Surface Decontamination of Fresh, Whole Peaches (Prunus persica) Using Sodium Hypochlorite or Acidified Electrolyzed Water Solutions

ABSTRACT

Nearly half (45%) of the U.S. peach production is sold through the fresh market sector, making post-harvest disinfectant washing treatments a critical step for reducing surface microorganisms. This study was conducted to determine the effects of various disinfectant washing treatments on the surface decontamination of fresh whole freestone peaches. Minimal application of disinfectant washing treatments (five-second dip) reduced numbers of Listeria innocua recovered from inoculated peaches by 93% (acidified electrolyzed water containing 22 ppm sodium hypochlorite, pH 2.8) or 96% (chlorine containing 22 ppm sodium hypochlorite, pH 8.2) compared to cell counts on unwashed or water-only washed peaches. Increasing the wash time to 40 minutes resulted in a greater reduction in numbers of Listeria innocua recovered from the surfaces of peaches (99.95% and 99.9% for acidified electrolyzed water and chlorine treatments, respectively). All of the washing treatments decreased counts of total aerobic microorganisms on peaches (>92% reduction) when applied for 40 minutes. The results of this study demonstrate that washing peaches reduced but did not completely eliminate surface microorganisms.

KEYWORDS:

• Listeria
• peaches
• surface decontamination
• fresh produce
• produce washing

Introduction

According to the Centers for Disease Control and Prevention (CDC), 48 million individuals become sick each year from foodborne illness, but only 20% (9.4 million) of those illnesses have been directly linked to a known pathogen in food (Centers for Disease Control and Prevention, 2018; Painter et al., 2013; Scallan et al., 2011). The annual economic burden of foodborne disease in the U.S. has been estimated to be 15.5 USD billion and includes lost wages, medical expenses, and loss of life (Hoffman et al., 2015). One of the major sources of foodborne illness outbreaks is consumption of fresh produce (Centers for Disease Control and Prevention, 2018; Murray et al., 2017; Painter et al., 2013). Painter et al. (2013) reported that 46% of the foodborne illness outbreaks occurring from 1998 to 2008 were associated with the consumption of fresh produce. Fruits-tree nuts consumption was linked to 11.7% of the illnesses (1,123,808), 10.1% of the hospitalizations (5,829), and 6.4% of the deaths (Painter et al., 2013). Despite the association with foodborne illness outbreaks, consumption of fresh produce, other than orange juice, potatoes, and lettuce, has continuously increased over the past decade, prompting implementation of adequate preventive measures (Lin and Morrison, 2016). Preventive measures for foodborne illness were mandated by the FDA in the Food Safety Modernization Act (FSMA), and specifically for fresh produce in 2016 in the FSMA Produce Safety Rule (US FDA, 2018a, 2018b).

One of the most widely consumed, low-risk produce commodities are fresh peaches (Prunus persica) as they are grown as a tree fruit with minimal direct contact to soil and irrigation water (Duvenage and Korsten, 2017). In the U.S., approximately 652 thousand metric tons of peaches, valued at nearly 522 USD million dollars, are grown and harvested each year (USDA, National Agriculture Statistics Service, 2019). Forty-five percent of the peaches produced in the U.S. are sold in the fresh-market sector, while 55% of the peaches produced in the U.S. are either canned, frozen, dried, or further processed into other products for use as an ingredient (USDA, Agriculture Marketing Service, 2019). In 2014, the CDC reported the first link between human listeriosis and consumption of stone fruit. Whole peaches, nectarines, plums, and pluots were voluntarily recalled in California based on internal company testing for Listeria spp. (Jackson et al., 2015). The CDC stated that although the exposure to the recalled product was widespread, disease was rare; however, the link demonstrated the need to strengthen prevention strategies associated with ready-to-eat fruit (Jackson et al., 2015).

