Antimicrobial Activity of Plant Essential Oils Against Escherichia coli O157:H7 and Salmonella on Lettuce

Abstract

Foodborne outbreaks associated with the consumption of fresh produce have increased. In an effort to identify natural antimicrobial agents as fresh produce-wash, the effect of essential oils in reducing enteric pathogens on iceberg and romaine lettuce was investigated. Lettuce pieces were inoculated with a five-strain cocktail of Escherichia coli O157:H7 or Salmonella enterica (5 log CFU/g) and then immersed in a treatment solution containing 5 ppm free chlorine, cinnamaldehyde, or Sporan^® (800 and 1000 ppm) alone or in combination with 200 ppm acetic acid (20%) for 1 min. Treated leaves were spin-dried and stored at 4°C. Samples were taken to determine the surviving populations of E. coli O157:H7, Salmonella, total coliforms, mesophilic and psychrotrophic bacteria, and yeasts and molds during the 14-day storage period. The effect of treatments on lettuce color and texture was also determined. Cinnamaldehyde-Tween (800 ppm, 800T) reduced E. coli O157:H7 by 2.89 log CFU/g (p<0.05) on iceberg lettuce at day 0; Sporan^®–acetic acid (1000SV) reduced E. coli O157:H7 and Salmonella on iceberg and romaine lettuce by 2.68 and 1.56 log CFU/g (p<0.05), respectively, at day 0. The effect of essential oils was comparable to that of 5 ppm free chlorine in reducing E. coli O157:H7 and Salmonella populations on iceberg and romaine lettuce throughout the storage time. The natural microbiota on treated lettuce leaves increased during the storage time, but remained similar (p>0.05) to those treated with chlorine and control (water). The texture and the color of iceberg and romaine lettuce treated with essential oils were not different from the control lettuce after 14 days of storage. This study demonstrates the potential of Sporan^® and cinnamaldehyde as effective lettuce washes that do not affect lettuce color and texture.

Introduction

T he Center for Science in the Public Interest identified 365 outbreaks in the United States linked to leafy greens contaminated with Escherichia coli O157:H7, Norovirus, or Salmonella that resulted in 13,568 cases of illness (CSPI, 2009). Beyond their health effects, foodborne illnesses can cause emotional and economic hardship; Salmonella alone causes approximately 1 million foodborne infections that cost $365 million in direct medical expenditures annually (CDC, 2011). Such foodborne outbreaks cause economical hardship to the farmers and the fresh produce industry, and can contribute to the skepticism of the consumers in regards to the safety of food. For example, most consumers stopped eating spinach and buying other bagged produce after 2006 spinach outbreak (Cuite et al., 2007).

For both organic and conventional operations, chlorine based sanitizers are commonly used on produce surfaces and processing equipment (Suslow, 2000). Effectiveness of these sanitizers depends on their chemical and physical state, treatment conditions, resistance of pathogens, and the nature of the produce surface. Further, plant exudates released during slicing and shredding of fresh produce may react with the chlorine and neutralize its antimicrobial activity (FDA, 2009), requiring frequent monitoring and replenishing of chlorine (Sapers, 2009). Previous research has shown less than 2 log CFU reductions in enteric pathogens when chlorine was used as a produce wash (Lang et al., 2004; Singh et al., 2002). Chlorine may also form harmful chlorinated compounds such as chloramines and trihalomethanes in water (Dychdala, 2001; Lopez-Galvez et al., 2010). Due to these limitations, there is a need for alternative sanitizers in reducing or eliminating pathogens and microbial loads from produce.

Moreover, consumer awareness and concern regarding synthetic chemical additives have led researchers and food processors to look for natural food additives with a broad spectrum of antimicrobial activity (Marino et al., 2001). Essential oils have been evaluated in various foods for their antimicrobial and preservative properties (Burt, 2004; Du et al., 2009; Obaidat and Frank, 2009). The antimicrobial effect of basil oil on spoilage bacteria on lettuce was comparable to washing with 125 ppm of chlorine (Wan et al., 1998). Singh et al. (2002) reported the antimicrobial effect of thyme oil in reducing Salmonella on romaine lettuce. Gunduz et al. (2009, 2010) found that iceberg lettuce washes with 75 ppm oregano oil and 1000 ppm myrtle oil were comparable to 50 ppm chlorine in reducing Salmonella Typhimurium on iceberg lettuce. Lemongrass oil (0.5%) reduced Salmonella Newport by 1.5 and 2 log CFU/g on organic romaine and iceberg lettuce, respectively (Moore-Neibel et al., 2012). The present study is the first report on antimicrobial activity of cinnamaldehyde and Sporan^® (a proprietary antimicrobial containing clove, rosemary, and thyme oil) on romaine and iceberg lettuce. The objectives of this study were to compare the antimicrobial effects of cinnamaldehyde or Sporan^® alone and in combination with acetic acid against E. coli O157:H7, Salmonella and the native microbiota. The quality parameters of essential oils treated lettuce leaves were also analyzed.

Materials and Methods

Antimicrobials used and preparation of antimicrobial suspensions

Cinnamaldehyde (Sigma-Aldrich, St. Louis, MO) and Sporan^® obtained from EcoSMART Technologies (Alpharetta, GA) were used as antimicrobials in this study. Antimicrobial suspensions were prepared as follows: 800 and 1000 ppm cinnamaldehyde (800C, pH 4.17; 1000C, 4.16) and Sporan^® (800S, pH 4.70; 1000S, pH 4.62) in sterile distilled water (w/v), 800 ppm cinnamaldehyde in 0.5% Tween 20 (800T, pH 3.83), and combined with acetic acid (20%) as 800 ppm cinnamaldehyde + 200 ppm acetic acid (1000CV, pH 4.00) and 800 ppm Sporan^® + 200 ppm acetic acid (1000SV, pH 4.21). A 5 ppm free chlorine solution (pH 6.5) was freshly made by dissolving sodium hypochlorite in deionized water. Chlorine was chosen at 5 ppm as recommended by the Safe Drinking Water Act (4 ppm) (USDA, 2011).

