Antimicrobial Efficacy of Cinnamaldehyde Against Escherichia coli O157:H7 and Salmonella enterica in Carrot Juice and Mixed Berry Juice Held at 4°C and 12°C

Abstract

The effectiveness of cinnamaldehyde for inactivating Salmonella enterica and Escherichia coli O157:H7 in carrot juice (CRJ) and mixed berry juice (MBJ) was investigated. Brain heart infusion broth (BHI), CRJ, and MBJ, with concentrations of added cinnamaldehyde ranging from 0.15 to 1.5 μL/mL, 0.25 to 2.0 μL/mL, and 0.25 to 1.5 μL/mL, respectively, were each inoculated with a 5-strain mixture of Salmonella enterica or Escherichia coli O157:H7 to give an initial viable count of 5.07 log₁₀ colony-forming units/mL. Inoculated BHI or juices without cinnamaldehyde served as controls. Growth of the pathogens in BHI (35°C) was monitored by taking absorbance readings (optical density [OD] 600 nm) for 24 h. The inoculated juices were held at 4°C or 12°C for 24 h, and numbers of viable pathogens were determined at 0, 2, 4, 8, and 24 h by plating samples on selective agar followed by incubation (35°C) and counting bacterial colonies at 48 h. The minimum inhibitory concentration of cinnamaldehyde for both pathogens in BHI was 0.25 μL/mL. The pathogens were more sensitive to cinnamaldehyde in MBJ compared with CRJ, irrespective of storage temperature (p < 0.05). At 4°C, cinnamaldehyde (1.5 μL/mL) completely inactivated S. enterica and E. coli in MBJ (negative by enrichment) within 2 h and 8 h, respectively; whereas both pathogens were detected in CRJ (4°C; with 2.0 μL/mL cinnamaldehyde) at 8 and 24 h. At 12°C, S. enterica and E. coli were undetected in MBJ (1.5 μL/mL cinnamaldehyde) within 2 and 4 h, respectively; however, in CRJ (12°C; 2.0 μL/mL cinnamaldehyde), complete inactivation of S. enterica and E. coli occurred within 4 and 24 h, respectively. Cinnamaldehyde is an effective antimicrobial from natural sources that can be used for inactivating bacterial pathogens in fruit and vegetable juices to enhance microbial safety of these nutritious food products.

Introduction

Rising consumer demand for nutritious, low-calorie foods with fresh-like characteristics and heightened public awareness of the health benefits of consuming fresh produce have contributed to increased production and consumption of fruits, vegetables, and fruit juices (Rico et al., 2007; Boeing et al., 2012). Many commercially available fruit and vegetable juices are heat pasteurized, ultra-high-temperature processed, or canned to ensure microbiological safety and extended shelf-life. Although these processes are effective in destroying pathogenic microorganisms, they can negatively alter quality characteristics of juices such as color, flavor, and nutritional value (Mittal and Griffiths, 2005). In response to consumer demand for minimally processed foods with fresh-like attributes and growing public concern over perceived health risks posed by synthetic food preservatives, juice manufacturers continue to seek alternatives to traditional physical or chemical processes to enhance the microbial safety of juices and newly developed juice blends. One potential alternative is the use of plant essential oils (EOs) and their isolated antimicrobial components (Davidson et al., 1983; Davidson, 1997).

Plant EOs are aromatic, volatile extracts from various plant parts that have long been used for food preservation as well as for improving food flavor (Burt, 2004). Although the antimicrobial properties of EOs have been widely investigated, food applications for EOs have been limited by certain factors, including variability in EO composition and antimicrobial activity, high concentrations of EO required for microbial control (Hyldgaard et al., 2012), and the undesirable alterations of food flavor from high levels of added EO (Yamazaki et al., 2004). One approach with the potential to alleviate these challenges and to allow better control over concentrations of natural antimicrobials added to foods involves using the extracted antimicrobial component(s) from EOs.

