed for microbiological analysis, and (C) after each soaking process, water samples (100 mL and 100 µL aliquots) were collected for microbiological analysis. 4 Y. Jung et al. submerged together in the 76 L of TW, EW, CA, or LPA for in case too many colonies were recovered on the filter. All 5 min; floating bunches were pushed down into the soaking plates were incubated at 37 °C for 18–36 h. water with sterile tongs. After the 5-min soaking, cilantro bunches were removed from the water using a tong and Statistical analysis placed in perforated crisping trays to allow excess water to All measurable data (pH values, free chlorine concentration, drain; inoculated or non-inoculated cilantro were placed onto temperature of the soaking water, and APC) were recorded separate trays. Then, two subsequent soaking processes with ® in a spreadsheet (Microsoft Excel, Redmond, WA, USA). eight bunches of non-inoculated cilantro were performed in The mean values of the three trials and their standard devi- the same soaking water used for the first soaking. After the ation were calculated in Microsoft Excel. The reduction of second or third soakings, the eight bunches of non-inoculated foodborne pathogen levels on inoculated cilantro after the cilantro were placed in separate perforated trays for micro- first soaking process was determined as the mean of two sam - biological analysis. ples at each sampling for each of the three trials. The mean of log CFU data among different soaking treatments (TW, Microbiological analysis of cilantro EW, CA, and LPA) were compared by analysis of variance (ANOVA)–Duncan’s posthoc analysis using a SAS Education After the first soaking, as shown in Figure 1B, cilantro Analytical Suite 9.4 package (SAS Institute, Cary, NC, USA). bunches were chopped into 3-cm long pieces using a sterile The P-value was set at 0.05. knife and mixed well in a sterile tray. Two samples (25 g each) were collected from the inoculated bunch, and four samples (25  g each) were collected from the seven non-inoculated Results and Discussion cilantro bunches. Then, after the second or third soakings, four samples (25 g each) were obtained from the eight non- APC of untreated cilantro and changes in free inoculated cilantro bunches for microbiological analysis. chorine and pH during soaking processes Each 25  g sample treated with TW or EW was then trans- Because cilantro is often harvested with roots intact and field- ferred to a sampling bag containing 100  mL of D/E broth packed, it typically carries a high population of bacteria com- to neutralize the antimicrobials; double-strength D/E broth pared to other leafy greens. In the present study, the APC of was prepared for the samples treated with CA or LPA to neu- cilantro was (7.4±0.8) log CFU/g before the soaking/crisping tralize acid-based antimicrobials effectively (Park and Chen, process. This result is in line with other studies with cilantro 2011). Then, the sample in the D/E broth was homogenized having an APC of 6.7 log CFU/g for unwashed cilantro leaves for 1 min (easyMIX™, BioMerieux, Marcy-l’Etoile, France). and aerobic bacterial count of (7.0±0.1) log CFU/g for fresh- The undiluted and diluted (1:10) homogenized samples in cut cilantro (Wang et al., 2004; Allende et al., 2009). Stefano 0.10% sterile peptone water were surface plated in duplicate and colleagues (2016) reported fresh cilantro purchased from onto selective agar plates containing 100 µg/mL of nalidixic farmer’s markets in Virginia and North Carolina (USA) had acid to recover inoculated foodborne pathogens; XLT4-N APCs of 7.7 log CFU/g, which was the highest counts among for S. enterica, SMAC-N for E. coli O157:H7, and Palcam-N 14 different commodities of leafy greens and herbs evaluated. for L. monocytogenes. The selective plates were incubated Korir et al. (2016) indicated that cilantro sold in the retail at 37 °C for 24 h to enumerate the typical colonies of each markets in Maryland (USA) had an APC of 9.0 log CFU/g. pathogen. The detection limit of this direct plating method Parameters of soaking water such as temperature, pH, was 1.7 log CFU/g. The presence of inoculated foodborne contact time, the ratio of produce and water, residual concen- pathogens below the detection limit was determined by en- tration of active compounds (i.e. free chlorine), and amount richment; all homogenized samples in D/E broth were further of organic matter from produce are important factors for the incubated at 37 °C for 18–20 h, and aliquots were streaked efficacy