Bacterial culture preparation

Five nalidixic acid–resistant E. coli O157:H7 strains RM 4406, RM 4688, RM 1918, RM 4407, and RM 5279, and five Salmonella enterica serovars described previously (Yossa et al., 2012) were used in the study. Actively growing cells of Salmonella and E. coli O157:H7 strains in tryptic soy broth (TSB; Acumedia, Lansing, MI) and TSB supplemented with 50 ppm nalidixic acid (TSBN), for Salmonella and E. coli O157:H7 strains, respectively, were harvested by centrifugation and adjusted to OD₆₀₀ of 1 (approximately 8 log CFU/mL). Equal volumes of individual strains were mixed to prepare E. coli O157:H7 or Salmonella cocktails for inoculation studies. Three independent trials were carried out either with E. coli O157:H7 or Salmonella cocktail before each inoculation.

Lettuce inoculation and treatment

The outer leaves of iceberg and romaine lettuce obtained from retail grocer were removed and discarded. Cut lettuce pieces (3×2 cm) were each inoculated with E. coli O157:H7 or Salmonella cocktail (7 log CFU/mL, five spots of 10 μL) on the adaxial surface of the pieces and then air dried for 30 min. Approximately 20 g of air-dried lettuce pieces were placed in a beaker containing 100 mL of treatment solution and washed for 1 min with manual agitation. Treated lettuce pieces were processed in a salad spinner for 1 min and then placed in sterile whirl-pak filter bags (Nasco, Fort Atkinson, WI); the bags were sealed and stored at 4°C for 14 days. Samples washed with sterile water served as control.

Enumeration of E. coli O157:H7, Salmonella, and native microbiota

Lettuce leaves (5 g) were analyzed after inoculation and during incubation period at 0 (after wash treatment), 2, 7, and 14 days for surviving populations of E. coli O157:H7 and Salmonella by spiral plating on Sorbitol MacConkey agar (Acumedia, MI) supplemented with 0.05 mg/L of cefixime, 2.5 mg/L of potassium tellurite and 50 ppm nalidixic acid (Sigma-Aldrich, MO), and xylose lysine tergitol agar (Acumedia, MI) for E. coli O157:H7 and Salmonella, respectively (Yossa et al., 2012).

Uninoculated lettuce pieces (40 g) were washed in 400 mL of the different treatment solutions as described above and stored at 4°C for 14 days. At days 0, 2, 7, and 14, serially diluted suspensions of 10 g of lettuce pieces were spiral plated onto TSA (Acumedia) to enumerate mesophilic (incubation at 37°C/24 h) and psychrotrophic (incubation at 4°C/5–8 days) bacteria; MacConkey Agar (Acumedia; incubation at 35°C/24 h) for total coliforms, and Dichloran Rose Bengal chloramphenicol agar (Acumedia; incubation at 23°C/2–5 days) for yeasts and molds. Colonies were counted using Protocol colony counter (Microbiology International Inc., Frederick, MD).

Color and texture measurement of treated lettuce

Color values (L, a, b) of lettuce leaves treated with selected essential oils were determined using a CR-400 chroma meter (Minolta, Inc., Tokyo, Japan) as described by Park et al. (2001). Texture was measured as the force required to shear 5 g lettuce leaves using the TA-XT2i texture analyzer (Texture Technology Corp., Scarsdale, NY) equipped with Kramer shear cell. Lettuce leaves were placed into the press holder perpendicular to the five shear blades. The force (Newton [N]) of 10 samples was recorded using Texture Expert software (version 1.22; Texture Technology Corp.).

Statistical analysis

The experiment was repeated three times for each treatment and storage period. Reduction in E. coli O157:H7, Salmonella, and background microflora (log CFU/g) from initial populations were compared among treatment-time combinations by a three-way ANOVA using “proc-mixed” procedure (SAS 9.2, Cary, NC). Color and texture data were analyzed similarly by the proc mixed procedure. The level of statistical significance was set at p<0.05 in all cases.

Results

Antimicrobial effects of cinnamaldehyde and Sporan^® against E. coli O157:H7 or Salmonella on iceberg lettuce

The initial E. coli O157:H7 populations of inoculated iceberg leaves was 4.39 log CFU/g (data not shown). At day 0, a treatment with 800 ppm cinnamaldehyde+Tween 20 (800T) significantly reduced E. coli O157:H7 by 2.89 log CFU/g compared to that of 5 ppm chlorine (1.49 log CFU/g reduction) and water (0.76 log CFU/g reduction) (Fig. 1). A treatment with 1000SV and 1000S significantly reduced E. coli O157:H7 (2.88 and 2.65 log CFU/g, respectively) in lettuce compared to chlorine (1.20 log CFU/g) and control (0.71 log CFU/g) after 7 days of storage. E. coli O157:H7 were undetectable (<1 log CFU/g) in most treatments after 14 days of storage; more than 2.5 log reductions were reported with all treatments except 1000CV and control.

FIG. 1.

Reductions (log CFU/g) of mixed strains of Escherichia coli O157:H7 (top) and Salmonella (bottom) after treatment of fresh-cut iceberg lettuce stored for 14 days at 4°C. Values (log CFU/g) are the mean of three replicates and vertical bars represent the standard errors. 800C–800 ppm cinnamaldehyde, 800T–800 ppm cinnamaldehyde in 0.5% Tween 20, 800S–800 ppm Sporan^®, 1000C–1000 ppm cinnamaldehyde, 1000CV–800 ppm cinnamaldehyde+200 ppm acetic acid, 1000S–1000 ppm Sporan^®, 1000SV–800 ppm Sporan^®+200 ppm acetic acid.

Initial Salmonella populations on inoculated iceberg lettuce were 4.62 log CFU/g (data not shown). More than 2.5 log reductions in Salmonella were observed with most treatments on day 0; reductions were significant in lettuce treated with 1000S (2.67 log CFU/g) compared to that of 800T (1.73 log CFU/g) and control (1.06 log CFU/g). Likewise, significant reductions in Salmonella were reported with 1000SV treatment compared to 800S, 1000C, and control on day 2. Salmonella were further reduced during storage in all treated samples. Significant reductions were observed on day 14 in lettuce treated with chlorine and 800T (2.87- and 2.65 log reductions, respectively) compared to other treatments and control.