Cinnamaldehyde constitutes ∼1–8%, 55–75%, and 70–95% of the EO from cinnamon leaf, cinnamon bark, and cassia cinnamon bark, respectively (Ross, 1976). Cinnamaldehyde is generally recognized as safe by the U.S. Food and Drug Administration and is approved for food use (21 CFR 182.60). According to the U.S. Flavoring Extract Manufacturers Association, toxicity studies (sub chronic and chronic) revealed that cinnamaldehyde has a broad margin of safety (Adams et al., 2004). Although cinnamaldehyde has exhibited antimicrobial activity (Bilgrami et al., 1992; Burt, 2004; Holley and Patel, 2005), its effectiveness in enhancing the microbial safety of juices needs to be validated. Apart from inactivation of Escherichia coli O157:H7 by cinnamaldehyde added to apple juice and apple cider (Baskaran et al., 2010), to our knowledge, there are no published reports on the antibacterial efficacy of cinnamaldehyde against enteric pathogens in juices such as carrot juice (CRJ) and mixed berry juice (MBJ). Accordingly, the objective of this study was to investigate the efficacy of low concentrations of cinnamaldehyde for inactivating Salmonella enterica and Escherichia coli O157:H7 in CRJ and MBJ at refrigeration temperature (4°C) and at an abusive temperature (12°C).

Materials and Methods

Bacterial strains and culture conditions

Five serotypes of Salmonella enterica (Enteritidis-ATCC13076, Heidelberg, Typhimurium-ATCC 14802, Gaminara-8324, and Oranienburg-9329), and five strains of Escherichia coli O157:H7 (FRIK125, ATCC 35150, ATCC 43894, ATCC 43895, and 93-062) were obtained from the culture collection of the Microbial Food Safety Laboratory, Iowa State University, Ames, IA. Stock cultures were kept frozen (−70°C) in brain heart infusion (BHI) broth (Difco; Becton Dickinson, Sparks, MD) that was supplemented with 10% (v/v) glycerol. Frozen stock cultures, thawed under cold running water, were activated in tryptic soy broth (TSB; pH 7.2; Difco; Becton Dickinson) at 35°C. At least two consecutive 24-h transfers of each stock culture were performed before using the cells to inoculate BHI or juices for each experiment.

Preparation of inoculum

Equal volumes (6 mL per culture) of each of the working cultures of Salmonella enterica and Escherichia coli O157:H7 were combined in a sterile centrifuge tube. Cells were harvested by centrifugation (10,000 × g, 10 min, 4°C) by using a Sorvall Super T21 centrifuge (American Laboratory Trading, Inc., East Lyme, CT), and they were washed once in 0.85% (w/v) saline. Pelleted cells were suspended in fresh saline to obtain a final viable cell concentration of 9.0 log₁₀ colony-forming unit (CFU)/mL. Colony counts of the washed cell suspensions were evaluated by serially diluting (10-fold) and surface plating samples on tryptic soy agar (Difco; Becton Dickinson) that was supplemented with 0.6% yeast extract (TSAYE) followed by counting bacterial colonies on TSAYE after incubation (35°C) for 24 h.

Preparation of treatment solutions for Bioscreen C assay

Certified foodgrade cinnamaldehyde (Sigma-Aldrich, Milwaukee, WI) was added to BHI broth. Portions of BHI broth with added cinnamaldehyde (0, 0.15, 0.25, 0.5, 1.0, 1.25, and 1.5 μL/mL), and adjusted to pH 7.4 by using 1 M sodium hydroxide or 1 M hydrochloric acid, were filter sterilized by using 0.22 μm pore size filters (Fisher Scientific). The BHI broth samples (2.5 mL) with the added cinnamaldehyde, including control (BHI with no cinnamaldehyde) in test tubes, were each inoculated with 25 μL of diluted Salmonella enterica or Escherichia coli O157:H7 cell suspensions to obtain a final cell concentration of 5.07 log₁₀ CFU/mL.

Minimum inhibitory concentration

Inoculated samples (200 μL each) of BHI broth (pH 7.4 ± 0.2) were added in triplicate to a 100-well microtiter plate for the Bioscreen C Turbidometer (Growth Curves USA, Piscataway, NJ) and incubated at 35°C for 24 h. OD measurements were taken at 600 nm every 30 min, with shaking of samples for 10 s before each measurement. Minimum inhibitory concentration (MIC) was the lowest cinnamaldehyde concentration that inhibited (<0.05 OD unit increase) growth for 24 h in BHI broth (35°C).