of antimicrobials in washing fresh produce ( Gil et al., onto XLT4-N, SMAC-N, or Palcam-N plates. The number of 2009; Banach et al., 2015). In this study, the temperature of positive samples after enrichment of non-inoculated cilantro the water was not adjusted to simulate standard retail prac- samples was recorded to determine the cross-contamination tice where water for crisping/soaking is used directly from of inoculated foodborne pathogens (present below the de- the tap without temperature adjustment. The temperature tection limit) from the inoculated cilantro to non-inoculated of tap water was (17.7±2.9) °C in this study. Fluctuation in cilantro. water temperature was minimal even though experiments were conducted during the summer and winter seasons. Water quality during soaking processes Hsu (2005) reported that the impact of water temperature Four water samples (100 mL each) were collected from each in the range of 10–40 °C was minor on the total chlorine batch of soaking water (TW, EW, CA, or LPA) after every concentration in EW. Table 1 shows measurements of pH soaking process (Figure 1C). The collected water sample was and concentration of free chlorine in the soaking water be- passed through a filtering apparatus with a 0.2- μm pore size fore and after soaking processes. Before soaking the cilantro filter (Analytical Filter Units, Nalgene™, Rochester, NY, USA) bunches, the pH of TW, EW, CA, and LPA were 7.3±0.3, followed by rinsing the filter with 100 mL of sterile distilled 6.7±0.1, 2.7±0.1, and 2.9±0.1, respectively. The pH of acid- water to aid in the removal of residual sanitizing agents. based chemical antimicrobials (CA and LPA) was adjusted Then, the filters were aseptically placed onto D/E neutralizing to below pH 3.0 based on the manufacturer’s instruction agar (Difco, BD) and selective media (XLT4-N, SMAC-N, or to achieve optimal efficacy. Antimicrobials with low pH Palcam-N) to determine total APC and the presence of inocu- can cause some quality issues (e.g. discoloration) of fresh lated foodborne pathogens in the soaking water, respectively. produce, but no visible quality change was observed during Additionally, two aliquots (100 μL) of each soaking water soaking processes with CA and LPA. As shown in Table 1, were spread out onto D/E agar to enumerate viable total APC the concentration of free chlorine in the tap water was at Antimicrobial interventions of cilantro 5 0. The initial concentration of free chlorine in EW was in chlorine due to a lower oxidation strength (Gombas et al., the range of 61–64 (62±2) mg/L, and this dropped to 38–56 2017). The contribution of organic matter to processing (47±13) mg/L after three soaking events. Our previous re- waters can also be the result of cellular exudate leakage from search (Jung et al., 2017) showed less than 5 mg/L decreases the cut surfaces of vegetables and fruits. in free chlorine concentration after three soakings of lettuce heads (8 heads/soaking). The variability in the concentration APCs of the soaking water of free chlorine is likely associated with the levels of organic matter from the cilantro during soaking. The cilantro used in Table 2 shows the APCs of the soaking water following cil- this study carried a considerable amount of soil and organic antro processing. After the first soaking of cilantro bunches matter debris because the roots were intact when purchased. (one inoculated bunch and seven non-inoculated bunches), This suggests that the concentration of antimicrobials should the level of aerobic bacteria exceeded 80 CFU/100  mL of be monitored to maximize the efficacy of antimicrobials de- TW by a filter method. Thus, for better quantification, we pending on the commodities. In a study conducted by Shen plated out the aliquots of 100 µL soaking water. No signifi - (2014), the concentration of free chlorine decreased from 40 cant difference was observed on the APCs of the acid-based to 15 mg/L after the third cycle among seven washing cycles antimicrobials (LPA and CA) soaking water compared to TW of spinach and lettuce and further declined to 0.5  mg/L (P>0.05). In contrast, the APC levels of EW soaking water after the completion of seven cycles of washing. Ampiaw was significantly lower than TW, CA, and LPA ( P<0.05), and et al. (2021) and Luo et al. (2012) also reported that the ef- there was no significant difference among the three consecu - ficacy of antimicrobials (e.g. hypochlorous acid) in soaking tive cycles of the process (P>0.05). This indicates that