Antimicrobial effects of cinnamaldehyde and Sporan^® against E. coli O157:H7 or Salmonella on romaine lettuce

The initial E. coli O157:H7 populations on unwashed romaine lettuce were 5.22 log CFU/g (data not shown). A treatment with Sporan^® plus acetic acid (1000SV) significantly reduced E. coli O157:H7 (1.56 log CFU/g) on romaine lettuce compared to Sporan^® alone (800S, 1.14 log CFU/g), 5 ppm free chlorine (1.07 log CFU/g), or water (0.87 log CFU/g) (Fig. 2). E. coli O157:H7 were further reduced at day 2 in most treated samples. The treatments with 1000S, 800T, 1000CV, and 1000SV significantly reduced E. coli O157:H7 on romaine lettuce compared to that with water or Sporan^® (800S) after 7 days. An increase in Sporan^® concentration from 800 to 1000 ppm resulted in an additional approximately 2 log reduction of E. coli O157:H7 on 14 days. Similarly, addition of acetic acid enhanced the antimicrobial effect of Sporan^® against E. coli O157:H7 as evident from significant reductions in 1000SV-treated iceberg lettuce during storage compared to that of Sporan^® alone.

FIG. 2.

Reductions (log CFU/g) of mixed strains of Escherichia coli O157:H7 (top) and Salmonella (bottom) after treatment of fresh-cut romaine lettuce stored for 14 days at 4°C. Values (log CFU/g) are the mean of three replicates and vertical bars represent the standard errors. 800C–800 ppm cinnamaldehyde, 800T–800 ppm cinnamaldehyde in 0.5% Tween 20, 800S–800 ppm Sporan^®, 1000C–1000 ppm cinnamaldehyde, 1000CV–800 ppm cinnamaldehyde+200 ppm acetic acid, 1000S–1000 ppm Sporan^®, 1000SV–800 ppm Sporan^®+200 ppm acetic acid.

Salmonella populations were significantly reduced in romaine lettuce following treatment with 5 ppm chlorine and 1000SV (2.58- and 2.28 logs, respectively). Use of acetic acid with Sporan^® (1000SV) resulted in significantly higher Salmonella reduction (2.28 log CFU/g) than the treatment with Sporan^® alone (800S, 0.96 log CFU/g). Salmonella were reduced during storage in all samples irrespective of treatment. More than 3 log reductions were reported in 5 ppm chlorine-treated romaine lettuce on 2, 7, and 14 days from their respective initial concentrations. Likewise, 2.38–2.99 log reductions in 1000SV-treated romaine lettuce were observed during 2–14 days storage period from their initial concentrations. The antimicrobial effects of chlorine and 1000SV were significantly different from control and all other treatments during 14-day storage period in most cases.

Antimicrobial effects of cinnamaldehyde and Sporan^® against native microbiota on iceberg and romaine lettuce

Populations of mesophilic and psychrotrophic bacteria, total coliforms and yeasts and molds on iceberg lettuce were influenced by the treatment and storage period (Table 1). In general, populations of native microbiota from treated samples were not different from control or untreated lettuce leaves. Mesophilic bacterial populations increased significantly on all iceberg lettuce samples by day 7 except on those samples treated with 1000SV and 5 ppm chlorine. Mesophilic bacterial populations on chlorine-treated iceberg lettuce remained similar (p>0.05) throughout the storage time. Total coliforms, psychrotrophic bacterial populations, and yeasts and molds also increased significantly at day 7 in most treated iceberg lettuce.

Table 1.

Effect of Essential Oils on Native Microbiota Populations of Iceberg Lettuce Stored for 14 Days at 4°C