Beauchat (1996) reported that microorganisms on fresh fruits and vegetables may originate from feces, soil, irrigation water, aerosols, wild and domestic animals, improperly composted manure, harvesting and processing equipment, and pre- and post-harvest human handling. Research has shown that washing is critical for reducing the numbers of microorganisms on the surfaces of fresh produce (Abadias et al., 2008; Beauchat, 1996; Bell and Kyriakides, 2005; Kim et al., 2012; Lee et al., 2014; Natvig et al., 2002). According to the U.S. Food and Drug Administration (FDA), the effectiveness of antimicrobial chemicals at reducing surface microbial contamination of fresh produce depends on the chemical and physical state of the treatment (water temperature, contact time, and acidity), resistance of the pathogen, and nature of the fruit or vegetable surface (U.S. Food and Drug Administration, 1998). Regardless of the above conditions, the FDA states that washing fresh produce is an important step since most pathogens are located on the surface skin or tissue of produce (U.S. Food and Drug Administration, 1998).

The most common method of processing fresh produce involves the use of a chlorinated water spray or chlorinated immersion wash (Beuchat, 1998; Graca et al., 2011). Research on chlorine has shown that it may inactivate microorganisms by the following methods: 1) alteration of nucleic acids within the cells; 2) the inactivation of cellular enzymes; and 3) the disruption of the cell membrane (Fair et al., 1947; Green and Stumpf, 1946; Knox et al., 1948; Marks and Strandskov, 1950; Sconce, 1962; White, 2010; Wyss, 1962). Chlorine is most active against microorganisms when it is in the hypochlorous acid form, and this species is predominant at pH 4 to 6 in a water-chlorine mixture (Marriott and Gravani, 2006; White, 2010). Hypochlorous acid also readily migrates into bacterial cells and disrupts enzymatic processes in the cells (Sconce, 1962; White, 2010).

Studies on the effectiveness of chlorine as an antimicrobial treatment for fresh produce have given mixed results. Several researchers have shown that chlorinated water at concentrations of 20 to 200 ppm produced similar reductions in numbers of Escherichia coli recovered from lettuce when compared to non-chlorinated tap water (Beuchat, 1999; Lin and Morrison, 2016; Park et al., 2001), and this was not improved with longer exposure times of 1 to 5 min (Beuchat, 1999). Two hundred ppm of chlorine was found to reduce the numbers of E. coli by 2 log cfu/cm² on the surface of oranges after 8 min of exposure time at 30°C (Pao and Davis, 1999). Numbers of Listeria monocytogenes were reduced on lettuce and cabbage by 1.7 and 1.2 log cfu/g, respectively, when treated with 200 ppm chlorine for 10 min at 22°C (Zhang and Farber, 1996). Inconsistencies in the efficacy of chlorine as an antimicrobial treatment may be attributed to managing the multiple factors that affect the concentration of hypochlorous acid in treatment solutions such as pH, amount of organic material present, temperature, concentration, quality of incoming water (water hardness), interactions with other chemical treatments, internalization of pathogens within the plant tissue, biofilm formation on plant surfaces, hydrophobicity of plant surfaces, and susceptibility of the target microorganism (Centers for Disease Control and Prevention, 2012). In spite of the aforementioned disadvantages to chlorine, it remains the most popular antimicrobial treatment for washing fresh produce.

Electrolyzed oxidizing water has gained in popularity as an alternative to traditional chlorine because it has several advantages to chlorine. Advantages of electrolyzed oxidizing water include on-site generation, relatively low-cost, one- to two-year shelf-life when properly stored, environmentally friendly for discharge when converted to non-electrolyzed water and potential for reducing enzymatic browning in cut-fruit (Shiroodi and Ovissipour, 2018). The acidic portion of electrolyzed water (AEW) contains hypochlorous acid and small amounts of hydrochloric acid, hydrogen peroxide, ozone, and chlorine oxides (Hricova et al., 2008; Kim et al., 2000; Koseki and Itoh, 2001; Machado et al., 2016; Northcutt et al., 2007; Park et al., 2001). AEW inhibits bacteria because of the synergistic effects of the low pH, high oxidation reduction potential, and high chlorine content of the water (Northcutt et al., 2007; Park et al., 2001). The popularity of AEW among food manufacturers has been attributed to reduced water and chlorine consumption while avoiding disinfection by-products such as organochlorinated compounds (Machado et al., 2016). Additionally, because it is produced from salt water, AEW may appear to be a more “natural” preservative than traditional chlorine for concerned consumers.