Juice preparation and inoculation

Commercially available pasteurized CRJ and MBJ with no added preservatives, and each juice from the same production lot, were purchased from a local grocery store. The juices were transported in a portable cooler to the laboratory and stored at 4 ± 0.2°C until they were used. The juices were tested for Salmonella, Escherichia coli O157:H7, and other microflora, respectively, by surface plating on Xylose lysine tergitol agar (XLT), Sorbitol MacConkey agar (SMA), and TSAYE. Specifically, 1.0-mL aliquots of each juice were added to 1.0-mL portions of sterile buffered peptone water (BPW; Becton Dickinson) at pH 7.2 ± 0.2. The entire 2.0-mL mixture was surface plated on 8 plates (0.25 mL per plate) of SMA or XLT. Bacterial colonies were counted after 48 h of incubation (35°C). In addition, 1.0-mL aliquots of each juice were added to tubes of sterile TSB (10 mL each), followed by incubation (35°C) for 24 h. After incubation, looped samples of TSB were streak-plated on appropriate selective agar, which was then incubated (35°C) and examined for bacterial colonies at 24 and 48 h.

A portion (400 mL) of each juice type was aseptically transferred into a separate sterile screw cap glass bottle and held at 4°C. Filter-sterilized cinnamaldehyde was added to each type of juice to give the following concentrations: 0 (control), 0.25, 0.5, 1.5, and 2.0 μL/mL. Bottles of juice were capped, vigorously shaken, and inoculated with 4.0 mL of a diluted (1:100 in 0.85% saline) cell suspension of Salmonella enterica or Escherichia coli O157:H7 to give a final cell concentration of 5.07 log₁₀ CFU/mL for each pathogen. Each bottle of juice was swirled to mix its contents and stored at 4°C or 12°C.

Measurement of pH and degrees BRIX

Measurements of pH were taken by using an Orion Model 525 pH meter (Orion Research, Inc., Boston, MA) that was fitted with a glass electrode. Before performing the pH measurements, all juice samples were tempered to 23 ± 1°C. A digital Pocket Refractometer PAL (ATAGO, USA, Inc., Bellevue, WA) was used to take degrees BRIX measurements.

Microbiological analysis

Inoculated juices stored at 4°C and 12°C were tested for viable pathogens at 1, 2, 4, 8, and 24 h. Ten-fold serial dilutions of the juice were prepared by using sterile BPW (pH 7.2). Aliquots (1.0 or 0.1 mL) of juice were surface plated (in duplicate) on XLT and SMA. Agar plates were incubated at 35°C, and bacterial colonies were counted at 48 h. All agar media were purchased from Difco (Becton Dickinson).

Presumptive Escherichia coli O157:H7 or Salmonella enterica colonies (∼2 to 3 colonies per agar plate) were confirmed by using biochemical test kits obtained from Fisher Scientific (Remel Products, Lenexa, KS). Confirmation of Escherichia coli O157:H7 was performed by testing for the presence of O157 and H7 antigens by using the Escherichia coli O157:H7 agglutination test (RIM^®, Escherichia coli O157:H7 Latex Test Kit). The OXOID Salmonella Latex Test Kit was used to confirm presumptive Salmonella enterica colonies.

The inoculated juices were enriched by aseptically transferring them to an enrichment broth and incubation (35°C) for 48 h. Looped samples of enrichment broth were streak-plated on SMA and XLT and incubated (35°C) for 24 h. Bacterial colonies were confirmed as Escherichia coli O157:H7 or Salmonella enterica as previously described.

Statistical analysis

Three replications of each experiment were performed. Mean numbers of pathogen survivors were analyzed by using SAS statistical software version 9.3 (SAS Institute, Inc., Cary, NC). Treatment means were evaluated for significant differences (p < 0.05) by using the Waller–Duncan test.