		Populations (log cfu/g)^a
Treatment	Concentration (ppm)	Day 0	Day 2	Day 7	Day 14
Mesophilic bacteria
Unwash		3.47±0.47ax	4.74±0.29axy	6.51±0.26ay	6.58±0.35ay
Control (water)		3.14±0.58ax	4.46±1.00axy	6.24±0.66ay	6.56±0.65ay
Chlorine	5	3.52±0.81ax	4.07±0.17ax	6.14±0.31ax	6.39±0.83ax
Cinnamaldehyde	800C	4.13±1.23ax	4.70±0.14axy	7.23±0.31ay	7.55±0.45ay
Cinnamaldehyde+Tween	800T	3.14±0.94ax	4.30±0.57axy	7.17±0.61ay	7.13±0.58ay
Sporan^®	800S	3.59±0.34ax	5.06±0.52axy	6.80±0.23ay	6.88±0.46ay
Cinnamaldehyde	1000C	3.43±0.52ax	4.39±0.49axy	7.21±0.34ayz	7.59±0.21az
Cinnamaldehyde+acetic acid	1000CV	2.94±0.14ax	4.30±0.19axy	7.01±0.41ayz	7.29±0.67az
Sporan^®	1000S	3.95±0.79ax	4.54±0.09axy	6.91±0.49ay	7.38±0.31ay
Sporan^®+acetic acid	1000SV	3.43±0.35ax	4.23±0.19axy	6.37±0.53axy	6.93±0.16ay
Total coliform
Unwashed		3.35±0.50ax	4.51±0.52axy	6.00±0.06axy	6.80±0.12ay
Control (water)		2.96±0.83ax	4.40±0.75axy	6.24±0.94ay	6.60±0.60ay
Chlorine	5	3.11±0.40ax	4.01±0.16axy	6.59±0.59ay	6.59±0.89ay
Cinnamaldehyde	800C	4.12±1.12ax	4.56±0.21axy	7.08±0.37ayz	7.53±0.55az
Cinnamaldehyde+Tween	800T	3.04±0.86ax	4.06±0.63axy	7.22±0.60ayz	6.95±0.39az
Sporan^®	800S	3.50±0.49ax	5.01±0.58axy	6.75±0.13ay	7.26±0.18ay
Cinnamaldehyde	1000C	3.40±0.58ax	4.32±0.52axy	6.54±0.52ayz	7.75±0.35az
Cinnamaldehyde+acetic acid	1000CV	2.98±0.19ax	4.10±0.13ax	7.12±0.30ay	7.13±0.29ay
Sporan^®	1000S	3.85±0.78ax	4.35±0.24ax	6.68±0.48axy	7.40±0.29ay
Sporan^®+acetic acid	1000SV	3.18±0.50ax	4.21±0.21axy	6.49±1.05ay	6.84±0.17ay
Psychrotrophic bacteria
Unwashed		3.33±0.51ax	5.08±0.41axy	6.60±0.13ay	6.75±0.18ay
Control (water)		3.33±0.37ax	4.55±0.82axy	6.30±0.63ay	6.77±0.51ay
Chlorine	5	3.55±0.79ax	4.02±0.19axy	6.25±0.31axy	6.63±0.91ay
Cinnamaldehyde	800C	4.13±1.12ax	4.68±0.13axy	7.37±0.15ayz	7.85±0.03az
Cinnamaldehyde+Tween	800T	3.23±0.76ax	4.33±0.44axy	7.13±0.62ayz	7.39±0.75az
Sporan^®	800S	3.56±0.59ax	5.00±0.58axy	6.90±0.08ay	7.31±0.22ay
Cinnamaldehyde	1000C	3.50±0.54ax	4.53±0.49axy	7.28±0.26ayz	7.69±0.41az
Cinnamaldehyde+acetic acid	1000CV	2.93±0.30ax	4.06±0.46axy	7.01±0.41ayz	7.43±0.63az
Sporan^®	1000S	4.08±0.50ax	4.58±0.28axy	6.82±0.76axy	7.52±0.32ay
Sporan^®+acetic acid	1000SV	3.27±0.53ax	3.89±0.31axy	6.30±1.04ayz	7.03±0.18az
Yeasts and molds
Unwashed		1.83±1.61ax	1.90±1.65abx	4.56±0.91abxy	5.36±1.03ay
Control (water)		2.29±0.59ax	2.93±0.40abxy	3.38±2.97abxy	5.45±0.59ay
Chlorine	5	2.20±0.46ax	1.90±1.65abx	2.67±2.61bxy	5.34±0.86ay
Cinnamaldehyde	800C	2.78±0.50ax	4.13±1.55axy	4.18±3.62abxy	6.02±0.82ay
Cinnamaldehyde+Tween	800T	2.75±0.23ax	1.80±1.56abxy	5.11±1.38abyz	6.52±0.64az
Sporan^®	800S	2.70±0.45ax	2.59±2.24abx	5.01±0.52abxy	6.16±0.13ay
Cinnamaldehyde	1000C	3.14±1.09ax	3.57±0.44abxy	5.63±0.66axy	6.34±0.28ay
Cinnamaldehyde+acetic acid	1000CV	2.09±0.35ax	3.18±0.48abxy	5.36±1.25abyz	6.46±0.31az
Sporan^®	1000S	3.08±0.55ax	2.10±1.87abxy	5.39±1.23abyz	6.24±0.22az
Sporan^®+acetic acid	1000SV	2.47±0.12ax	1.00±1.73bxy	5.20±0.89abyz	6.11±0.29az

Values are mean±SD. Each experiment was replicated three times.

Values in the same row not followed by the same letters (xyz) are significantly different; values in the same column not followed by the same letters (ab) are significantly different (p<0.05).

800C–800 ppm cinnamaldehyde, 800T–800 ppm cinnamaldehyde in 0.5% Tween 20, 800S–800 ppm Sporan^®, 1000C–1000 ppm cinnamaldehyde, 1000CV–800 ppm cinnamaldehyde+200 ppm acetic acid, 1000S–1000 ppm Sporan^®, 1000SV–800 ppm Sporan^®+200 ppm acetic acid.

Mesophilic, total coliforms, psychrotrophic, and yeasts and molds populations on treated romaine lettuce were not significantly different from untreated romaine lettuce on day 0 (Table 2). However, populations of this native microbiota increased with storage. Mesophilic bacteria on untreated romaine lettuce, control, and lettuce treated with 5 ppm chlorine, 800S, and 1000SV remained similar (p>0.05) throughout the storage period but increased significantly in 1000S-treated samples at day 14. Similarly, total coliforms recovered from untreated romaine lettuce, control, and lettuce treated with chlorine were not different (p<0.05) throughout the storage time.

Table 2.

Effect of Essential Oils on Native Microbiota Populations of Romaine Lettuce Stored for 14 Days at 4°C