Comprehensive reviews on the sanitation of fresh-cut produce have been published by Beauchat (2002), Gill et al. (2002), and Ramos et al. (2013). The FDA (U.S. Food and Drug Administration, 2018a; U. S. Food and Drug Administration, 2018b) also provides guidance on methods to reduce or eliminate pathogens from fresh produce. In addition, Shiroodi and Ovissipour (2018) have reported on electrolyzed water application in fresh produce sanitation. However, since few studies have focused on cleaning and sanitizing non-citrus or ‘stone fruit’ during processing, the reviews and guidance documents provide little or no information on fruits such as peaches. Thus, the purpose of this study was to determine the efficacy of various antimicrobial treatments on reducing the numbers of native microflora on commercially produced unwashed, fresh, whole peaches and then to determine if these same treatments provided surface decontamination of peaches inoculated with Listeria innocua.

Materials and Methods

Approximately 300 freestone peaches of the ‘Sweet September’ cultivar were obtained within 24 hours of harvest and before processing. Peaches were manually harvested by a commercial crew as “firm mature,” and were packed before washing into new corrugated boxes for transportation to the laboratory. Color of peaches as an index of maturity was measured aseptically at four equidistant points around the midsection of each peach using a HunterLab Ultrascan Pro Spectrocolorimeter set for a 2^⸰ and 10^⸰ observer in illuminate C light. C.I.E. L* a* and b* measurements of peach skin color were taken after standardizing the spectrocolorimeter using a light trap and standard white tile (EVU 000746), and the aperture on the spectrocolorimeter was sanitized between each peach. Average color readings for all peaches was L* = 55.9 ± 1.1; a* = 19.9 ± 0.9; b* = 35.5 ± 1.2. Peaches were visually inspected for defects (skin discoloration, skin tears, mold growth, or similar) using aseptic techniques, and only those peaches found to be free of defects were used in the study. The peaches that were selected for the study were aseptically placed onto numbered weigh boats and the individual weight of each peach was recorded. Weight of peaches ranged from 297 g to 349 g per peach. Weigh boats were marked using a treatment code to allow tracking. Three experiments were conducted during this study, and four replications were performed for each experiment.

Experiment 1: Five- Second Wash of Non-Inoculated and Listeria-Inoculated Peaches

Unwashed peaches were divided into two treatment groups: Non-inoculated and Inoculated. On the day before the experiment, peaches in the inoculated group were inoculated with Listeria innocua (single strain, ATCC 33090), packed into new corrugated commercial boxes, and held for 24 hours at 4°C. The other half of the peaches (non-inoculated) were packed into new corrugated boxes that were separate from the inoculated treatment, and these peaches were also held for 24 hours at 4°C. On the day of each inoculation, Listeria innocua inoculum was freshly prepared immediately before inoculation. To prepare the inoculum, a Listeria innocua culture was plated and grown on PALCAM agar containing the PALCAM Listeria selective supplement. Colonies were removed from the agar after 48 hours incubation at 30°C and diluted in 0.1% (w/v) sterile peptone (Bacto Peptone, BD Diagnostics, Sparks, MD) to make a stock solution. The absorbance of the stock solution of inoculum was measured at 600 nm (Thermo Scientific Genesys 10S UV-Vis spectrophotometer) to estimate the number of cells per mL at approximately 10⁶ to 10⁸ cells per mL. The exact number of cells per mL was confirmed by plating the stock solution onto PALCAL agar containing the selective Listeria supplement (Hitchins et al., 2017). Each peach was inoculated with 0.1 mL of the Listeria innocua inoculum containing between 10⁶ to 10⁸ cells per mL. Inoculation was performed by two individuals – while one person was dispensing small droplets of inoculum around the top of the peach in approximately 5 locations away from the stem, the other individual was spreading the inoculum on the skin surface using a sterile loop. A new sterile loop was used for each peach. After inoculation and before packing into boxes, peaches were held at room temperature for approximately 1 hour to allow the inoculation to dry. Peaches were then packed into boxes and held overnight at 4°C.