Results

Growth inhibition of pathogens by cinnamaldehyde in BHI broth

Cinnamaldehyde concentrations from 0.25 to 1.5 μL/mL completely inhibited the growth of Salmonella enterica and Escherichia coli O157:H7; absolutely no increase in OD_600nm for any of the two pathogens was observed at concentrations that were higher than 0.15 μL/mL for 24 h. The MIC of cinnamaldehyde for both pathogens in BHI broth (35°C) was 0.25 μL/mL.

Background microflora, juice characteristics, and initial viable count

No Salmonella, generic Escherichia coli, or Escherichia coli O157:H7 was detected in noninoculated CRJ or MBJ juice; however, there was a low level of background microflora in CRJ (1–3 CFU/mL) and MBJ (0–1 CFU/mL). The initial average pH of CRJ and MBJ was 6.25 and 3.59, respectively. The initial °Brix values for the juices were 8.5 (for CRJ) and 12.3 (for MBJ). The addition of cinnamaldehyde did not significantly change the pH or °BRIX in either of the two juices (data not shown). The average initial viable count for Salmonella enterica or Escherichia coli O157:H7 in artificially inoculated control juice and in juice with added cinnamaldehyde was 5.07 (±0.2) log₁₀ CFU/mL.

Viability of pathogens in CRJ at 4°C and 12°C

Viable counts of Salmonella enterica after 24 h were 3.93 and 4.83 log₁₀ CFU/mL in CRJ held at 4°C and 12°C, respectively (Table 1). At 4°C, significant (p < 0.05) reductions of the pathogen first occurred at 8 and 24 h in juice containing 2.0 and 1.5 μL/mL, respectively, representing the two highest concentrations of cinnamaldehyde tested (Table 1A). At 12°C, significant (p < 0.05) reductions occurred after 1 h in juice with cinnamaldehyde at 0.5, 1.5, or 2.0 μL/mL (Table 1B), indicating a greater sensitivity of the pathogen to cinnamaldehyde at the higher temperature (12°C). At 4°C, no complete inactivation (based on enrichment tests) of S. enterica occurred in CRJ with any of the cinnamaldehyde concentrations evaluated (Table 1A). In contrast, at 12°C, cinnamaldehyde (2.0 μL/mL) completely inactivated Salmonella enterica at 4, 8, and 24 h of storage (Table 1B) based on results of selective plating and enrichment.

Table 1.

Antibacterial Efectiveness of Cinnamaldehyde Against Salmonella enterica in Carrot Juice Held at 4°C (A) or 12°C (B) for 24 Hours

Treatment (μL mL⁻¹)	1 h	2 h	4 h	8 h	24 h
A. Viable count (log₁₀ CFU mL⁻¹)^a at 4°C
Control	4.45 ± 0.64^b	4.21 ± 0.66^b	4.09 ± 0.68^b	4.40 ± 0.61^b	3.93 ± 0.71^b
Cinnamaldehyde (0.25)	4.12 ± 0.73^b	4.40 ± 0.51^b	4.06 ± 0.80^b	4.39 ± 0.71^b	3.94 ± 0.56^b
Cinnamaldehyde (0.5)	4.15 ± 0.78^b	4.13 ± 0.75^b	4.21 ± 0.82^b	4.27 ± 0.77^b	3.56 ± 0.39^b
Cinnamaldehyde (1.5)	4.17 ± 0.71^b	4.10 ± 0.80^b	3.83 ± 0.44^b	3.40 ± 0.47^b	1.47 ± 1.31^b
Cinnamaldehyde (2.0)	3.48 ± 0.36^b	3.87 ± 0.39^b	3.26 ± 0.30^b	2.45 ± 0.32^b	0.59 ± 1.03^b
B. Viable count (log₁₀ CFU mL⁻¹)^a at 12°C
Control	4.14 ± 0.34^b	4.24 ± 0.23^b	4.20 ± 0.48^b	4.39 ± 0.58^b	4.83 ± 0.42^b
Cinnamaldehyde (0.25)	3.79 ± 0.29^b	3.98 ± 0.11^b	4.33 ± 0.46^b	4.09 ± 0.42^b	3.06 ± 0.58^b
Cinnamaldehyde (0.5)	3.65 ± 0.20^b	3.81 ± 0.17^b	3.92 ± 0.30^b	3.68 ± 0.21^b	1.48 ± 0.27^b
Cinnamaldehyde (1.5)	3.31 ± 0.19^b	2.83 ± 0.94^b	1.54 ± 0.23^b	0.62 ± 0.56^b	0.16 ± 0.28^b
Cinnamaldehyde (2.0)	3.28 ± 0.25^b	2.72 ± 0.01^b	ND; −ve	ND; −ve	ND; −ve