		Populations (log cfu/g)^a
Treatment	Concentration (ppm)	Day 0	Day 2	Day 7	Day 14
Mesophilic bacteria
Unwash		3.83±0.70ax	4.82±0.60ax	5.64±0.31ax	6.12±0.77ax
Control (water)	0	3.74±1.26ax	4.89±0.86ax	6.87±1.75ax	6.39±0.69ax
Chlorine	5	3.88±0.90ax	4.78±1.45ax	5.90±0.69ax	5.62±1.57ax
Cinnamaldehyde	800C	3.75±0.32ax	4.36±0.49axy	7.35±0.26ay	7.72±0.31ay
Cinnamaldehyde+Tween	800T	3.69±0.68ax	4.02±0.61axy	7.24±0.24ay	7.33±0.33ay
Sporan^®	800S	3.24±0.76ax	4.92±0.15ax	5.87±0.39ax	6.54±0.96ax
Cinnamaldehyde	1000C	3.30±1.51ax	4.14±0.65axy	7.14±0.27ay	7.33±1.05ay
Cinnamaldehyde+acetic acid	1000CV	3.66±1.10ax	4.25±0.61axy	7.35±0.49ayz	7.76±0.29az
Sporan^®	1000S	2.79±0.94ax	4.08±1.20axy	6.14±1.44axy	6.46±1.20ay
Sporan^®+acetic acid	1000SV	2.94±0.72ax	4.33±0.61ax	5.69±0.64ax	6.14±0.96ax
Total coliform
Unwashed		4.01±0.36ax	5.08±0.43ax	5.90±0.71ax	6.45±0.88ax
Control (water)	0	4.37±0.43ax	4.72±1.11ax	6.76±0.75ax	6.59±1.25ax
Chlorine	5	3.95±0.95ax	5.13±0.82ax	6.29±0.74ax	6.60±1.18ax
Cinnamaldehyde	800C	3.99±0.10ax	4.42±0.62axy	6.00±0.67ayz	7.95±0.35az
Cinnamaldehyde+Tween	800T	3.85±0.72ax	4.24±0.67ax	7.05±1.40axy	7.82±0.72ay
Sporan^®	800S	3.21±0.80ax	5.10±0.68axy	6.75±0.13axy	7.13±1.31ay
Cinnamaladehyde	1000C	3.93±0.92ax	4.39±0.55axy	7.56±0.74ay	7.76±1.14ay
Cinnamaldehyde+acetic acid	1000CV	3.79±0.84ax	4.33±0.50axy	7.67±0.53ayz	8.27±0.65az
Sporan^®	1000S	2.58±1.05ax	4.35±0.81axy	7.24±0.52ay	6.78±1.42ay
Sporan^®+acetic acid	1000SV	2.79±0.96ax	4.70±0.54axy	5.99±0.97axy	6.35±0.92ay
Psychrotrophic bacteria
Unwashed		4.01±0.56ax	5.25±0.45ax	6.06±0.84ax	6.58±0.61ax
Control (water)	0	4.39±0.30ax	5.22±0.51ax	6.91±0.94ax	7.18±0.79ax
Chlorine	5	3.64±0.81ax	5.12±0.54ax	6.46±1.32ax	7.09±1.58ax
Cinnamaldehyde	800C	3.71±0.39ax	4.77±0.91axy	7.69±0.55ayz	8.30±0.56az
Cinnamaldehyde+Tween	800T	3.35±0.30ax	4.32±0.40axy	7.65±0.70ayz	8.06±0.64az
Sporan^®	800S	2.20±1.95ax	5.00±0.83axy	6.75±0.92ay	7.31±0.22ay
Cinnamaldehyde	1000C	3.85±0.99ax	4.64±0.47axy	7.84±0.61ayz	8.22±1.20az
Cinnamaldehyde+acetic acid	1000CV	3.42±0.47ax	4.42±0.52axy	7.92±0.47ayz	8.43±0.70az
Sporan^®	1000S	2.27±2.03ax	4.28±1.54axy	7.02±1.09ay	7.42±1.46ay
Sporan^®+acetic acid	1000SV	2.50±2.21ax	4.75±0.85axy	6.14±1.02ay	7.12±1.44ay
Yeasts and molds
Unwashed		3.49±0.26ax	3.83±0.07ax	4.75±0.98ax	5.01±0.28ax
Control	0ppm	3.40±0.70ax	3.57±0.54ax	5.58±0.77ax	5.46±0.47ax
Chlorine	5ppm	2.76±1.14ax	3.64±0.40ax	5.66±1.23ax	5.42±0.78ax
Cinnamaldehyde	800C	3.31±0.40ax	3.06±0.39ax	6.20±0.96ax	6.27±0.36ax
Cinnamaldehyde+Tween	800T	3.34±0.16ax	3.29±0.10ax	6.22±0.53ax	6.34±0.50ax
Sporan^®	800S	2.03±1.78ax	3.43±0.14axy	5.58±1.10ay	5.89±0.81ay
Cinnamaldehyde	1000C	3.09±1.02axy	2.28±1.99ax	6.42±0.91ay	6.46±0.68ay
Cinnamaldehyde+acetic acid	1000CV	2.51±2.18ax	3.19±0.44axy	6.58±0.94ay	6.40±0.32ay
Sporan^®	1000S	1.87±1.63ax	2.27±2.00axy	5.54±1.16ay	5.67±1.42ay
Sporan^®+acetic acid	1000SV	2.91±0.86ax	2.04±1.80ax	5.47±1.34ax	5.10±0.96ax

Values are mean±SD. Each experiment was replicated three times.

Values in the same row not followed by the same letters (xyz) are significantly different; values in the same column followed by the same letter (a) are not significantly different (p<0.05).

Effect of cinnamaldehyde and Sporan^® on texture and color of iceberg and romaine lettuce

Texture of treated iceberg lettuce was not different (p>0.05) from control with the exception of 800C treatment (Table 3). After 14 days, the force measurement of 800 ppm cinnamaldehyde-treated iceberg lettuce were higher (p≤0.05) than those recorded at day 0. Likewise, initial force values of iceberg lettuce treated with 1000 ppm cinnamaldehyde were significantly higher than those stored at 2 days (Table 4). The texture measurements of treated romaine lettuce were not significantly different from that of control. In addition, no differences were observed in the force of treated samples and the control throughout the 14-day storage time at 4°C.

Table 3.

Maximal Force (N) Required for Breakage for Iceberg Lettuce Treated with Essential Oils and Stored for 14 Days at 4°C

		Maximum force (N)^a
Treatment	Concentration	Day 0	Day 2	Day 7	Day 14
Control	0	193±13^abx	184±12^abcx	192±16^abx	190±13^ax
Cinnamaldehyde	800C	164±14^cy	169±18^cxy	176±05^bxy	182±10^ax
Sporan^®	800S	187±08^abx	194±19^ax	188±19^abx	197±13^ax
Cinnamaldehyde	1000C	195±11^abx	173±15^bcy	185±17^abxy	180±14^axy
Sporan^®	1000S	180±19^bcx	180±16^abcx	186±09^abx	182±11^ax
Sporan^®+acetic acid	1000SV	200±24^ax	187±22^abx	199±12^ax	197±12^ax

Values are mean±SD. Each experiment was replicated 10 times.

