Synergistic Effects of Lauric Arginate and Peracetic Acid in Reducing Listeria monocytogenes on Fresh Apples

Apples are naturally coated with a water-repelling hydrophobic wax layer, which may limit the antimicrobial efficacies of surface sanitizer solutions. Lauric arginate (LAE) is a cationic surfactant with antimicrobial efficacy against Listeria monocytogenes. In this study, we investigated the antimicrobial and the wettability effects of LAE in enhancing anti-L. monocytogenes efficacy of peracetic acid (PAA) and further verified the optimized treatment combinations in a pilot spray-bar brush bed system. Apples after 48 h of inoculation were treated with PAA surface sanitation in combination with different concentrations of LAE at 22 or 46°C. The effectiveness of PAA with LAE solutions in decontaminating L. monocytogenes significantly increased with the increased concentration of PAA (60–80 ppm) or LAE (0.01–0.05%) or the treatment temperature (from 22 to 46°C). A 30–120-sec wash by 80 ppm PAA with 0.01 and 0.05% LAE at 22°C reduced L. monocytogenes on apples by 2.10–2.25 and 2.48–2.58 log₁₀ CFU/apple, respectively. Including LAE in the PAA solution decreased contact angles on apple surfaces. However, the increased wettability of the sanitizer solution may not be the main contributor to the enhanced antimicrobial efficacy of the PAA solution, given that the addition of Tween 80 or Tween 85 only slightly boosted the anti-L. monocytogenes efficacy of PAA solutions though both increased the wettability of the PAA solutions. The synergistic effects of PAA and LAE were further validated in a pilot spray-bar brush bed packing system, where a 30-sec spray wash with 80 ppm PAA and 0.05% LAE at 22 and 46°C caused 1.68 and 2.08 log reduction of Listeria on fresh apples, respectively. This study provides an improved PAA process/preventive strategy for ensuring microbial food safety of fresh apples that is applicable to commercial apple packing lines.

Introduction

Listeria monocytogenes can potentially transfer to fresh produce including apples during the postharvest handling, which results in deadly outbreaks (McCollum et al., 2013; Angelo et al., 2017). Although listeriosis is rare, the mortality rate of listeriosis is very high (∼ 21%) (Silk et al., 2013). The recent foodborne outbreak of L. monocytogenes linked to caramel apples (Angelo et al., 2017) and multiple recall linked to fresh apples due to the potential contamination of L. monocytogenes (FDA, 2015, 2017b) highlight the importance of effective control strategies to minimize contamination risk of L. monocytogenes on fresh apples.

Antimicrobial wash interventions including chlorine-based sanitizers (Du et al., 2002; Rodgers et al., 2004; Sheng et al., 2020a), organic acids such as lactic acid and citric acid (Park et al., 2011), and peracetic acid (PAA) (Rodgers et al., 2004; Shen et al., 2019) were used to decontaminate L. monocytogenes on fresh apples. Of these antimicrobials, PAA is the most widely used sanitizer in apple packing lines during the spray-bar brush bed intervention (Zhu et al., 2020). PAA has broad-spectrum antimicrobial activity (Baert et al., 2009) and does not produce toxic by-products (Monarca et al., 2002). PAA applied at 80 ppm, a concentration approved by the Food and Drug Administration (FDA) to wash fresh produce without further rinsing requirement (FDA, 2017a), is more efficient in decontaminating L. monocytogenes on fresh apples compared with 100 ppm chlorine-based sanitizers (Shen et al., 2019; Sheng et al., 2020a). However, spray wash of 80 ppm PAA at the current industry practice (at ambient temperature for 30–120 sec contact time) only resulted in about one log reduction of L. monocytogenes (Shen et al., 2020), indicating the need to boost the anti-Listeria efficacy of PAA.

Fresh apples are naturally covered with a hydrophobic wax layer (Dong et al., 2012; Veraverbeke et al., 2001), which might limit the direct contact of sanitizer solutions on apple surfaces. Surfactants are amphipathic molecules that exist in hydrophobic/hydrophilic interfaces resulting in easy spreading of a liquid solution (Nakama, 2017). Lauric arginate (LAE) is a cationic surfactant derived from lauric acid, L-arginine, and ethanol (Infante et al., 1984), which was approved by the FDA as Generally Recognized as Safe (GRAS) when used at the concentration of 0.02% for direct addition to food products such as meat, cheese, and fruit juice (FDA, 2005). LAE has antimicrobial activity against Listeria when used alone or in combination with other sanitizers. Application of 0.01% LAE for 120 sec caused 3.1 log reduction of L. monocytogenes on lettuces at an initial bacterial population of 6.6 log₁₀ CFU/g (Nübling et al., 2017). A 20-min treatment of 0.10% LAE reduced Listeria innocua by 2.3 log₁₀ CFU/cm² on lettuce surfaces (Huang and Nitin, 2017). A 3-min of 0.02% LAE exposure resulted in 1 log₁₀ CFU/cm² reduction of L. monocytogenes biofilm on lettuce surfaces (Sadekuzzaman et al., 2017). LAE was reported to enhance the anti-Listeria efficacy of potassium lactate and sodium diacetate on dairy and meat products (Stopforth et al., 2010; Soni et al., 2012). Existing data collectively suggest that LAE is a promising antimicrobial substance for the control of L. monocytogenes on food products. However, a recent laboratory scale study reported that the antimicrobial efficacy of 80 ppm PAA with 0.10% LAE against L. innocua on apple surfaces was not significantly different from that of 80 ppm PAA alone (Pietrysiak et al., 2019). Currently, no information is available about the strengthening effects of LAE in PAA solutions against L. monocytogenes on fresh apples.