Initial viable count of Salmonella enterica: 5.04 ± 0.03 log₁₀ CFU mL⁻¹.

Each value for viable count is the mean (standard deviation) of three replicate experiments.

Means with a different letter within a column differ significantly (p < 0.05).

CFU, colony-forming unit; ND, no colonies detected on agar plates with the lowest dilution (1:3) of the sample; −ve, negative enrichment test.

Increased pathogen sensitivity to cinnamaldehyde in CRJ at 12°C compared with at 4°C was also observed for Escherichia coli O157:H7 in CRJ (Table 2A, B). At 24 h, cinnamaldehyde (1.5 μL/mL) completely inactivated (negative enrichment test) Escherichia coli O157:H7 in CRJ at 12°C; whereas at 4°C, the pathogen was detected (positive enrichment) in juice containing that same concentration of cinnamaldehyde (Table 2A, B).

Table 2.

Antibacterial Effectiveness of Cinnamaldehyde Against Escherichia coli O157:H7 in Carrot Juice Held at 4°C (A) or 12°C (B) for 24 Hours

Treatment (μL mL⁻¹)	1 h	2 h	4 h	8 h	24 h
A. Viable count (log₁₀ CFU mL⁻¹)^a at 4°C
Control	4.29 ± 0.20^b	4.34 ± 0.26^b	4.26 ± 0.19^b	4.04 ± 0.03^b	3.75 ± 0.38^b
Cinnamaldehyde (0.25)	4.08 ± 0.35^b	4.31 ± 0.40^b	4.48 ± 0.15^b	3.73 ± 0.25^b	3.71 ± 0.22^b
Cinnamaldehyde (0.5)	4.26 ± 0.20^b	4.25 ± 0.49^b	4.29 ± 0.11^b	3.67 ± 0.17^b	3.20 ± 0.25^b
Cinnamaldehyde (1.5)	4.14 ± 0.08^b	4.23 ± 0.16^b	3.87 ± 0.09^b	3.09 ± 0.37^b	0.52 ± 0.45^b
Cinnamaldehyde (2.0)	4.11 ± 0.16^b	4.22 ± 0.33^b	3.86 ± 0.36^b	2.62 ± 0.44^b	ND; +ve
B. Viable count (Log₁₀ CFU mL⁻¹)^a at 12°C
Control	4.74 ± 0.02^b	4.75 ± 0.08^b	4.51 ± 0.43^b	4.80 ± 0.30^b	5.26 ± 0.43^b
Cinnamaldehyde (0.25)	4.50 ± 0.11^b	4.19 ± 0.30^b	4.04 ± 0.60^b	4.16 ± 0.44^b	3.58 ± 0.29^b
Cinnamaldehyde (0.5)	4.37 ± 0.27^b	3.88 ± 0.19^b	3.48 ± 0.64^b	3.98 ± 0.56^b	2.68 ± 0.18^b
Cinnamaldehyde (1.5)	3.59 ± 0.25^b	3.73 ± 0.13^b	2.73 ± 0.89^b	1.10 ± 0.59^b	ND; −ve
Cinnamaldehyde (2.0)	3.66 ± 0.33^b	3.21 ± 0.54^b	2.29 ± 0.31^b	0.16 ± 0.28^b	ND; −ve

Initial viable count of Escherichia coli O157:H7: 5.04 ± 0.02 log₁₀ CFU mL⁻¹.