Values in the same row for the same parameter not followed by the same letters (xy) are significantly different; values in the same column not followed by the same letters (ab) are significantly different (p<0.05).

Table 4.

Maximal Force (N) Required for Breakage for Romaine Lettuce Treated with Essential Oils and Stored for 14 Days at 4°C

		Maximum force (N)^a
Treatment	Concentration	Day 0	Day 2	Day 7	Day 14
Control	0ppm	109±12^abx	114±11^ax	104±11^ax	113±09^abx
Cinnamaldehyde	800C	114±13^ax	107±10^ax	111±13^ax	120±09^ax
Sporan^®	800S	95±08^bx	105±13^ax	95±08^ax	109±12^abx
Cinnamaldehyde	1000C	101±11^abx	104±12^ax	104±15^ax	100±08^bx
Sporan^®	1000S	99±16^abx	110±08^ax	101±06^ax	113±07^abx
Sporan^®+acetic acid	1000SV	95±14^abx	107±11^ax	102±08^ax	107±10^abx

Values are mean±SD. Each experiment was replicated 10 times.

Values in the same row for the same parameter followed by the same letter x are not significantly different; values in the same column not followed by the same letters (ab) are significantly different (p<0.05).

Color measurements showed no significant differences (p>0.05) in color coordinate values a (greenness) and b (yellowness) between control and treated iceberg lettuce (Table 5). However, significant differences (p≤0.05) were observed in the lightness (L) values. At day 0, the lightness values of the iceberg samples treated with cinnamaldehyde or Sporan^® at 800 and 1000 ppm concentrations were different from control iceberg. During 14 days of storage at 4°C, the lightness values of iceberg samples treated with 800S and 1000SV were similar (p>0.05) to the control. Color coordinate values (L, a, b) of control and treated romaine lettuce were not significantly different throughout the 14 days of storage (Table 6).

Table 5.

Color Measurements (L, a, b Values) of Iceberg Lettuce Treated with Essential Oils and Stored for 14 Days at 4°C

Color values^a
	Day 0			Day 2			Day 7			Day 14
Treatment	L^*	a^*	b^*	L^*	a^*	b^*	L^*	a^*	b^*	L^*	a^*	b^*
0ppm	51±2^cxy	−19±1^ax	33±2^ax	54±3^cy	−18±2^ax	34±3^abx	54±5^cxy	−20±1^ax	33±6^abx	56±2^cdx	−20±1^abx	36±2^abx
800C	62±2^axy	−20±2^ax	36±3^axy	61±3^ay	−17±1^ax	35±2^ay	61±4^axy	−20±1^ax	37±3^axy	65±3^ax	−20±1^abx	39±4^ax
800S	55±3^bx	−20±2^ax	33±5^ax	52±3^cx	−17±1^ax	30±3^bx	55±4^bcx	−18±2^ax	31±5^bx	53±1^dx	−19±1^ax	32±2^cx
1000C	59±4^abx	−19±2^ax	33±4^ay	58±2^abx	−17±1^ax	33±3^aby	61±3^ax	−19±1^ax	34±3^abxy	62±3^abx	−21±2^abx	37±4^abx
1000S	58±2^abx	−21±1^ax	36±1^ax	59±3^ax	−18±1^ax	35±2^ax	59±2^abx	−20±1^ax	35±3^abx	59±4^bcx	−21±1^bx	37±3^ax
1000SV	54±1^cx	−19±4^ax	32±5^ax	54±4^bcx	−17±2^ax	32±4^abx	55±4^bcx	−19±1^ax	32±3^bx	55±7^cdx	−20±4^abx	34±6^bcx

Values are mean±SD. Each experiment was replicated 10 times.

Table 6.

Color Measurements (L, a, b Values) of Romaine Leaves Treated with Essential Oils and Stored for 14 Days at 4°C

Color values^a
	Day 0			Day 2			Day 7			Day 14
Treatment	L^*	a^*	b^*	L^*	a^*	b^*	L^*	a^*	b^*	L^*	a^*	b^*
0ppm	50±5^ax	−19±2^ax	30±5^ax	48±6^ax	−19±2^ax	29±6^ax	48±8^ax	−19±3^ax	30±7^ax	49±8^ax	−19±2^ax	32±7^ax
800C	49±6^ax	−20±3^ax	31±6^ax	51±6^ax	−20±3^ax	33±6^ay	51±7^ax	−21±4^ax	36±1^axy	50±6^ax	−21±2^ax	36±2^ax
800S	46±2^ax	−18±1^ax	28±3^ax	46±4^ax	−19±2^ax	30±4^ax	45±1^ax	−18±1^ax	28±2^ax	44±3^ax	−19±1^ax	29±2^ax
1000C	47±4^ax	−19±2^ax	31±4^ax	48±4^ax	−20±2^ax	32±5^ay	48±2^ax	−21±1^ax	33±3^axy	49±4^ax	−21±2^ax	35±3^ax
1000S	46±2^ax	−19±3^ax	30±2^ax	45±4^ax	−19±1^ax	30±3^ax	46±3^ax	−18±2^ax	28±3^ax	47±3^ax	−20±1^ax	32±3^ax
1000SV	44±3^ax	−19±2^ax	29±4^ax	45±2^ax	−18±2^ax	28±4^ax	45±2^ax	−19±2^ax	30±3^ax	46±2^ax	−20±1^ax	31±3^ax

Values are mean±SD. Each experiment was replicated 10 times.

Values in the same row for the same parameter not followed by the same letters (xy) are significantly different; values in the same column followed by the same letter (a) are not significantly different (p<0.05).