The objectives of this study were to evaluate and optimize the effects of LAE in strengthening the anti-Listeria efficacy of PAA on fresh apples and further verify the optimized PAA and LAE treatments in a pilot spray-bar brush bed apple processing system. This study provides a practical and optimized PAA intervention strategy for the apple industry and other produce industries with similar postharvest handling and processing for their food safety programs.

Materials and Methods

Bacterial Inoculum Preparation

Three L. monocytogenes strains, NRRL B-57618 (1/2a, 2011 cantaloupe outbreak isolate), NRRL-33466 (1/2b, processing plant isolate), and NRRL B-33053 (4b, 1983 coleslaw outbreak isolate) and L. innocua strains (NRRL B-33197, NRRL B-33314, and NRRL B-33554) were obtained from USDA-ARS culture collection [National Center for Agricultural Utilization Research (NRRL), Peoria, IL, United States). Stock cultures were stored at −80°C in trypticase soy broth [Becton, Dickinson and Company (BD), Sparks, MD, United States) supplemented with 6 g/L yeast extract (Fisher Scientific, Fair Lawn, NJ, United States) (TSBYE) and 20% (v/v) glycerol (J. T. Baker, Philipsburg, NJ, United States). Each frozen stock culture was subcultured in TSBYE at 37°C for 24 h and transferred into fresh TSBYE for a second 24-h subculture. Following incubation, the cultures were centrifuged at 8,000 × g for 5 min at 4°C and the resulting pellets were washed once and resuspended in sterile phosphate-buffered saline (PBS, pH 7.4) (EMD Millipore, Billerica, MA, United States). For apple inoculation, a three-strain L. monocytogenes or L. innocua cocktail was used, and each strain suspension was mixed in equal proportions and diluted to achieve ∼ 10⁶ CFU/ml in sterile PBS solutions.

Preparation of Sanitizer Solutions

Cationic surfactant LAE (CytoGuard^TM) was kindly provided by A&B Ingredients, Inc. (Field, NJ, United States). Nonionic surfactants of Tween 80 and Tween 85 were purchased from Sigma (St. Louis, MO, United States). Bioside HS (a stabilized mixture of 15% PAA and 22% hydrogen peroxide) (Pace International Inc., Wapato, WA, United States) was used to prepare the 60–80 ppm PAA solutions. The concentration of PAA in the respective solution was verified using a titration kit (Aquaphoenix Scientific, Hanover, PA, United States). LAE (0.001–0.10%, v/v), Tween 80 (0.10–0.20%, v/v) or Tween 85 (0.10–0.20%, v/v) were added to 80 ppm PAA solution to the specified concentration.

The Intervention of LAE With PAA Solutions Against L. monocytogenes in Water

The above prepared PAA with LAE solutions were individually inoculated with three-strain L. monocytogenes cocktail at 5 × 10⁸ CFU/ml, where 1 ml of three-strain L. monocytogenes cocktail suspension was added to 9 ml of respective PAA with LAE solution, treated for 30 sec, then the 1.0 ml of solution was sampled and immediately neutralized with 9.0 ml of D/E neutralizing broth (BD). The survivals were enumerated by 10-fold serial dilution with sterile PBS and plating on duplicate TSAYE (TSBYE with 1.5% agar) plates and incubated at 37°C for 24 h. The detection limit of L. monocytogenes is 10 CFU/ml.

Apple Inoculation

Medium-sized (210–230 g) unwaxed Granny Smith apples (GSA) and Fuji apples devoid of cuts, bruising, or scars were used for this study. Before inoculation, 30 apples were randomly sampled for background microflora enumeration, which is about 3.3–4.0 log₁₀ CFU/apples. To inoculate, apples were washed with cold tap water, equilibrated to room temperature (∼22°C, RT) overnight, then dip inoculated with the three-strain L. monocytogenes or L. innocua cocktail inoculum (∼ 6.8 Log₁₀ CFU/ml) solution to have ∼ 6 log₁₀ CFU/apple inoculation level (Sheng et al., 2017). The inoculated apples were held at RT for 48 h when apples were treated with different sanitizer solutions. For each batch of the inoculated apples, 10 inoculated apples were randomly sampled 0 and 48 h after inoculation to evaluate the initial L. monocytogenes or L. innocua population and the uniformity on apples.

Sanitizing Treatment of Apples

The inoculated apples after 48 h inoculation were subjected to 80 ppm PAA solutions in the presence of 0.001–0.10% LAE at RT for a 2-min exposure unless specified. PAA with 0.10–0.20% Tween 80 or Tween 85 were included as a control.

We previously reported that PAA solution when applied at 46°C had enhanced antimicrobial activity against L. monocytogenes on apples compared with that at 22°C without compromising the quality of fruit (Shen et al., 2019). Therefore, the antimicrobial efficacies of PAA in combination with 0.01 or 0.05% LAE solutions were further evaluated at 46°C. Each experiment was independently repeated three times; 10 apples per treatment within an independent study. There were 30 apples in total for a selected treatment.