Each value for viable count is the mean (standard deviation) of three replicate experiments.

Means with a different letter within a column differ significantly (p < 0.05).

CFU, colony-forming unit; ND, no colonies detected on agar plates with the lowest dilution (1:3) of the sample; −ve, negative enrichment test; +ve = positive enrichment test.

Viability of pathogens in MBJ at 4°C and 12°C

Viable counts of Salmonella enterica after 1 h of exposure to the control MBJ were 3.92 (at 4°C) and 3.58 (at 12°C) log₁₀ CFU/mL, representing 1.15 and 1.49 log reduction, respectively (Table 3). At 4°C and 12°C, cinnamaldehyde (1.5 μL/mL) completely inactivated Salmonella enterica in MBJ within 2 h; no salmonellae were detected by plating or enrichment. In contrast, at 2 h, viable salmonellae were 4.0 and 3.63 log₁₀ CFU/mL, in control MBJ at 4°C and 12°C, respectively.

Table 3.

Antibacterial Effectiveness of Cinnamaldehyde Against Salmonella enterica in Mixed Berry Juice Held at 4°C (A) or 12°C (B) for 24 Hours

Treatment (μL mL⁻¹)	1 h	2 h	4 h	8 h	24 h
A. Viable count (log₁₀ CFU mL⁻¹)^a at 4°C
Control	3.92 ± 0.62^b	4.00 ± 0.34^b	3.62 ± 0.07^b	3.62 ± 0.19^b	3.64 ± 0.02^b
Cinnamaldehyde (0.25)	3.61 ± 0.13^b	3.89 ± 0.26^b	3.62 ± 0.18^b	3.46 ± 0.43^b	3.16 ± 0.21^b
Cinnamaldehyde (0.5)	3.61 ± 0.34^b	3.62 ± 0.17^b	3.27 ± 0.22^b	2.59 ± 0.46^b	0.56 ± 0.97^b
Cinnamaldehyde (1.5)	2.56 ± 1.20^b	ND; −ve	ND; −ve	ND; −ve	ND; −ve
B. Viable count (log₁₀ CFU mL⁻¹)^a at 12°C
Control	3.58 ± 0.09^b	3.65 ± 0.31^b	3.74 ± 0.15^b	3.81 ± 0.28^b	3.39 ± 0.26^b
Cinnamaldehyde (0.25)	3.61 ± 0.14^b	3.75 ± 0.23^b	3.65 ± 0.29^b	3.49 ± 0.14^b	2.61 ± 0.05^b
Cinnamaldehyde (0.5)	3.48 ± 0.13^b	3.45 ± 0.53^b	3.07 ± 0.10^b	2.25 ± 0.30^b	0.16 ± 0.28^b
Cinnamaldehyde (1.5)	2.51 ± 0.54^b	ND; −ve	ND; −ve	ND; −ve	ND; −ve

Initial viable count of Salmonella enterica: 5.04 ± 0.03 log₁₀ CFU mL⁻¹.

Each value for viable count is the mean (standard deviation) of three replicate experiments.

Means with a different letter within a column differ significantly (p < 0.05).

CFU, colony-forming unit; ND, no colonies detected on agar plates with the lowest dilution (1:3) of the sample; −ve, negative enrichment test; +ve, positive enrichment test.

The survival of Escherichia coli O157:H7 in the more acidic MBJ was greater than that of Salmonella enterica, irrespective of storage temperature (Table 4). Reductions of Escherichia coli O157:H7 after a 1 h exposure to control MBJ at 4°C and 12°C were only 0.95 and 1.02 log₁₀ CFU/mL, respectively. In comparison to Salmonella enterica, a longer time elapsed before complete inactivation of Escherichia coli O157:H7 was observed in MBJ with cinnamaldehyde (1.5 μL/mL); Escherichia coli O157:H7 was not detected (by plating or enrichment) within 8 and 4 h in MBJ held at 4°C and 12°C, respectively (p < 0.05).

Table 4.