Discussion

Essential oils have been evaluated to reduce pathogen population on fresh produce. Kim et al. (2011) found significant effect of clove extracts in reducing E. coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium on fresh lettuce. We found that the antimicrobial efficacy of cinnamaldehyde and Sporan^® was dependent on pathogen, type of produce, and storage period. In this study, the washing step was not followed by the rinsing; the presence of residual antimicrobials on the leaves could have contributed to further reduction of these pathogens throughout the storage period. The antimicrobial effect of essential oils against native microflora was not evident in our study. The results are in agreement with Ponce et al. (2011) study who did not observe antimicrobial effect of clove, tea tree and rosemary oils on native microflora of lettuce. Several substances have been used to dissolve the essential oils or to stabilize it in water-based culture media, such as ethanol, Tween 20, acetone, polyethylene glycol, dimethyl sulfoxide (Burt, 2004). A cinnamaldehyde-Tween treatment (800T) significantly reduced E. coli O157:H7 populations on iceberg lettuce at day 2; however, its effect was not evident against E. coli O157:H7 on romaine lettuce. While some researchers have recommended additives to dissolve or stabilize the essential oils in water based culture (Hammer et al., 1999), others have reported reduced activity of oils when emulsifiers and solvents are used (Hili et al., 1997; Mann and Markham 1998; Remmal et al., 1993). Furthermore, a number of potential synergists have been suggested for use with essential oils, such as low pH, low water activity, chelators, low oxygen tension, mild heat and raised temperature (Burt, 2004). In this study, adding acetic acid (200 ppm) to cinnamaldehyde (800 ppm) lowered the pH from 4.17 to 4.00; however, the inhibitory effect of cinnamaldehyde was not significant when acetic acid was added. On the other hand, 200 ppm acetic acid in 800 ppm Sporan^® (1000SV) lowered the pH from 4.7 to 4.21 and contributed to increased antimicrobial activity of Sporan^® in some cases. Juven et al. (1994) stated that the susceptibility of bacteria to essential oils might increase with lower pH values, since the hydrophobicity of the oils increases at low pH, consequently enabling easier dissolution in the lipids of the cell membrane of Salmonella Typhimurium. This result implies that the efficacy of acetic acid added to essential oils may be dependent on the type of oils, strains, and produce surface.

In previous studies, antibacterial activity of Sporan^® against E. coli O157:H7 and Salmonella in organic soil was dose dependent (Yossa et al., 2010, 2011). Other researchers have achieved a higher inactivation of enteric bacteria on fresh lettuce with higher concentration of essential oils (Gunduz et al., 2010; Kim et al., 2011). However, the effect of essential oil concentration was not significant on iceberg and romaine lettuce in this study. These results indicated that the antimicrobial effects of these oils were also dependent on bacterial strain, storage period, and type of produce. The antimicrobial activity of essential oils has been attributed to more than one mechanism (Burt, 2004; Moreira et al., 2005). It has been demonstrated that cinnamaldehyde disrupts cell membrane causing leakage of small ions (Gill and Holley, 2004). The mechanism of action of Sporan^® on bacteria is unknown, but since it is made up of clove, rosemary, and thyme oil, the mode of action could be disintegration of the cellular membrane, followed by leakage of cellular components (Burt 2004; Devi et al., 2010). Throughout the storage period, the texture of cut iceberg and romaine washed with 800C, 800S, 1000C, and 1000SV was not significantly different from control lettuce, with the exception of iceberg samples treated with 800C at day 0. Likewise, color characteristics of treated romaine lettuce were not significantly different from control; however, the lightness of iceberg lettuce was affected by cinnamaldehyde.

Conclusion

Recovery of E. coli O157:H7 populations from lettuce treated with essential oils were significantly lower than in chlorine-treated samples at days 0 and 7 for iceberg and days 0 and 14 for romaine lettuce stored at 4°C. The effect of these essential oils was comparable to chlorine in reducing Salmonella populations on iceberg and romaine lettuce throughout the storage period. In addition, the texture and the color of iceberg and romaine leaves treated with essential oils were not different from control lettuce. The results of this study suggest that Sporan^® plus acetic acid has the potential to be used as a produce wash treatment to control enteric pathogens in fresh produce provided that sensory characteristics of treated lettuce are acceptable. Further studies simulating industrial settings will be helpful.

Footnotes

Acknowledgments

We thank Eunhee Park for texture analysis of samples and Dr. Bryan Vinyard for statistical analysis. The mention of trade names or commercial products does not imply recommendation or endorsement to the exclusion of other products by the U.S. Department of Agriculture.

Disclosure Statement

No competing financial interests exist.

References

Burt

. Essential oils: Their antimicrobial properties and potential applications in foods—A review. Int J Food Microbiol, 2004; 94:223–253.

[CDC] Centers for Disease Control and Prevention. Estimates of foodborne illness in the United States. 2011. www.cdc.gov/foodborneburden/trends-in-foodborne-illness.html. 2011 October 25.

Chang

, Fang

. Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7. Food Microbiol, 2007; 24:745–751.

[CSPI] Center for Science in the Public Interest. Leafy greens, eggs, and tuna: Top list of riskiest FDA-regulated foods. 2009. http://cspinet.org/new/200910061.html. 2011 October 25.

Cuite

, Condry

, Nucci

, Hallman

. Public response to the contaminated spinach recall of 2006. 2007. http://fpi.rutgers.edu/docs/pubs/2007_Public%20Response%20to%20the%20Contaminated%20Spinach%20Recall%20of%202006.pdf. 2011 October 25.

Devi

, Nisha

, Sakthivel

, Pandian

. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol, 2010; 130:107–115.

, Olsen

, Avena-Bustillos

, McHugh

, Levin

, Friedman

. Effects of allspice, cinnamon, and clove bud essential oils in edible apple films on physical properties and antimicrobial activities. J Food Sci, 2009; 74:374–378.

Dychdala

. Chlorine and chlorine compounds. Disinfection, Sterilization, and Preservation. Block

. Philadelphia: Lippincott Williams and Wilkins, 2001; 135–158.

[FDA] U.S. Food and Drug Administration. Analysis and evaluation of preventive control measures for the control and reduction/elimination of microbial hazards on fresh and fresh-cut produce, 2009. www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses/ucm091363.htm. 2011 October 25.

10.

Gill

, Holley

RA.

Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei. Appl Environ Microbiol, 2004; 70:5750–5755.