Evaluation of Antimicrobial Efficacy in a Pilot Scale Spray-Bar System

The spray wash of apples was conducted in a pilot spray-bar processing system equipped with a brush bed at Washington State University. L. innocua, a well-defined surrogate of L. monocytogenes (Sheng et al., 2020b; Zhu et al., 2020), was used in the pilot spray bar intervention. GSA inoculated with L. innocua were spray washed by 80 ppm PAA with or without LAE at 0.01 and 0.05% (v/v) at the specific temperature (22 or 46°C) for 30 or 120 sec. The flow rate of the spray bar was 0.977 L/min, and the brush bed is rotating at 47 revolutions per min. Each experiment was independently repeated three times with 10 inoculated apples per treatment within each independent study.

Microbiological Analysis

Immediately after sanitizer interventions, each apple was transferred into a stomacher bag with the addition of 10 ml of D/E neutralizing broth (BD). Each apple was hand-rubbed for 90 s as previously reported (Shen et al., 2019). Rub solutions were 10-fold serially diluted with sterile PBS, and the appropriate dilutions were plated in duplicate on TSAYE plates. The above TSAYE plates were first incubated at 35 ± 2°C for 4 h and then overlaid with a thin layer of modified Oxford agar (MOX, BD) to facilitate the recovery of the injured Listeria and to discern Listeria from apple background microflora (Kang and Fung, 1999; Shen et al., 2019).

Contact Angle Measurement

The contact angles of each wash solution on apple surfaces were measured using the Theta Lite Tensiometer (Biolin Scientific, Stockholm, Sweden) at RT. Apple disks with a 2-cm diameter were cut from fresh apples using a sharp knife. To measure the contact angle, 10 μl of wash solution was deposited onto the above-prepared apple disk using a microliter syringe needle, then the static contact angle was recorded within 5-sec contact. To accurately measure the wettability, the contact angle of each solution was measured on 20 apple disks. Mean values of 20 replicates were reported. The larger water contact angle represents the lower wettability.

Statistical Analysis

Mean differences were compared by one-way analysis of variance (ANOVA) and analyzed by Tukey’s multiple-comparison test (P < 0.05) using IBM SPSS 19.0 (Chicago, IL, United States). Microbiological data were reported as mean ± SEM (standard error mean) averaged from three independent experiments with 10 apples per treatment in each independent study, n = 30. Contact angles were presented as mean ± SEM, calculated from 20 apple disks, n = 20.

Results

Efficacies of Peracetic Acid With Lauric Arginate Against L. monocytogenes in Water

Adding LAE as low as 0.001% significantly (P < 0.05) enhanced anti-Listeria efficacy of 80 ppm PAA, which resulted in an additional ∼ 1.48 log₁₀ CFU/ml reduction of L. monocytogenes in water compared with that treated with 80 ppm PAA alone (caused 3.12 ± 0.02 log₁₀ CFU/ml) (Figure 1). The addition of 0.01% LAE in 80 ppm PAA solution caused more than a 5-log reduction of L. monocytogenes in water (Figure 1).

FIGURE 1

Figure 1. Antimicrobial efficacy of 80 ppm peracetic acid with different concentrations of lauric arginate against L. monocytogenes in water. The treatment time is 30 sec. The initial L. monocytogenes population was ∼ 7.06 log₁₀ CFU/ml. In all treatments, peracetic acid (PAA) was used at 80 ppm. Data are reported as mean ± SEM from three independent studies; there are three replicates per treatment within an independent study. ^a–fMean among bars without common letters differ significantly (P < 0.05). LAE, lauric arginate.

Antimicrobial Efficacy and Wettability of Peracetic Acid Solutions With or Without Surfactant Against L. monocytogenes on Fresh Apples

The addition of LAE as low as 0.001% enhanced (P < 0.05) the effectiveness of PAA against L. monocytogenes on fresh apples (Figure 2A). A 2-min exposure of 80 ppm PAA in combination with 0.05 or 0.10% LAE reduced L. monocytogenes by 2.68 ± 0.02 or 2.81 ± 0.01 log₁₀ CFU/apple (Figure 2A). However, including Tween 80 or Tween 85, up to 0.20% only slightly improved the efficacy of 80 ppm PAA solution against L. monocytogenes on apples, though they were statistically different (P < 0.05) (Figure 2B).

FIGURE 2

Figure 2. The effects of lauric arginate in enhancing the efficacies of peracetic acid against L. monocytogenes on Granny Smith apples may not be due to its wettability. Reduction of L. monocytogenes on apples treated with 80 ppm PAA in the presence of lauric arginate (LAE) (A) or Tween 80 or Tween 85 (B) for 2 min. The wettability of 80 ppm PAA solution with LAE (C) or Tween 80 or Tween 85 (D) as indicated by their contact angle on apple surfaces. Mean ± SEM, n = 30 for the reduction of L. monocytogenes and 20 for the contact angle. ^a–eMean among bars without common letters differ significantly (P < 0.05).

Including either LAE, Tween 80, or Tween 85 significantly increased (P < 0.05) the wettability of PAA solution on apple surfaces as indicated by decreased contact angles (Figures 2C,D). The PAA solutions with 0.001–0.005% and 0.01% LAE had a contact angle of 76–77° and 68.2°, respectively, which did not differ from those with 0.1 or 0.2% Tween 80/85 (Figures 2C,D). Data indicated that the enhanced anti-Listeria efficacy of LAE in PAA solutions might be mainly due to its antimicrobial activity instead of wettability.