Antibacterial Effectiveness of Cinnamaldehyde Against Escherichia coli O157:H7 in Mixed Berry Juice Held at 4°C (A) or 12°C (B) for 24 Hours

Treatment (μL mL⁻¹)	1 h	2 h	4 h	8 h	24 h
A. Viable count (log₁₀ CFU mL⁻¹)^a at 4°C
Control	4.12 ± 0.41^b	4.11 ± 0.39^b	3.99 ± 0.43^b	3.86 ± 0.40^b	3.65 ± 0.15^b
Cinnamaldehyde (0.25)	4.00 ± 0.27^b	4.08 ± 0.39^b	3.93 ± 0.33^b	3.71 ± 0.42^b	3.38 ± 0.07^b
Cinnamaldehyde (0.5)	4.00 ± 0.32^b	3.88 ± 0.20^b	3.81 ± 0.30^b	3.56 ± 0.15^b	3.09 ± 0.47^b
Cinnamaldehyde (1.5)	3.57 ± 0.27^b	3.58 ± 0.81^b	1.25 ± 1.21^b	ND; −ve	ND: −ve
B. Viable count (log₁₀ CFU mL⁻¹)^a at 12°C
Control	4.05 ± 0.02^b	4.08 ± 0.15^b	3.97 ± 0.13^b	4.19 ± 0.24^b	4.02 ± 0.28^b
Cinnamaldehyde (0.25)	4.04 ± 0.07^b	4.03 ± 0.11^b	3.91 ± 0.02^b	3.79 ± 0.06^b	3.59 ± 0.08^b
Cinnamaldehyde (0.5)	4.00 ± 0.05^b	3.98 ± 0.10^b	3.72 ± 0.14^b	3.59 ± 0.10^b	2.63 ± 0.19^b
Cinnamaldehyde (1.5)	3.46 ± 0.12^b	3.23 ± 0.29^b	ND; −ve	ND; −ve	ND; −ve

Initial viable count of Escherichia coli O157:H7: 5.04 ± 0.02 log₁₀ CFU mL⁻¹.

Each value for viable count is the mean (standard deviation) of three replicate experiments.

Means with a different letter within a column differ significantly (p < 0.05).

CFU, colony-forming unit; ND, no colonies detected on the agar plates with the lowest dilution (1:3) of the sample; −ve, negative enrichment test; +ve, positive enrichment test.

Discussion

Several published reports describe the use of plant-based components, including EOs (Friedman et al., 2004), herb extract (Wu et al., 2008), caffeic acid (Reinders et al., 2001), cinnamon (Yutse and Fung, 2003; Yuste and Fung, 2004), and cinnamaldehyde (Baskaran et al., 2010), for inactivating Escherichia coli O157:H7 in apple juice or apple cider; however, this study investigated the effectiveness of cinnamaldehyde for inactivating Salmonella enterica and Escherichia coli O157:H7 and in two types of fruit juices with substantially different pH and °Brix values. Those pathogens would typically be the most resistant organisms in juices (USFDA, 2001).

The extent of inactivation of both pathogens in CRJ and MBJ increased with higher concentrations of cinnamaldehyde. For example, at 24 h, log₁₀ CFU/mL reductions of viable Salmonella enterica in CRJ (4°C) with cinnamaldehyde at 0.25, 0.5, 1.5, and 2.0 were 1.13, 1.51, 3.60, and 4.48, respectively (Table 1A). At those same cinnamaldehyde concentrations in CRJ, log₁₀ CFU/mL reductions of viable Escherichia coli O157:H7 were 1.36, 1.87, 4.55, and 5.07, respectively (Table 2A). Based on these results, cinnamaldehyde is a concentration-dependent antimicrobial that increases bacterial kill with increasing concentrations. In addition to cinnamaldehyde concentration, temperature was another factor that impacted the antibacterial activity of cinnamaldehyde in the juices.