the EW water is highly affected by the organic and inorganic sub- was not able to inactivate microorganisms completely, but stances during the processing of fresh and fresh-cut produce mitigated the population of the aerobic bacteria from cilantro (Gil et  al., 2009; Luo et al., 2012). Peracetic acid, ozone, during three soaking cycles compared to TW, LPA, and CA. A and chlorine dioxide are less sensitive to organic load than high APC in food/water samples does not necessarily indicate Table 1. The pH and concentrations of free chlorine in soaking water Parameter Soaking event Soaking water TW EW CA LPA pH Before soaking 7.3±0.3 6.7±0.1 2.7±0.1 2.9±0.1 After 1st soaking 7.4±0.2 6.7±0.2 2.7±0.1 2.9±0.1 After 2nd soaking 7.4±0.3 6.7±0.1 2.8±0.1 2.9±0.1 After 3rd soaking 7.4±0.3 6.7±0.1 2.8±0.1 3.0±0.1 Free chlorine (mg/L) Before soaking ND 62±2.1 ND ND After 1st soaking ND 56±1.5 ND ND AB After 2nd soaking ND 49±7.6 ND ND After 3rd soaking ND 47±12.5 ND ND Soaking event: before and after the soaking event, each soaking water in a sink was vigorously stirred and then collected to measure pHs and the concentrations of free chlorine. Values are the mean of at least three replicates±standard deviation. Different capital letters (subscript) in free chlorine level indicate statistical difference (P<0.05). TW, tap water alone; EW, electrolyzed water; CA, citric acid-based antimicrobial; LPA, lactic acid and phosphoric acid-based antimicrobial; ND, not detected. Table 2. Microbial quality of soaking water Soaking cycle Aerobic plate count Soaking water TW EW CA LPA 1st soaking Filter (CFU/100 mL) >80 1.8±1.7 >80 >80 Direct plating (log CFU/mL) 2.4±1.3 BD 2.5±1.4 2.4±1.2 2nd soaking Filter (CFU/100 mL) > 80 2.0±2.8 >80 >80 Direct plating (log CFU/mL) 2.9±1.3 BD 2.8±1.6 2.9±1.0 3rd soaking Filter (CFU/100 mL) >80 3.2±3.9 >80 >80 Direct plating (log CFU/mL) >3.4 BD 2.9±1.4 2.9±1.5 Aerobic plate count: filter method (average of total colony number on the filter after 100 mL soaking water passed through); direct plating method (average of total colony number of 100 µL soaking water); values are the mean of at least three replicates±standard deviation. Aerobic plate count of EW was significant lower than in TW, CA, and LPA (P<0.05). No significant difference was observed among three consecutive soaking steps (P>0.05). TW, tap water alone; EW, electrolyzed water; CA, citric acid-based antimicrobial; LPA, lactic acid and phosphoric acid-based antimicrobial; BD, below detection limit (<1 log CFU/mL). 6 Y. Jung et al. fecal contamination or the presence of foodborne pathogens; treated with 200  mL of 200  mg/L chlorine, achieving a however, the APC level may be used for the quality control 2.6 log CFU/g S. enterica reduction, while a 1.0 log CFU/g re- of fresh produce (Mendonca et al., 2020; Su et al., 2021). duction was achieved with water. Other researchers reported Compared to our previous study (Jung et al., 2017), the APC that immersion of 100  g of fresh-cut cilantro in 300  mL of the EW soaking water for soaking cilantro bunches was of slightly acidic electrolyzed water (pH (5.85±0.05)) and higher than that for soaking lettuce heads, indicating the ini- acidic electrolyzed water (pH (2.48±0.07)) for 5  min re- tial microbial load of cilantro affects the number of aerobic sulted in reductions of 1.5 log CFU/g and 2.5 log CFU/g bacteria in water. of E. coli O78, respectively (Hao et al., 2011). The anti- microbial effect of acid-based sanitizers was limited; the reductions of foodborne pathogens achieved by TW, CA, Pathogen reduction on cilantro following 5-min and LPA were in the range of 1.0–1.6 log CFU/g. No sig- soaking nificant difference between TW and acid-based chemical Spot-inoculation on cilantro achieved populations of antimicrobials (CA and LPA) was noted (P>0.05; Figure 2). S. enterica, E. coli O157:H7, and L. monocytogenes López-Gálvez et al. (2009) also demonstrated that commer- of (4.9±0.3), (5.1±0.5), and (5.4±0.2) log CFU/g, re- cial acid-based chemical antimicrobials in 5  L wash water spectively. Prior to inoculation, we screened for natur- ® ® (Citrox , London, UK; Purac , Lund, Sweden) were not ef- ally occurring foodborne pathogens resistant to nalidixic fective in reducing the E. coli population on fresh-cut let- acid (100 µg/mL) in non-inoculated cilantro and found tuce or in preventing cross-contamination of E. coli from no viable colonies in the representative samples (2 of 24 inoculated fresh-cut lettuce (100 g) to non-inoculated lettuce