Carvacrol antimicrobial wash treatments reduce Campylobacter jejuni and aerobic bacteria on broiler chicken skin

Carvacrol antimicrobial wash treatments reduce Campylobacter jejuni and aerobic bacteria on broiler chicken skin S. Shrestha,∗ B. R. Wagle,∗ A. Upadhyay,† K. Arsi,∗ D. J. Donoghue,∗ and A. M. Donoghue‡,1 ∗

enumeration of C. jejuni and aerobic bacterial counts (n = 5/treatment/time point). In addition, the effect of treatments on the color of chicken skin was evaluated. Data were analyzed using PROC MIXED procedure of SAS. In the first study, all the tested doses of CR suspension consistently reduced C. jejuni counts across all time points. The 2% CR suspension wash reduced C. jejuni counts by ∼2.4 to 4 log10 cfu/sample (P < 0.05). In addition, 1% and 2% CR suspensions significantly reduced aerobic counts at all the time points. The results from the second study suggest that antiCampylobacter efficacy of CR emulsion or nanoemulsion treatments was not improved compared to CR suspension. Several CR suspension treatments were more effective than corresponding emulsion or nanoemulsion treatments. No significant differences were observed in the color of the samples between treatments (P > 0.05). The results suggest that CR could potentially be used as an antimicrobial wash treatment in postharvest poultry.

ABSTRACT Campylobacter jejuni, a major cause of human gastroenteritis worldwide, is often associated with the consumption of contaminated poultry products. With increasing consumer preference to natural and minimally processed foods, interventions utilizing natural antimicrobials for controlling C. jejuni on poultry products are gaining popularity. This study investigated the efficacy of the generally recognized as safe compound carvacrol (CR) as a wash treatment in reducing C. jejuni and aerobic bacteria on chicken skin. Two separate studies, each with 2 trials, were conducted. In the first study, the efficacy of CR suspension (0, 0.25, 0.5, 1, and 2%) was investigated, whereas in the second the efficacy of CR as suspension, emulsion, and nanoemulsion was studied. In both studies, skin samples were inoculated with 50 μL (∼8 log10 cfu/sample) of a cocktail of 4 wild strains of C. jejuni. After 30 min of attachment, samples were washed with the respective treatments for 1 min, drip dried for 2 min, and processed at 0, 8, 24, h post-treatment for

Key words: Campylobacter jejuni, carvacrol, emulsion, nanoemulsion, chicken skin 2019 Poultry Science 0:1–11 http://dx.doi.org/10.3382/ps/pez198

INTRODUCTION

lead to death. It was estimated that the annual cost associated with campylobacteriosis is approximately 1.9 billion dollars (Hoffmann et al., 2015). Surveys of raw agricultural products support epidemiologic evidence implicating undercooked and contaminated chicken meat as one of the primary sources for human C. jejuni infection (Kramer et al., 2000). Poultry are the reservoir for C. jejuni wherein the pathogen colonizes at high level (108 cfu/g of cecal material) in the lower gastrointestinal tract (Blaser et al., 1983; Evans, 1991; Wagenaar et al., 2015) leading to product contamination during slaughter and poultry processing. Commercial poultry processors have implemented various Hazard Analysis and Critical Control Points and good manufacturing practices to lower the pathogen load on carcasses; however, potential for contamination exists in the farm to fork supply chain (Elvers et al., 2011; Alonso-Hernando et al., 2013). Campylobacter spp. was isolated from up to 76% of retail organic chicken meat and 74% of conventional

Campylobacter infection in humans is one of the most common bacterial foodborne diseases worldwide (WHO, 2017). In the United States, Campylobacter causes an estimated 1.3 million illnesses each year. However, the actual figure might be higher as many cases go undiagnosed or unreported (CDC, 2017). Campylobacter jejuni accounts for 90% of human campylobacteriosis cases characterized by vomiting, bloody diarrhea, abdominal cramps, and fever (WHO, 2017). In certain cases, campylobacteriosis leads to serious sequelae, such as reactive arthritis and Guillain-Barr´e syndrome (Spiller, 2007; Gradel et al., 2009), that could Published by Oxford University Press on behalf of Poultry Science Association 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US. Received November 26, 2018. Accepted March 21, 2019. 1 Corresponding author: [email protected]

1

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Department of Poultry Science, University of Arkansas, AR 72701, USA; † Department of Animal Science, University of Connecticut, CT 06269, USA; and ‡ Poultry Production and Product Safety Research Unit, USDA-ARS, AR 72701, USA

2

SHRESTHA ET AL.

our lab and collaborators have demonstrated the efficacy of CR against C. jejuni in vitro studies on cecal contents (Kollanoor Johny et al., 2010) as well as in vivo studies in 10-day-old broiler chickens (Arsi et al., 2014); however, CR as an antimicrobial wash treatment for reducing C. jejuni in postharvest poultry has not been investigated. Despite multiple benefits, the use of essential oils as a natural antimicrobial in food systems is limited primarily due to flavor concern and need for high concentrations to exert their antimicrobial effect. Moreover, CR has low water solubility. Albeit, the total surface area, solubility, and the antimicrobial property of CR could be enhanced by using emulsion/nanoemulsion technology (Landry et al., 2014, 2015; Yadav et al., 2014). We hypothesized that CR could reduce both C. jejuni and aerobic counts on poultry products and could be used as an effective antimicrobial treatment to improve food safety. In addition, we also hypothesized that CR emulsion and nanoemulsion would result in greater bacterial reductions than CR suspension alone. The present study investigated the efficacy of CR (suspension, emulsion, nanoemulsion) as an antimicrobial wash to reduce C. jejuni and aerobic bacteria on chicken skin. In addition, the effect of aforementioned treatments on the color of chicken skin was investigated.