On the next day, non-inoculated and inoculated peaches were removed from cold storage, allowed to warm at room temperature for 1 hour, and were then assigned to one of the following four washing treatments: No Wash (NW); Tap Water Wash (TW); Acidified Electrolyzed Water Wash (AEW); and Chlorinated Water Wash (CL). NW (Control) peaches did not undergo a wash treatment. TW peaches were dipped into municipal tap water (pH 6.4 to 7.0 and ORP of 526 to 591 mV), with no additional antimicrobial treatment. AEW peaches were dipped into acidified electrolyzed water (AEW) that was generated using a Hoshizaki ROX-10WA-E Water Electrolyzer that was set to a voltage of 10 Amps before a 100 g/L NaCl solution was pumped through the system. AEW was generated immediately before it was used to treat peaches, and the chlorine concentration of the AEW was 22.0 ± 0.2 mg/L for each replication. The CL treatment was also prepared immediately before use by diluting non-floral, over-the-counter commercial bleach (8.25% sodium hypochlorite) previously purchased from a local retail store, with tap water to create a chlorine concentration that was similar to the AEW chlorine concentration for each replication. Chlorine concentration of both the AEW and chlorine (CL) treatments was determined using a single wavelength spectrophotometer (530 nm) and the CHEMetrics CHLORINE 2 SAM (Single Analyte Method) test kit with Vacu-vials to analyze N,N diethyl-p-phenylenediamine (DPD) colorimetric reactions. The pH of each solution was measured in duplicate using two identical Accumet Model 10 pH meters that were standardized using pH 4.0 and 7.0 standard solutions. Oxidation Reduction Potential (ORP) for each solution was measured using the same pH meter affixed with a platinum ORP combination electrode (Ag/AgCl combination). The ORP probe was standardized each time using a + 600 mV standard solution.

For all treatments, excluding NW, 500 mL of one of the freshly prepared solutions at approximately 2°C (TW, AEW, and CL) was placed into individual 1000 mL beakers. One peach was dipped into one beaker for 5 seconds. Five hundred mL of solution per peach and 2°C water temperature were chosen based on estimates of loads and water flow in a commercial peach operation (Carr, 2016). Treatment solutions were not reused during any of the experiments.

After treatment, peaches were removed from the solutions and allowed to air dry for 1 minute in a clean, labeled weigh boat. Peaches were then aseptically transferred into clean bags (1 gallon) with 100 mL of sterile 0.1% (w/v) peptone solution (Bacto Peptone, BD Diagnostics, Sparks, MD). Peaches and peptone were shaken for approximately 1 min to remove surface microorganisms. NW peaches were also shaken in 0.1% (w/v) sterile peptone (Bacto Peptone, BD Diagnostics, Sparks, MD) for 1 min. Peaches were aseptically removed from the rinsate, and the rinsate was used to prepare serial dilutions for plating onto media.

Dilutions of the peach rinsate were plated on to aerobic plate count (APC) agar plates to enumerate total aerobic microorganisms. After plating, APC plates containing the sample were inverted and incubated for 48 hours at 37°C. Visible colony forming units (CFUs) were counted, converted to log₁₀CFU/g peach, statistically analyzed, and reported.

Similarly, Listeria innocua was recovered from the surfaces of peaches after treatment by shaking individual peaches in sterile 0.1% (w/v) peptone (Bacto Peptone, BD Diagnostics, Sparks, MD) in a clean plastic bag for 1 min. Serial dilutions of the rinsate were prepared and plated onto PALCAM agar with the selective Listeria supplement. Plates were inverted and incubated for 48 hours at 30°C, and visible colonies were counted, converted to log₁₀ cfu/g peach for analysis and reporting.

After all the rinsates had been plated for APC or Listeria, the rinsates were tested for chlorine level using the same method as described above. This was done to determine potential carry-over from the peach to the rinsate. In all cases, chlorine was not detected in any of the rinsates.