Factors Impact Antimicrobial Strengthening Effects of Lauric Arginate in Peracetic Acid Solutions

Extending the contact time from 30 to 120 sec significantly enhanced (P < 0.05) anti-L. monocytogenes efficacies of PAA with or without LAE (Figure 3A). Regardless of contact time, the antimicrobial efficacies of PAA solutions against L. monocytogenes on GSA increased with elevated LAE concentrations (P < 0.05). Increasing LAE concentration to 0.05% caused 2.48–2.58 log₁₀ CFU/apple reductions of L. monocytogenes on apples (Figure 3A). Decreasing the concentration of PAA from 80 to 60 or 70 ppm diminished (P < 0.05) the efficacy of PAA with LAE treatment against L. monocytogenes on GSA, regardless of LAE concentration (Figure 3B). The efficacies of PAA with 0.01 and 0.05% LAE against L. monocytogenes on Fuji apples were not significantly different from those on GSA; a 2-min exposure to PAA with 0.01–0.05% LAE reduced L. monocytogenes on Fuji apples by 2.40–2.62 log₁₀ CFU/apple (Figure 3C).

FIGURE 3

Figure 3. Efficacy of peracetic acid with lauric arginate in reducing L. monocytogenes on apples under different concentrations and contact times at 22°C. (A) Reduction of L. monocytogenes on Granny Smith apples subjected to 80 ppm peracetic acid (PAA) with or without lauric arginate (LAE) for a 30-sec or 2-min contact. (B) Reduction of L. monocytogenes on Granny Smith apples treated with different PAA and LAE combinations for 2 min. (C) Reduction of L. monocytogenes on Fuji apples after a 2-min exposure of 80 ppm PAA with different concentrations of LAE. The initial L. monocytogenes population on apple surfaces was ∼ 6.8 log₁₀ CFU/apple. Mean ± SEM, averaged from three independent experiments; 10 apples per treatment within an independent study. ^a–fMean among bars without common letters differ significantly (P < 0.05).

Peracetic acid with LAE solutions at 46°C showed higher effectiveness against L. monocytogenes on GSA than that at 22°C (P < 0.05) (Figure 4). A 30–120-sec treatment of PAA at 80 ppm with 0.05% LAE at 46°C reduced L. monocytogenes by 2.90–2.95 log₁₀ CFU/apple (Figure 4). The contact time has a diminished effect on enhancing effects of LAE in PAA solutions at 46°C compared with that at 22°C (Figures 3A, 4). The strengthening effects of 0.05% LAE on anti-Listeria efficacy of PAA, when applied at 46°C, provided an additional 0.54 log₁₀ CFU/apple reduction compared with that at 22°C (Figures 3A, 4).

FIGURE 4

Figure 4. Efficacy of peracetic acid with lauric arginate against L. monocytogenes on apples at 46°C. Granny Smith apples subjected to 80 ppm peracetic acid (PAA) alone or in the presence of lauric arginate (LAE) for 30-sec or 2-min contact time. The initial L. monocytogenes level on apple surfaces was ∼ 6.5 log₁₀ CFU/apple. Mean ± SEM, averaged from three independent experiments; each independent experiment has 10 apples per treatment. ^a–cMean among bars without common letters differ significantly (P < 0.05).

Verification of Anti-Listeria Efficacies of Peracetic Acid With Lauric Arginate Solutions in a Pilot Spray-Bar Brush Bed System

The antimicrobial efficacies of PAA in combination with LAE against Listeria on fresh apples were further verified in a pilot spray-bar brush bed system. Spray wash of PAA with 0.01 and 0.05% LAE at RT for 30–120 sec reduced Listeria by 1.47–1.64 and 1.68–1.76 log₁₀ CFU/apple, respectively (Table 1). Consistently with the lab-scale testing, the addition of LAE significantly improved the antimicrobial efficacy of PAA solution (P < 0.05) (Table 1). Increasing the temperature of PAA with 0.01 and 0.05% LAE solution from 22 to 46°C further increased (P < 0.05) the efficacies and resulted in 1.83–1.94 and 2.08–2.14 log₁₀ CFU/apple reductions of Listeria for 30–120 sec contact, respectively (Table 1).

TABLE 1

Table 1. Antimicrobial effectiveness of peracetic acid with lauric arginate solution against L. innocua on Granny Smith apples applied in a pilot spray-bar system.

Discussion

Roles of Lauric Arginate in Enhancing Antimicrobial Efficacy of Peracetic Acid Solution Against Listeria monocytogenes on Fresh Apples