MATERIALS AND METHODS Bacterial Strains and Culture Conditions Four wild-type strains (S1, S3, S4, and S8) of C. jejuni originally isolated from commercial broilers were used for this study. The C. jejuni inoculum for each trial was prepared as described by Shrestha and colleagues (2017). Briefly, 1 loopful of glycerol stock of each strain was inoculated into separate 15 mL tube containing 5 mL of sterile Campylobacter Enrichment Broth (catalogue no. 7526A, Neogen Corp, Lansing, MI) followed by incubation at 42◦ C under microaerophilic atmosphere (5% O2 , 10% CO2 , and 85% N2 ) for 48 h. After incubation, C. jejuni strains were sub-cultured for 24 h and centrifuged at 3,500 × g for 10 min. The cell pellet was resuspended in 20 mL of Butterfield’s Phosphate Diluent (BPD; 0.625 mM potassium dihydrogen phosphate, pH 6.67) and used as inoculum.

Study 1: Evaluation of Antimicrobial Activity of CR Suspension on Chicken Skin Preparation of CR Suspension Treatments For the preparation of CR suspension, 2% (vol/vol) stock suspension of CR (catalogue no. W224502, SigmaAldrich Co., St. Louis, MO) was made in sterile BPD followed by stirring at 300 rpm for 15 min. Two-fold concentrations of the stock solution were made in BPD to obtain 0.25, 0.5, and 1% CR suspension. The pHs of all the treatment solutions were ∼6.6.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

chickens collected from retail stores in Maryland between September 2002 and August 2003 (Cui et al., 2005). A more recent study by Williams and Oyarzabal (2012) observed that the average prevalence of Campylobacter spp. in skinless boneless retail meat from 2005 through 2011 in Alabama, USA was 41%. With a growing population and increasing consumer awareness, the consumption of poultry meat has tripled over the last 5 decades (USDA-FAS, 2018). As a result of the high consumption rate of poultry meat and greater prevalence of C. jejuni on retail poultry products, the risk of C. jejuni infections in humans is substantial (Nauta et al., 2007; Marder et al., 2017). A number of studies have been conducted for controlling C. jejuni in poultry with varied degree of success (Byrd et al., 2001; Cole et al., 2006; Arsi et al., 2014; Guyard-Nicodeme et al., 2015; Shrestha et al., 2017; Wagle et al., 2017; Huneau-Sala¨ un et al., 2018). As part of multihurdle approach to control foodborne pathogens, research is being focused on developing safer and effective postharvest interventions for controlling C. jejuni on poultry products. Various interventions including physical methods, such as freezing, chilling, hot, steam and electrolyzed water treatments, ultrasound, and irradiation, have been tested to reduce the pathogen counts on carcasses (Patterson, 1995; Park et al., 2002; Corry et al., 2007; James et al., 2007; Musavian et al., 2014). In addition, chemicals such as organic acids, chlorine or phosphate-based compounds have been used as chemical decontaminant with pathogen reduction ranging from 1 to 2.2 log10 (Zhao and Doyle, 2006; Bauermeister et al., 2008; Riedel et al., 2009; Birk et al., 2010; Loretz et al., 2010; Thormar et al., 2011). However, potential concerns with regards to change in the meat quality, reduced consumer acceptance, and cost of treatments have made most of the aforementioned treatments less applicable for decontaminating poultry carcasses at industrial setting (Dawson et al., 1963; Cox et al., 1974; Bilgili et al., 1998; Whyte et al., 2003). To address this, researchers are focusing on using natural plant-derived antimicrobials for carcass decontamination (Smith-Palmer et al., 1998). The antimicrobial properties of various essential oils in the food system is well documented in the literature (Chouliara et al., 2007; Sharma et al., 2017). Carvacrol [2-Methyl-5-(1-methylethyl) phenol] is a monoterpenic phenol biosynthesized from γ -terpinene. This phenolic compound is the major component of the essential oil fraction of oregano (Arrebola et al., 1994). Carvacrol (CR) is currently listed as generally recognized as safe (GRAS) by the US Food and Drug Administration (Code of Federal Regulations 21 Part 172). CR exhibits a plethora of biological activities including broad spectrum antimicrobial action (Ultee et al., 2000; Xu et al., 2008; Kollanoor Johny et al., 2010), antioxidant (Aeschbach et al., 1994; Ramos et al., 2014), anticancer (Arunasree, 2010; Mehdi et al., 2011; Luo et al., 2016), and antifungal properties (Lima et al., 2013; Chavan and Tupe, 2014). Previous studies from