Experiment 2: Forty-minute Wash of Non-Inoculated Peaches

Unwashed peaches were obtained from a local farmer immediately after harvest and were transported to the laboratory in corrugated boxes where they were held at 4°C for 24 hours. After the 24 hours storage period, peaches were randomly divided into the same four washing treatments as reported in Experiment 1 (NW, TW, AEW, and CL). All washing treatments were prepared using the same procedures as outlined in Experiment 1. Experiment 2 focused solely on non-inoculated peaches (native total aerobic microorganisms only) and the washing exposure time was increased to 40 min. Forty min was selected as the unagitated wash exposure time for Experiment 2 because this is the dwell time that is commonly used in commercial peach hydrocoolers (Carr, 2016). After 40 min, peaches were removed from the individual beakers containing the wash treatment, placed into clean, pre-labeled weigh boats, and allowed to air dry for 1 min. Peaches were then placed into clean bags (1 gallon) with 100 mL of sterile 0.1% (w/v) peptone solution and were shaken for approximately 1 min to remove surface microorganisms. NW peaches were also shaken in 0.1% (w/v) sterile peptone (Bacto Peptone, BD Diagnostics, Sparks, MD) for 1 min. Peaches were aseptically removed from the rinsate, and the rinsate was used to prepare serial dilutions for plating.

Dilutions of the peach rinsate were plated on to aerobic plate count (APC) agar plates to enumerate total aerobic microorganisms. After plating, APC plates containing the sample were inverted and incubated for 48 hours at 37°C. After incubation, visible colony forming units (CFUs) were counted, converted to log₁₀CFU/g peach, statistically analyzed, and reported.

Experiment 3: Forty-minute Wash of Listeria Innocua Inoculated Peaches

Unwashed peaches were obtained immediately after harvest, packed into clean corrugated boxes, and transported to the laboratory where they were inoculated with Listeria innocua. After inoculation, peaches were packed into new corrugated boxes and held for 24 hours at 4 C. Listeria innocua inoculum was prepared fresh on each day that it was used to inoculate peaches. The inoculum was prepared using the same procedures that were outlined in Experiment 1. The exact number of Listeria innocua cells per mL was confirmed by plating the stock solution onto PALCAL agar containing the selective Listeria supplement (Hitchins et al., 2017). Each peach was inoculated with 0.1 mL of the Listeria innocua inoculum containing between 10⁶ to 10⁸ cells per mL. Similar to Experiment 1, inoculated peaches were held at room temperature for approximately 1 hour to allow the inoculation to dry before they were packed into boxes and stored overnight at 4^◦C. On the next day, inoculated peaches were removed from cold storage, allowed to warm at room temperature for 1 hour, and were then washed as described in Experiment 2. Peaches were washed using the extended dwell time of 40 min in each of the treatment solutions.

After the 40 min wash, peaches were aseptically removed from the solutions, placed into clean, pre-labeled weigh boats for 1 min. Listeria innocua was recovered from the surfaces by shaking in sterile 0.1% (w/v) peptone (Bacto Peptone, BD Diagnostics, Sparks, MD) in a clean plastic bag for 1 min. Serial dilutions of the rinsate were prepared and plated onto PALCAM agar with the selective Listeria supplement. Plates were inverted and incubated for 48 hours at 30°C and visible colonies were counted, converted to log₁₀ cfu/g peach for analysis and reporting.

Statistical Analyses

Experiments 1, 2, and 3 were repeated four times. During each replication of the three experiments, four peaches were used for each washing treatment (N = 16). For the statistical analyses, numbers of total aerobic microorganisms or Listeria innocua were converted to log₁₀ cfu/g peach. Log counts were then analyzed using the general linear model procedure of SAS statistical analysis (SAS Institute, 2002). The main effects in the model were antimicrobial treatment and replication. Tukey–Kramer statistics adjusted for multiple comparisons and determined where differences were found. The level of significance was assessed using a Type I error (alpha) of 5%.

Results and Discussion

Recovery of microorganisms from surfaces of unwashed commercial peaches was evaluated without a disinfectant wash or after washing in tap water, AEW or CL for 5 sec or 40 min. Municipal tap water was used as one of the treatments and it also served as the basis for preparing the AEW and CL solutions. The chemical characteristics of tap water varied slightly (pH 6.4 to 7.0 and ORP of 526 to 591 mV), but it had consistently negligible chlorine levels (0–0.1 ppm which is below the accuracy of the machine of 0.2 ppm). In all three experiments, AEW solutions had similar pH values (pH 2.8–2.9), chlorine concentrations (approximately 22 ppm), and ORP (approximately 1130–1160 mV). Similarly, the CL solutions, which were prepared by diluting concentrated commercial sodium hypochlorite, had chlorine concentrations of 21–22 ppm with pH values of 8.2 to 8.4 and ORP range of 744 to 773 mV. The acidity of the CL treatments was not adjusted to optimum for disinfection (< pH 7.5) prior to use since this is not a common practice employed by most commercial produce operations.