Lauric arginate has received increasing attention in the fresh produce industry due to its antimicrobial activity against L. monocytogenes, used alone or in combination with other antimicrobials. However, the reported effectiveness of LAE against Listeria varied among different studies. For example, a 2-min exposure of 0.01% LAE resulted in 3.1 log₁₀ CFU/g log reduction of L. monocytogenes spot-inoculated on lettuce surfaces with an initial bacterial population of 6.6 log₁₀ CFU/g and treated 15-min after inoculation (Nübling et al., 2017). The application of 0.10% LAE for 20 min reduced L. innocua by 2.3 log₁₀ CFU/cm² on lettuce surfaces, where L. innocua was spot inoculated on the lettuce surfaces with an initial bacterial load of 6–8 log₁₀ CFU/ml and exposure to LAE treatment 1 h after inoculation (Huang and Nitin, 2017). The antimicrobial strengthening effects of LAE when used with other antimicrobials have been mostly conducted on dairy and meat products (Stopforth et al., 2010; Soni et al., 2012; Ma et al., 2013). The addition of 0.075% LAE with essential oils (cinnamon, eugenol, and thymol oils) caused an additional 2–4 log₁₀ CFU/ml reduction of L. monocytogenes in 2% reduced-fat milk compared with adding essential oils alone after 4-h treatment (Ma et al., 2013). The addition of 0.07% LAE in a mixture of 1.68% potassium lactate and 0.12% sodium diacetate caused an additional ∼ 1.0 log₁₀ CFU/g reduction of L. monocytogenes on cooked cured ham after 24-h exposure at 4°C compared with no LAE control (Stopforth et al., 2010). Nonetheless, a recent study on fresh apples reported that the antimicrobial efficacy of 80 ppm PAA with 0.10% LAE against L. innocua on apple surfaces was not different from no LAE control (Pietrysiak et al., 2019), which was likely due to large standard deviations among replicates associated with that study. Consistent with studies on dairy and meat products, we herein showed that the addition of LAE significantly enhanced the antimicrobial efficacies of PAA solutions against L. monocytogenes in water or on apple surfaces in a concentration-dependent manner. This strengthening effect was further verified in a pilot apple spray-bar brush bed processing line.

The enhanced efficacy of LAE with PAA treatment could be due to their synergic antimicrobial activities. Both PAA and LAE disrupt the lipoprotein cytoplasmic membranes of bacteria and cause the leakage of intercellular components (Maris, 1995; Rodriguez et al., 2004; Becerril et al., 2013). PAA also oxidizes proteins, enzymes, lipids, and DNA by inducing the release of intracellular reactive oxygen species (Leaper, 1984; Maris, 1995; González-Aguilar et al., 2012). Additionally, LAE has an oxidizing property and has been shown to generate oxidative stress against Escherichia coli (Yang et al., 2019), which might be synergistic with PAA in releasing bactericidal reactive oxygen species to activate membrane lipid peroxidation and amplify oxidative damage of DNA, ultimately causing cell death. LAE also can interact with bacterial DNA and result in DNA secondary structure changes (Ma et al., 2016).

Lauric Arginate Wettability in Enhancing the Antimicrobial Efficacy of Peracetic Acid Against L. monocytogenes on Apple Surfaces

Lauric arginate, Tween 80, and Tween 85 are surface-active agents that lower the interfacial tension between a liquid and solid surface and increase the spreading property of a liquid solution (Yu et al., 2001; Huang and Nitin, 2017). Consistently, we showed that including LAE, Tween 80, or Tween 85 in PAA solution increased the spreading property on apple surfaces. Of note, the wettability of LAE is stronger than those of Tween 80 or Tween 85; the PAA solutions with 0.10% LAE and 0.10% Tween 80 or Tween 85 reduced contact angle from 99.1 to 41.6, 76.6, or 72.6°, respectively. PAA with 0.10% Tween 80 or Tween 85 solution had a comparable wettability as PAA with 0.005% LAE, but the antimicrobial strengthening effects of 0.005% LAE was much higher than those of PAA with 0.10% Tween 80 or Tween 85, indicating the enhanced anti-Listeria efficacies of LAE in PAA solutions should be mainly due to bactericidal reactive oxygen species instead of its wettability (Yang et al., 2019). In support of our findings, LAE is more effective against L. innocua on lettuce surface than that of Tween 20 (Huang and Nitin, 2017). Inclusion of 0.10% of Tween 80 in 2.0% citric acid solution failed to increase its antimicrobial efficacy against Salmonella on alfalfa seeds (Weissinger and Beuchat, 2000). In contrast, 80 ppm PAA solution with 0.10% Tween 20 and 0.10% LAE had a similar contact angle on Gala apple surface, but 80 ppm PAA with 0.10% Tween 20 exhibited a better antimicrobial efficacy against L. innocua on apples than that with 0.10% LAE (Pietrysiak et al., 2019).

Factors Influence the Antimicrobial Efficacy of Peracetic Acid With Lauric Arginate Solution Against L. monocytogenes on Apples

The extended contact time from 30 sec to 2 min significantly increased the efficacy of PAA with LAE solution at RT, but the magnitude of increment was smaller than that of 80 ppm PAA only (Shen et al., 2019). Increased PAA concentration significantly increased the antimicrobial activities of PAA with LAE solutions against L. monocytogenes on fresh apples, and the effects of PAA concentration are more pronounced in PAA with LAE solutions compared with PAA alone (Shen et al., 2019). This is possibly due to the increased production of bactericidal reactive oxygen species in PAA with LAE solutions compared with PAA alone.