11.

Gunduz

, Gonul

, Karapinar

. Efficacy of myrtle oil against Salmonella Typhimurium on fresh produce. Int J Food Microbiol, 2009; 130:147–150.

12.

Gunduz

, Gonul

, Karapinar

. Efficacy of oregano oil in the inactivation of Salmonella Typhimurium on lettuce. Food Control, 2010; 21:513–517.

13.

Hammer

, Carson

, Riley

. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol, 1999; 86:985–990.

14.

Hili

, Evans

, Veness

. Antimicrobial action of essential oils: The effect of dimethylsulphoxide on the activity of cinnamon oil. Lett Appl Microbiol, 1997; 24:269–275.

15.

Juven

, Kanner

, Schved

, Weisslowicz

. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol, 1994; 76:626–631.

16.

Kim

, Kang

, Kim

, Ha

, Hwang

, Kim

, Lee

. Antimicrobial activity of plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on fresh lettuce. J Food Sci, 2011; 76:41–46.

17.

Lang

, Harris

, Beuchat

. Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water. J Food Prot, 2004; 67:1092–1103.

18.

López-Gálvez

, Allende

, Truchado

, Martínez-Sánchez

, Tudela

, Selma

, Gil

. Suitability of aqueous chlorine dioxide versus sodium hypochlorite as an effective sanitizer for preserving quality of fresh-cut lettuce while avoiding by-product formation. Postharvest Biol Technol, 2010; 55:53–60.

19.

Mann

, Markham

. A new method for determining the minimum inhibitory concentration of essential oils. J Appl Microbiol, 1998; 84:538–544.

20.

Marino

, Bersani

, Comi

. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. Int J Food Microbiol, 2001; 67:187–195.

21.

Moore-Neibel

, Gerber

, Patel

, Friedman

, Ravishankar

. Antimicrobial activity of lemongrass oil against Salmonella enterica on organic leafy greens. J Appl Microbiol, 2012; 112:485–492.

22.

Moreira

, Ponce

, Del Valle

, Roura

. Inhibitory parameters of essential oils to reduce a foodborne pathogen. Food Sci Technol, 2005; 38:565–570.

23.

Obaidat

, Frank

. Inactivation of Salmonella and Escherichia coli O157:H7 on sliced and whole tomatoes by allyl isothiocyanate, carvacrol, and cinnamaldehyde in vapor phase. J Food Prot, 2009; 72:315–324.

24.

Park

, Hung

, Doyle

, Ezeike

GOI

, Kim

. Pathogen reduction and quality of lettuce treated with electrolyzed oxidizing and acidified chlorinated water. J Food Sci, 2001; 66:1368–1372.

25.

Ponce

, Roura

, Moreira

. Essential oils as biopreservatives: Different methods for the technological application in lettuce leaves. J Food Sci, 2011; 76:M34–M40.

26.

Remmal

, Bouchikhi

, Rhayour

, Ettayebi

. Improved method for the determination of antimicrobial activity of essential oils in agar medium. J Essential Oil Res, 1993; 5:179–184.

27.

Sapers

. Disinfection of contaminated produce with conventional washing. In: The Produce Contamination Problem—Causes and Solutions. Sapers

, Solomon

, Matthews

. New York: Academic Press, 2009; 393–407.

28.

Singh

, Singh

, Bhunia

, Stroshine

. Efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing Escherichia coli O157:H7 on lettuce and baby carrots. LWT Food Sci Technol, 2002; 35:720–729.

29.

Suslow

. Postharvest handling for organic crops. 2000. http://anrcatalog.ucdavis.edu/pdf/7254.pdf. 2011 October 25.

30.

[USDA] U.S. Department of Agriculture. Guidance. The use of chlorine materials in organic production and handling. 2011. www.ams.usda.gov/AMSv1.0/getfile?dDocName=STELPRDC5090760. 2012 July 1.

31.

Wan

, Wilcock

, Coventry

. The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens. J Appl Microbiol, 1998; 84:152–158.

32.

Yossa

, Patel

, Millner

, Lo

. Antimicrobial activity of essential oils against Escherichia coli O157:H7 in organic soil. Food Control, 2010; 21:1458–1465.

33.

Yossa

, Patel

, Millner

, Lo

. Inactivation of Salmonella in organic soil by cinnamaldehyde, ecotrol, eugenol, Sporan^® and vinegar. Foodborne Pathog Dis, 2011; 8:311–317.

34.

Yossa

, Patel

, Millner

, Lo

. Essential oils reduce Escherichia coli O157:H7 and Salmonella on spinach leaves. J Food Prot, 2012; 75:488–496.

Antimicrobial Activity of Plant Essential Oils Against Escherichia coli O157:H7 and Salmonella on Lettuce

Abstract

Introduction

Materials and Methods

Antimicrobials used and preparation of antimicrobial suspensions

Bacterial culture preparation

Lettuce inoculation and treatment

Enumeration of E. coli O157:H7, Salmonella, and native microbiota

Color and texture measurement of treated lettuce

Statistical analysis

Results

Antimicrobial effects of cinnamaldehyde and Sporan® against E. coli O157:H7 or Salmonella on iceberg lettuce

Antimicrobial effects of cinnamaldehyde and Sporan® against E. coli O157:H7 or Salmonella on romaine lettuce

Antimicrobial effects of cinnamaldehyde and Sporan® against native microbiota on iceberg and romaine lettuce

Effect of cinnamaldehyde and Sporan® on texture and color of iceberg and romaine lettuce

Discussion

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Antimicrobial effects of cinnamaldehyde and Sporan^® against E. coli O157:H7 or Salmonella on iceberg lettuce

Antimicrobial effects of cinnamaldehyde and Sporan^® against E. coli O157:H7 or Salmonella on romaine lettuce

Antimicrobial effects of cinnamaldehyde and Sporan^® against native microbiota on iceberg and romaine lettuce

Effect of cinnamaldehyde and Sporan^® on texture and color of iceberg and romaine lettuce