According to White (2010), the extent to which sodium hypochlorite (HOCl) dissociates in water is dependent upon the pH and temperature of the water. The concentration of HOCl in solutions is higher at lower temperatures and at lower pH values (White, 2010). Using the chlorine dissociation tables provided in White’s chlorination handbook, the percentage of chlorine present as HOCl at pH 8.2 and 0°C to 5°C may be estimated to be approximately 26–30%, and it decreases to 18–21% at pH 8.4 and 0–5°C (White, 2010). Chlorinated solutions that have pH of 5.0 or less at 0–5°C have will have 100% of their chlorine content in the HOCl form (White, 2010). AEW contains HOCl plus small amounts of hydrochloric acid, hydrogen peroxide, ozone, and chlorine oxides (Park et al., 2001). Similarly, sodium hypochlorite in water generates HOCl, hypochlorite ions, sodium hydroxide (or their ions), chloride ions, hydrogen ions, and other minor chlorinated species (White, 2010). Based on these references, the AEW solutions used in the present study had higher percentages of HOCl than the CL solutions used in this study.

Previous research studies have used ORP measurements to determine the disinfectant properties of chlorinated solutions (Chang, 1944; Schmelkes et al., 1939; Victorin et al., 1972). In their study of different food-related pathogens, Kim et al. (2000) stated that ORP was the primary factor affecting the efficacy of chlorinated solutions since it reflected the total oxidation capacity of the solution regardless of the pH and concentration of chlorine. Hricova et al. (2008) suggested that high ORPs inactivate bacteria by changing the electron flow in cell, damaging cell membranes, and disrupt metabolic processes. Other scientists have reported that the effectiveness of AEW and other chlorine-based antimicrobials is influenced by ORP, pH, and concentration of free available chlorine as all of these act synergistically to inactivate microorganisms (Al-Haq et al., 2005; Hricova et al., 2008; Northcutt et al., 2007; Park et al., 2001). White (2010) reported that ORP values increase with decreasing pH due to Nernst Law, and this is reflected in the pH and ORP values reported in the present study. ORP for AEW solutions was 46% to 56% higher than CL and was two-times higher than the ORP of tap water.

Total aerobic microorganisms recovered from unwashed peaches (NW treatment) were approximately 4.3 log cfu/g peach. Dipping peaches for 5 seconds or less (in and out) in disinfectant solutions had no effect on the number of total aerobic microorganisms recovered from the surfaces of peaches, and values ranged from 3.4 to 3.7 log₁₀ cfu/g peach [Table 1 near here]. However, dipping peaches for 5 seconds in the disinfectant solutions significantly reduced the numbers of Listeria innocua recovered from the surfaces of peaches (Table 1). Unwashed peaches (NW) were found to have approximately 4.9 log cfu/g peach of Listeria innocua on their surfaces and this was reduced when the peaches were washed with either AEW or CL by 1.2 and 1.4 log cfu/g peach, respectively. When compared to the NW treatment, dipping peaches in tap water did not reduce the level of Listeria innocua (4.3 log cfu/g peach) on peaches (Table 1).

Table 1. Chemical characteristics of treatment solutions and their effects on numbers (log₁₀ cfu per g peach) of total aerobic microorganisms (APC) and inoculated Listeria innocua recovered from surfaces of whole peaches after a 5 second exposure time

Treatment	pH of solution	Chlorine (ppm)	Oxidation Reduction Potential (mV)	APC log10 CFU/g peach	Listeria innocua log10 CFU/g peach
No Wash (NW)	N/A	N/A	N/A	4.3 ± 0.2	4.9^a ± 0.1
Tap Water Wash (TW)	7.00 ± 0.02	0.01 ± 0.1	526.3 ± 34.9	3.5 ± 0.2	4.3^ab ± 0.1
Acidified Electrolyzed Water (AEW)	2.86 ± 0.02	21.8 ± 0.3	1123.3 ± 8.0	3.4 ± 0.2	3.7^bc ± 0.2
Chlorine (CL)	8.21 ± 0.02	21.0 ± 0.1	773.3 ± 8.2	3.7 ± 0.3	3.5 ^c ± 0.1

N/A designation refers to ‘Not applicable.’