Our previous study showed that the efficacy of PAA against L. monocytogenes on fresh apples was enhanced when it was applied at 46°C, causing an additional 1.02 and 0.89 log₁₀ CFU/apple reduction at a 30-sec or 2-min treatment, respectively, compared with those at RT (Shen et al., 2019). The addition of 0.05% LAE in 80 ppm PAA solution at RT obtained a comparable log reduction of L. monocytogenes as PAA only solution applied at 46°C (Shen et al., 2019). Similarly, the addition of LAE significantly enhanced PAA anti-Listeria efficacy at 46°C, but the strengthening effects were smaller than that applied at RT. The mild heat (Ebrahimi et al., 2018) and LAE (Becerril et al., 2013; Pattanayaiying et al., 2014) caused cellular membrane damages. Thus, the effects of mild heat on the bacterial cell membrane might diminish the strengthening effects of LAE. Spray wash of PAA with LAE in a pilot facility at either 22°C or 46°C was less effective than those applied via lab-scale immersion intervention, which is consistent with our previous finding on PAA intervention alone (Zhu et al., 2020). Similar to our findings, LAE combined with eugenol was less effective in reducing Salmonella on spinach when conducted by spray wash compared with immersion intervention (Ruengvisesh et al., 2015).

Conclusion

Low concentrations of LAE significantly enhanced the effectiveness of PAA against L. monocytogenes on fresh apples. The antimicrobial efficacies of PAA with LAE treatments increased with the increased concentration of PAA or LAE. The most efficacious treatment was a combination of 80 ppm PAA with 0.05% LAE conducted at 46°C. In this scenario, a 2.90 log₁₀ CFU/apple reductions of L. monocytogenes on fresh apples was achieved. Data showed that LAE plus PAA is a practical and viable PAA spray-bar brush bed intervention strategy with enhanced anti-Listeria efficacy for the apple industry to prevent cross contamination of L. monocytogenes of fresh apples, facilitating their compliance with FSMA Preventive Controls requirements.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Author Contributions

XS conducted the experiment, analyzed the data, and wrote the manuscript. JC and JM helped with sample processing. M-JZ and IH revised the manuscript. M-JZ supervised the work, guided the experimental design, analyzed the data, and was in charge of the funding acquisition. All authors contributed to the article and approved the submitted version.

Funding

The research activity was partially supported by the Washington State Department of Agriculture (WSDA), the Center for Produce Safety 2017CPS10, and the Washington Tree Fruit Research Commission.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to acknowledge Guardian Manufacturing and Pace International for donation and setting up the pilot-scale spray-bar brush bed system, A&B Ingredients, Inc. for providing CytoGuard LA 20, and Pace International Inc. for providing Bioside HS. We acknowledge To Chiu and Tonia Green for their assistance.

References

Angelo, K. M., Conrad, A. R., Saupe, A., Dragoo, H., West, N., Sorenson, A., et al. (2017). Multistate outbreak of Listeria monocytogenes infections linked to whole apples used in commercially produced, prepackaged caramel apples: United States, 2014-2015. Epidemiol. Infect. 145, 848–856. doi: 10.1017/s0950268816003083

PubMed Abstract | CrossRef Full Text | Google Scholar

Baert, L., Vandekinderen, I., Devlieghere, F., Van Coillie, E., Debevere, J., and Uyttendaele, M. (2009). Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine Norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water. J. Food Prot. 72, 1047–1054. doi: 10.4315/0362-028x-72.5.1047

PubMed Abstract | CrossRef Full Text | Google Scholar

Becerril, R., Manso, S., Nerin, C., and Gomez-Lus, R. (2013). Antimicrobial activity of lauroyl arginate ethyl (LAE), against selected food-borne bacteria. Food Control 32, 404–408. doi: 10.1016/j.foodcont.2013.01.003

CrossRef Full Text | Google Scholar

Dong, X. Q., Rao, J. P., Huber, D. J., Chang, X. X., and Xin, F. C. (2012). Wax composition of ‘Red Fuji’ apple fruit during development and during storage after 1-methylcyclopropene treatment. Hort. Environ. Biotechnol. 53, 288–297. doi: 10.1007/s13580-012-0036-0

CrossRef Full Text | Google Scholar

Du, J. H., Han, Y., and Linton, R. H. (2002). Inactivation by chlorine dioxide gas (ClO2) of Listeria monocytogenes spotted onto different apple surfaces. Food Microbiol. 19, 481–490. doi: 10.1006/fmic.2002.0501

CrossRef Full Text | Google Scholar

Ebrahimi, A., Csonka, L. N., and Alam, M. A. (2018). Analyzing thermal stability of cell membrane of Salmonella using time-multiplexed impedance sensing. Biophys. J. 114, 609–618. doi: 10.1016/j.bpj.2017.10.032

PubMed Abstract | CrossRef Full Text | Google Scholar

FDA (2005). Agency Response Letter GRAS Notice No. GRN 000164. Available online at: https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=164. (accessed May 15, 2021)

Google Scholar

FDA (2015). Northstar Produce Inc. recalls Granny Smith size 175 apples because of possible health risk. Available online at: https://fdarecall.wordpress.com/2015/10/30/northstar-produce-inc-recalls-granny-smith-size-175-apples-because-of-possible-health-risk/. (accessed May 15, 2021).

Google Scholar

FDA (2017a). 21CFR173.315; Chemicals Used in Washing or to Assist in the Peeling of Fruits and Vegetables. Available online at: https://www.accessdata.fda.gov/SCRIPTs/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=173.315&SearchTerm=chemicals. (accessed May 15, 2021)

Google Scholar

FDA (2017b). Jack Brown Produce, Inc. recalls Gala, Fuji, Honeycrisp and Golden Delicious Apples due to Possible Health Risk. Available online at: https://www.fda.gov/Safety/Recalls/ucm589722.html. (accessed May 15, 2021)

Google Scholar

González-Aguilar, G., Ayala-Zavala, J. F., Chaidez-Quiroz, C., and Heredia, J. B. (2012). “Peroxyacetic acid,” in Decontamination of Fresh and Minimally Processed Produce, ed. V. M. Gómez-López (Hoboken, NJ: John Wiley & Sons), 215.