bunches). However, it should be noted that we may over- (900  g). Meta-analysis on the efficacy of different chemical estimate the number of foodborne pathogens if the cil- antimicrobials to reduce foodborne pathogens on a com- antro sample harbored any naturally occurring nalidixic modity suggests slightly acidic electrolyzed water possessed acid-resistant foodborne pathogens (viable at 100 µg/mL greater efficacy to inactivate foodborne pathogens than acid- nalidixic acid). Also, we inoculated foodborne pathogens based chemical antimicrobials (Prado-Silva et al., 2015). in pairs (S. enterica and E. coli O157:H7, S. enterica and L. monocytogenes, E. coli O157:H7 and L. monocytogenes) to mimic the situation of co-occurrence of foodborne patho- gens in the samples. We utilized selective media containing Cross-contamination during processing nalidixic acid and observed colonies characteristic of species Figure 3 presents the cross-contamination of E. coli when enumerating colonies. O157:H7, S. enterica, and L. monocytogenes from inocu- Figure 2 shows the reduction in the population of each lated cilantro bunches introduced in the first soaking event pathogen following five minutes of soaking in soaking water to non-inoculated cilantro bunches throughout the three (TW, EW, CA, and LPA). EW was significantly effective in soaking events. The results reported in Figure 3 are associated reducing the population of Salmonella ((2.9±0.5) log CFU/g with enrichment because no colonies were detected through reduction), E. coli O157:H7 ((3.0±0.1) log CFU/g reduc- direct plating (detection limit: 1.7 log CFU/g) of samples. tion), and L. monocytogenes ((2.7±0.3) log CFU/g reduc- These results indicate that cross-contamination does occur tion) on cilantro compared to TW, CA, and LPA (P<0.05). during the soaking/crisping of cilantro. Because enrichment Villagómez et al. (2010) investigated the efficacy of decon - was required for the detection of each foodborne pathogen, tamination treatments to reduce S. enterica on cilantro. the number of cells transferred was likely low, although a Whole leaf cilantro (10  g) inoculated with S. enterica was human health concern remains and could be exacerbated should the cilantro be temperature-abused. As shown in Figure 3, only EW completely inhibited the cross-contamin- ation of E. coli O157:H7, S. enterica, and L. monocytogenes on cilantro during three consecutive soaking processes. TW, CA, and LPA were ineffective at preventing cross-contam- ination of cilantro with E. coli O157:H7, S. enterica, and L. monocytogenes. Interestingly, no inoculated foodborne pathogens were detected in the 100  mL soaking water of EW, CA, and LPA (Figure 1). Shen (2014) evaluated the ef- ficacy of chlorine to prevent cross-contamination from in - oculated spinach (20  g) to non-inoculated shredded lettuce (1000 g) in 20 L of chlorinated water (40 mg/L of initial free chlorine) during seven consecutive cycles of 1-min washing. In Shen’s research, E. coli O157:H7 cross-contamination of non-inoculated lettuce occurred when residual free chlorine Figure 2. Reduction of Salmonella enterica, Escherichia coli O157:H7, was less than 4 mg/L. Luo et al. (2012) reported that E. coli and/or Listeria monocytogenes following a 5-min soaking of inoculated cilantro bunches during the soaking step of crisping: tap water alone O157:H7 was transferred from inoculated lettuce to non- (TW), electrolyzed water (EW; around 60 mg/L free chlorine), citric inoculated lettuce when the wash solution contained 5 mg/L acid-based antimicrobial (CA), and lactic acid and phosphoric acid- free chlorine; E. coli O157:H7 did not survive in the wash based antimicrobial (LPA). Bars represent the mean of three replicate water. These findings suggest that an active concentration of experiments (2 samples per experiment), and error bars indicate positive a chemical antimicrobial added to water is important in con- standard deviation. Different capital letters above the bars in the same trolling microbial populations in the water and minimizing foodborne pathogen represent significant differences among different cross-contamination. soaking/crisping water (P<0.05). 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Journal

Food Quality and Safety – Oxford University Press

Published: Mar 17, 2022

Keywords: Cross-contamination; retail settings; cilantro; antimicrobials; post-harvest washing; Escherichia coli O157:H7; Salmonella; Listeria monocytogenes

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