^a-cMeans ± standard error of means within a column with a common superscript are not significantly different (P > 0.05).

Using a longer dwell time in the disinfectant wash (40 min) resulted in a significant reduction in total aerobic microorganisms recovered from the surfaces of peaches (Table 2) [Table 2 near here]. Similar to experiment 1, the surfaces of unwashed peaches were found to have 4.3 log cfu/g peach of total aerobic microorganisms, while the surfaces of peaches washed with TW, AEW or CL had approximately 3.2, 2.6, and 2.3 log cfu/g peach of total aerobic microorganisms, respectively, on their surfaces (Table 2). No difference was observed in the number of total aerobic microorganisms recovered from peaches treated with AEW as compared to those treated with CL, and both treatments resulted in a 98–99% reduction in total aerobe counts (Table 2). Other studies have reported that increasing the exposure time to chlorine or AEW will reduce the numbers of surface microorganism on fresh produce (Graca et al., 2011; Hao et al., 2005; Rahman et al., 2011) Most of these other studies use short exposure times of <10 min. Rahman et al. (2011) published a table comparing various application methods of AEW for disinfecting various fruits and vegetables. Missing from their comprehensive summary is research describing the application of AEW on fresh peaches.

Table 2. Chemical characteristics of treatment solutions and their effects on numbers (log₁₀ cfu per g peach) of total aerobic microorganisms recovered from surfaces of whole peaches after 40 minutes of exposure.¹

Treatment	pH of solution	Chlorine (ppm)	Oxidation Reduction Potential (mV)	Log10 CFU/g peach	Reduction Log cfu/g peach
No Wash (NW)	N/A	N/A	N/A	4.3^a ± 0.2	-
Tap Water Wash (TW)	6.96 ± 0.05	0.01 ± 0.1	526.3 ± 34.9	3.2^b ± 0.2	1.1
Acidified Electrolyzed Water (AEW)	2.89 ± 0.02	21.9 ± 0.2	1126.3 ± 8.0	2.6 ^c ± 0.3	1.7
Chlorine (CL)	8.39 ± 0.02	21.5 ± 0.3	744.3 ± 12.4	2.3 ^c ± 0.4	2.0

N/A designation refers to ‘Not applicable.’

^a-cMeans ± standard error of means with a common superscript are not significantly different (P > 0.05)

¹Treatment exposure time of 40 minutes simulates dwell time in a commercial hydrocooler.

After 40 min of washing in TW, peaches had the same number of cells of Listeria innocua on their surfaces (4.7 log cfu/g peach) as the unwashed peaches (NW peaches at 4.9 log cfu/g peach) [Table 3 near here]. AEW and CL reduced the numbers of Listeria innocua on the surface of washed peaches by 3.5 and 3.0 log cfu/g peach, respectively, when compared to the NW peaches. None of the disinfectant treatments gave non-detectable counts of either total aerobic microorganisms or Listeria innocua. These data show that without agitation or scrubbing, an antimicrobial treatment such as AEW or CL is required to reduce the number of Listeria on the surface of fresh peaches.

Table 3. Chemical characteristics of treatment solutions and their effects on numbers (log₁₀ cfu per g peach) of Listeria innocua recovered from surfaces of whole peaches after 40 minutes of exposure.¹

Treatment	pH of solution	Chlorine (ppm)	Oxidation Reduction Potential (mV)	Log10 CFU/g peach	Reduction Log cfu/g peach
No Wash (NW)	N/A	N/A	N/A	4.9^a ± 0.1	-
Tap Water Wash (TW)	6.39 ± 0.11	0.01 ± 0.0	591.3 ± 30.8	4.7^a ± 0.1	0.2
Acidified Electrolyzed Water (AEW)	2.83 ± 0.02	21.8 ± 0.3	1159.7 ± 3.6	1.4^b ± 0.4	3.5
Chlorine (CL)	8.18 ± 0.03	20.9 ± 0.1	770.3 ± 8.2	1.9^b ± 0.3	3.0

N/A designation refers to ‘Not applicable.’