Google Scholar

Huang, K., and Nitin, N. (2017). Enhanced removal of Escherichia coli O157:H7 and Listeria innocua from fresh lettuce leaves using surfactants during simulated washing. Food Control 79, 207–217. doi: 10.1016/j.foodcont.2017.03.032

CrossRef Full Text | Google Scholar

Infante, R., Dominguez, J. G., Erra, P., Julia, R., and Prats, M. (1984). Surface active molecules: preparation and properties of long chain n-acyl-l-alpha-amino-omega-guanidine alkyl acid derivatives. Int. J. Cosmet. Sci. 6, 275–282. doi: 10.1111/j.1467-2494.1984.tb00385.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Kang, D. H., and Fung, D. Y. C. (1999). Thin agar layer method for recovery of heat-injured Listeria monocytogenes. J. Food Prot. 62, 1346–1349. doi: 10.4315/0362-028x-62.11.1346

PubMed Abstract | CrossRef Full Text | Google Scholar

Leaper, S. (1984). Influence of temperature on the synergistic sporicidal effect of peracetic acid plus hydrogen peroxide on Bacillus subtilis SA22 (NCA 72-52). Food Microbiol. 1, 199–203. doi: 10.1016/0740-0020(84)90034-0

CrossRef Full Text | Google Scholar

Ma, Q., Davidson, P. M., and Zhong, Q. (2013). Antimicrobial properties of lauric arginate alone or in combination with essential oils in tryptic soy broth and 2% reduced fat milk. Int. J. Food Microbiol. 166, 77–84. doi: 10.1016/j.ijfoodmicro.2013.06.017

PubMed Abstract | CrossRef Full Text | Google Scholar

Ma, Q. M., Davidson, P. M., Critzer, F., and Zhong, Q. X. (2016). Antimicrobial activities of lauric arginate and cinnamon oil combination against foodborne pathogens: improvement by ethylenediaminetetraacetate and possible mechanisms. LWT Food Sci. Technol. 72, 9–18. doi: 10.1016/j.lwt.2016.04.021

CrossRef Full Text | Google Scholar

Maris, P. (1995). Modes of action of disinfectants. Rev. Sci. Tech. 14, 47–55. doi: 10.20506/rst.14.1.829

PubMed Abstract | CrossRef Full Text | Google Scholar

McCollum, J. T., Cronquist, A. B., Silk, B. J., Jackson, K. A., O’Connor, K. A., Cosgrove, S., et al. (2013). Multistate outbreak of listeriosis associated with cantaloupe. N. Engl. J. Med. 369, 944–953.

Google Scholar

Monarca, S., Richardson, S. D., Feretti, D., Grottolo, M., Thruston, A. D., Zani, C., et al. (2002). Mutagenicity and disinfection by-products in surface drinking water disinfected with peracetic acid. Environ. Toxicol. Chem. 21, 309–318. doi: 10.1002/etc.5620210212

CrossRef Full Text | Google Scholar

Nakama, Y. (2017). “Chapter 15–Surfactants,” in Cosmetic Science and Technology, eds K. Sakamoto, R. Y. Lochhead, H. I. Maibach, and Y. Yamashita (Amsterdam: Elsevier), 231–244.

Google Scholar

Nübling, S., Wohlt, D., Saile, N., Weiss, A., and Schmidt, H. (2017). Antimicrobial effect of lauroyl arginate ethyl on Escherichia coli O157: H7 and Listeria monocytogenes on red oak leaf lettuce. Eur. Food Res. Technol. 243, 879–887. doi: 10.1007/s00217-016-2802-1

CrossRef Full Text | Google Scholar

Park, S. H., Choi, M. R., Park, J. W., Park, K. H., Chung, M. S., Ryu, S., et al. (2011). Use of organic acids to inactivate Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh apples and lettuce. J. Food Sci. 76, M293–M298.

Google Scholar

Pattanayaiying, R., H-Kittikun, A., and Cutter, C. N. (2014). Effect of lauric arginate, nisin Z, and a combination against several food-related bacteria. Int. J. Food Microbiol. 188, 135–146. doi: 10.1016/j.ijfoodmicro.2014.07.013

PubMed Abstract | CrossRef Full Text | Google Scholar

Pietrysiak, E., Kummer, J. M., Hanrahan, I., and Ganjyal, G. M. (2019). Efficacy of surfactant combined with peracetic acid in removing Listeria innocua from fresh apples. J. Food Prot. 82, 1965–1972. doi: 10.4315/0362-028x.jfp-19-064

PubMed Abstract | CrossRef Full Text | Google Scholar

Rodgers, S. L., Cash, J. N., Siddiq, M., and Ryser, E. T. (2004). A comparison of different chemical sanitizers for inactivating Escherichia coli O157:H7 and Listeria monocytogenes in solution and on apples, lettuce, strawberries, and cantaloupe. J. Food Prot. 67, 721–731. doi: 10.4315/0362-028x-67.4.721

PubMed Abstract | CrossRef Full Text | Google Scholar

Rodriguez, E., Seguer, J., Rocabayera, X., and Manresa, A. (2004). Cellular effects of monohydrochloride of L-arginine, N-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus. J. Appl. Microbiol. 96, 903–912. doi: 10.1111/j.1365-2672.2004.02207.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Ruengvisesh, S., Loquercio, A., Castell-Perez, E., and Taylor, T. M. (2015). Inhibition of bacterial pathogens in medium and on spinach leaf surfaces using plant-derived antimicrobials loaded in surfactant micelles. J. Food Sci. 80, M2522–M2529.