In CRJ and MBJ held at 4°C or 12°C, cinnamaldehyde exhibited increased antimicrobial effectiveness at 12°C compared with at 4°C. Our findings agree with reports on cinnamon or cinnamaldehyde in apple juice (Zhao et al., 1993; Yuste and Fung, 2004; Baskaran et al., 2010). For example, Baskaran et al. (2010) reported that at 4°C, viable Escherichia coli O157:H7 in apple juice (4°C) with cinnamaldehyde (0.25 μL/mL) decreased to undetectable levels at 14 d; however, at 23°C, the pathogen was undetectable at 5 d. Yuste and Fung (2004) observed that a combination of cinnamon and nisin caused greater inactivation of Salmonella Typhimurium and Escherichia coli O157:H7 in apple juice at 20°C compared with at 5°C. Mossel and de Bruin (1960) reported decreased viability of Salmonella Typhimurium and Escherichia coli in apple juice at 24°C compared with at 5°C. Also, in apple cider without preservatives, the decline in the viability of Escherichia coli O157:H7 was greater at 25°C compared with at 8°C (Zhao et al., 1993).

The enhanced antibacterial effect of cinnamaldehyde at higher temperatures is likely due to the increased rate of cellular metabolism, growth, and death that occur at an ambient temperature compared with those same cellular activities at refrigeration temperature (Yuste and Fung, 2004; Levin and Rosen, 2006). Bacterial sensitivity to certain antimicrobials may be attributed, in part, to increased fluidity of the cytoplasmic membrane that occurs at warmer temperatures (McElhaney, 1976). Stringent control of membrane fluidity is crucial for membrane-associated functions such as active transport of solutes, passive permeability to hydrophobic molecules, and protein–protein interactions (Zhang and Rock, 2008). The cytoplasmic membrane is the primary cellular site where cinnamaldehyde exerts its antibacterial action (Gill and Holley, 2006); therefore, cinnamaldehyde, by exerting a membrane-fluidizing effect (Di Pasqua et al., 2006), may, at warmer temperatures, further disrupt those previously stated membrane-associated functions. Although the precise mechanism of the antibacterial action of cinnamaldehyde is inconclusive, it is believed that cinnamaldehyde inactivates bacteria by inhibiting ATPase at sub-lethal concentrations and disrupting the cytoplasmic membrane at lethal concentrations (Hyldgaard et al., 2012).

Juice was held at 12°C to simulate a temperature-abused product that could result in the growth of foodborne pathogens. The temperature of 12°C is within the temperature danger zone (4.4–60°C) in which enteric bacteria can grow and to great numbers, thereby increasing the risk of foodborne illness. At 4°C and 12°C, cinnamaldehyde was effective in controlling both pathogens with a greater loss of pathogen viability in MBJ.

The intrinsic low pH (3.39) of MBJ could have also influenced the antibacterial activity of cinnamaldehyde by causing acid-induced sub-lethal injury in both pathogens, thus presenting additional cellular lesions for the pathogens to cope with in the presence of cinnamaldehyde. Considering that the cytoplasmic membrane is a primary target for cinnamaldehyde (Gill and Holley, 2006) and that low pH can denature membrane proteins, the acidity of MBJ could sensitize the pathogens to the membrane-damaging action of cinnamaldehyde. At sub-lethal concentrations, cinnamaldehyde inhibited ATPase activity in Escherichia coli (Helander et al., 1998). In this regard, it was concluded that cinnamaldehyde crosses the outer membrane of Gram-negative bacteria, enters the periplasm, and possibly interacts with the cytoplasmic membrane (Helander et al., 1998) where the ATPases are located (Barton, 2005).

Conclusions

Cinnamaldehyde (ranging from 0.25 to 2.0 μL/mL) has good potential for killing Salmonella enterica and Escherichia coli O157:H7 in CRJ and MBJ at 4°C and 12°C, respectively. Cinnamaldehyde concentrations (0.5 and 0.25 μL/mL or lower) in juices could potentially be used to enhance the antimicrobial effects of emerging food processing technologies such as high-pressure processing, high-pressure homogenization, ultra sound, and pulse electric fields. Further research is needed in those areas as well as on the effects of cinnamaldehyde concentrations on sensory characteristics of minimally processed juices.

Footnotes

Acknowledgment

The authors thank Emalie Thomas-Popo for technical assistance rendered.

Disclosure Statement

No competing financial interests exist.

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