^a-cMeans ± standard error of means with a common superscript are not significantly different (P > 0.05)

¹Treatment exposure time of 40 minutes simulates dwell time in a commercial hydrocooler.

In their study on sliced apples, Graca et al. (2011) demonstrated that AEW (pH 2.93; ORP 1128 mV) and CL (pH 11.03; ORP 583 mV) at 100 ppm and 30 min of exposure time reduced numbers of Listeria innocua by more than 1.2 log cfu/g apple. These authors showed that AEW at 50 (pH 3.08; ORP 1111) and 100 ppm gave similar reductions in counts of Listeria innocua on apples slices to CL at 100 ppm (Graca et al., 2011). Park et al. (2009) reported >5 log per g reduction in Listeria monocytogenes on tomatoes after 1 min of exposure to AEW (pH 2.0; 37.5 ppm free chlorine), but no reductions using similar treatments on green onion. These same authors along with other researchers indicated that decontamination of fresh produce is more difficult when it has a rough surface (Koseki et al., 2004; Park et al., 2009). Furthermore, it is also possible that the green onions tested by Park et al. (2009) internalized the Listeria monocytogenes or cells formed a biofilm protecting themselves from the disinfectant.

While the skin surfaces of peaches may appear to be smooth, they may have some defined and convoluted conformation that make them difficult to sanitize. Fernández et al. (2011) examined the chemical composition and morphology of peach skin and reported a dense indumentum of non-granular, unicellular trichomes (100 to 1000 μm) covered in a nonpolar layer (15% waxes and 19% cutin) and imbedded in the epidermis. Trichomes have been reported to restrict entry of microorganisms and toxic substances, along with reducing moisture loss and lessen the effects of radiation on plants and fresh produce (Sirinutsomboon et al., 2011). However, researchers have indicated that the base of the trichomes may serve as an attachment site once the microorganisms come in contact with the insertion point on the epidermis (Sirinutsomboon et al., 2011).

Listeria monocytogenes is considered to be an adulterant, with a zero-tolerance threshold in ready-to-eat foods, such as ‘covered’ fresh produce that is regulated under the FSMA Produce Safety Rule (U.S. Food and Drug Administration, 2018c). Few studies have examined the attachment of Listeria to fresh produce at the molecular level, and while the attachment mechanism is still unknown, scientists have demonstrated that Listeria spp. have specific genes that encode for cellulose-binding surface proteins that allow it to interact with plant surfaces thereby facilitating attachment (Bae et al., 2013). Additional research is needed to determine the interaction between fruit trichomes, bacterial attachment, and microbial surface proteins.

Conclusions

The results of the present study demonstrate that AEW gave similar results to CL for decontaminating the surface of fresh peaches. Results also demonstrate that fresh peaches may have up to 20,000 cells of total aerobic microorganisms on their surfaces prior to processing (washing) and washing in tap water can reduce these counts by more than 90%. Adding disinfectant treatments to the wash water further reduces total aerobic microorganisms counts by 98–99%.

Reductions in Listeria innocua counts on the surfaces of peaches requires a disinfectant treatment. When a disinfectant treatment is added to the wash water, counts of Listeria innocua may be reduced by 99.97% (AEW) or 99.9% (CL).

Notable advantages, besides those mentioned previously, for using AEW may be consistency and the acceptable consumer perception of a more ‘natural’ environmentally friendly disinfectant produced from salt solutions. Reported disadvantages include corrosiveness of processing plant surfaces due to AEW’s low pH and possible product quality defects associated with discoloration. It is for this reason that the authors of the present study conducted colorimetric analyses on peaches washed with various disinfectant treatments and the colorimetric results are published in the thesis.

Declaration Of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank Emily Moody, Kathryn Davis, Catherine Harvey, and Kimberly Baker for their technical assistance. The authors have no conflict of interest to declare.