Google Scholar

Sadekuzzaman, M., Yang, S., Kim, H. S., Mizan, M. F. R., and Ha, S. D. (2017). Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157: H7, Listeria monocytogenes, and Salmonella spp. Int. J. Food Sci. Tech. 52, 2058–2067. doi: 10.1111/ijfs.13484

CrossRef Full Text | Google Scholar

Shen, X., Sheng, L., Gao, H., Hanrahan, I., Suslow, T., and Zhu, M.-J. (2019). Enhanced efficacy of peroxyacetic acid against Listeria monocytogenes on fresh apples at elevated temperature. Front. Microbiol. 10:1196. doi: 10.3389/fmicb.2019.01196

PubMed Abstract | CrossRef Full Text | Google Scholar

Shen, X., Su, Y., Hua, Z., Cong, J., Dhowlaghar, N., Sun, Q., et al. (2020). Validation of peroxyacetic acid treatment against L. monocytogenes on fresh apples using E. faecium NRRL B-2354 as a surrogate in commercial spray-bar operations. Food Microbiol. 92:103590. doi: 10.1016/j.fm.2020.103590

PubMed Abstract | CrossRef Full Text | Google Scholar

Sheng, L., Edwards, K., Tsai, H. C., Hanrahan, I., and Zhu, M. J. (2017). Fate of Listeria monocytogenes on fresh apples under different storage temperatures. Front. Microbiol. 8:1396. doi: 10.3389/fmicb.2017.01396

PubMed Abstract | CrossRef Full Text | Google Scholar

Sheng, L., Shen, X., Ulloa, O., Suslow, T. V., Hanrahan, I., and Zhu, M.-J. (2020a). Evaluation of JC9450 and neutral electrolyzed water in controlling Listeria monocytogenes on fresh apples and preventing cross-contamination. Front. Microbiol. 10:3128. doi: 10.3389/fmicb.2019.03128

PubMed Abstract | CrossRef Full Text | Google Scholar

Sheng, L., Shen, X., and Zhu, M.-J. (2020b). Screening of non-pathogenic surrogates of Listeria monocytogenes applicable for chemical antimicrobial interventions of fresh apples. Food Control 110:106977. doi: 10.1016/j.foodcont.2019.106977

CrossRef Full Text | Google Scholar

Silk, B. J., Mahon, B. E., Griffin, P. M., Gould, L. H., Tauxe, R. V., Crim, S. M., et al. (2013). Vital signs: Listeria illnesses, deaths, and outbreaks—United States, 2009–2011. Morb. Mortal. Wkly. Rep. 62:448.

Google Scholar

Soni, K. A., Desai, M., Oladunjoye, A., Skrobot, F., and Nannapaneni, R. (2012). Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials. Int. J. Food Microbiol. 155, 82–88. doi: 10.1016/j.ijfoodmicro.2012.01.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Stopforth, J. D., Visser, D., Zumbrink, R., van Dijk, L., and Bontenbal, E. W. (2010). Control of Listeria monocytogenes on cooked cured ham by formulation with a lactate-diacetate blend and surface treatment with lauric arginate. J. Food Prot. 73, 552–555. doi: 10.4315/0362-028x-73.3.552

PubMed Abstract | CrossRef Full Text | Google Scholar

Veraverbeke, E. A., Lammertyn, J., Saevels, S., and Nicolai, B. M. (2001). Changes in chemical wax composition of three different apple (Malus domestica Borkh.) cultivars during storage. Postharvest Biol. Technol. 23, 197–208. doi: 10.1016/s0925-5214(01)00128-4

CrossRef Full Text | Google Scholar

Weissinger, W. R., and Beuchat, L. R. (2000). Comparison of aqueous chemical treatments to eliminate Salmonella on alfalfa seeds. J. Food Prot. 63, 1475–1482. doi: 10.4315/0362-028x-63.11.1475

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, X., Rai, R., Huu, C. N., and Nitin, N. (2019). Synergistic antimicrobial activity by light or thermal treatment and lauric arginate: membrane damage and oxidative stress. Appl. Environ. Microbiol. 85: e01033-19..

Google Scholar

Yu, K. H., Newman, M. C., Archbold, D. D., and Hamilton-Kemp, T. R. (2001). Survival of Escherichia coli O157 : H7 on strawberry fruit and reduction of the pathogen population by chemical agents. J. Food Prot. 64, 1334–1340. doi: 10.4315/0362-028x-64.9.1334

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhu, M.-J., Shen, X., Sheng, L., Mendoza, M., and Hanrahan, I. (2020). Validation of Enterococcus faecium NRRL B-2354 as a surrogate for Listeria monocytogenes on fresh apples during pilot spray-bar peroxyacetic acid intervention. Food Control 119:107472. doi: 10.1016/j.foodcont.2020.107472

CrossRef Full Text | Google Scholar