CAVACROL WASH REDUCES C. JEJUNI

Study 2: Comparative Antimicrobial Evaluation of CR Suspension, Emulsion, and Nanoemulsion on Chicken Skin Preparation of CR Emulsion and Nanoemulsion All the compounds used for the preparation of emulsion and nanoemulsion were designated as GRAS by the FDA. Emulsion was prepared by using spontaneous emulsification procedure as described previously (Ostertag et al., 2012) with minor modifications. Briefly, an organic phase was made by adding CR (30 mL) and Tween-80 (37.5 mL) (catalogue no. 01516, Chem-impex int’l inc., Wood Dale, IL) followed by stirring using a magnetic stirrer at 750 rpm for 30 min. Sodium phosphate buffer (5 mM; pH 7.0; SigmaAldrich) was added into organic phase at the rate of 4 mL/min with continuous stirring for 1 h. Emulsion solutions were stored at 4◦ C until needed. The final concentration of CR in the emulsion was 10% which was then diluted in BPD to prepare 0.25, 0.5, 1, and 2% CR emulsion. The preparation of nanoemulsion was based on the optimized system as described by Chang et al. (2013)

and Abd-Elsalam and Khokhlov (2015) with some modification. Briefly, a coarse emulsion of CR (15 mL), Tween-80 (30 mL), and buffer (255 mL) was prepared as described above and mixed for 30 min. The mixture was then sonicated using an ultrasonicator (Qsonica Q700, Newtown, CT, USA) for 10 min. The stock solution was 5% CR, which was further diluted in BPD to obtain 0.25, 0.5, 1, and 2% CR nanoemulsion. Characterization of Emulsion and Nanoemulsion The droplet size, zeta potential, and polydispersity index (PDI) of emulsion and nanoemulsion was measured using previously published method (Zainol et al., 2012; Abd-Elsalam and Khoklov, 2015). Briefly, 10 μL of emulsion or nanoemulsion was diluted with 1 mL of deionized water at room temperature. Three replicates of independent batches were analyzed by a dynamic light scattering method using Zeta-sizer Nano ZS (Malvern Instruments Ltd, Malvern, WR, UK). The thermodynamic stability of freshly prepared emulsion and nanoemulsion was determined using standard published method (Shafiq and Shakeel, 2010; Abd-Elsalam and Khoklov, 2015). Briefly, the solutions were centrifuged at 3,500× g for 20 min to evaluate the phase separation. In addition, 4 cycles of heating and cooling were performed between 4 and 40◦ C to study the effect of temperature on the stability of emulsion and nanoemulsion. Preparation, Inoculation, and Treatments of Chicken Skin Samples A total of 450 thigh skin samples were used for 2 replicate trials. These samples were collected as per study 1. For each trial, 225 skin samples were randomly divided into 15 treatment groups consisting of baseline, BPD, Tween-80, and 4 doses (0.25, 0.5, 1, and 2%) of CR suspension, emulsion, and nanoemulsion, respectively (n = 5 samples per treatment per time point). Chicken skin samples were prepared and inoculated followed by wash treatments as described in study 1. The sample processing and enumeration of C. jejuni and total aerobic bacteria were conducted according to the procedure described above. Additionally, C. jejuni counts in the wash solution (post-treatment) were determined by plating 250 μL wash treatment on CLA plates followed by incubation as described previously.

Determination of Color The color measurements of skin samples treated with CR suspension, emulsion, or nanoemulsion were carried out using a chroma meter (CR-300, Konica Minolta Sensing Inc., Japan), and this was used to objectively measure International Commission on Illumination (CIE) L∗ , a∗ , b∗ values (L∗ measure relative lightness, a∗ relative redness and b∗ relative yellowness). Two replicate trials were conducted. For each trial, 150 samples were randomly divided into 15 treatment groups (as described above) for 2 time points (0 and 24 h). The instrument was calibrated against white

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Preparation, Inoculation, and Treatments of Chicken Skin Samples Chicken thighs were procured form the University of Arkansas Poultry Pilot Processing Plant (Fayetteville, AR). The skin was removed from the muscle tissue, cut into pieces (4 × 4 cm) and ◦ stored at –20 C until the day of experiment. A total of 150 skin samples were used for 2 trials. For each trial, 75 skin samples were randomly allocated to 5 treatments (0, 0.25, 0.5, 1, and 2% CR; n = 5 samples per treatment per time point). Each skin sample was inoculated with 50 μL (∼8 log10 cfu/sample) of a cocktail of 4 wild strains of C. jejuni and allowed to adhere for 30 min. Inoculated skin samples were washed by transferring into centrifuge tube containing 25 mL of respective treatment solutions followed by gentle shaking for 1 min. The washed chicken skin samples were drip dried for 2 min and were processed either immediately (0 h) or after 8 or 24 h of storage in sterile sampling bags (catalogue no. 89085546, VWR International, Radnor, PA) at 4◦ C. Microbial Analysis For the sample processing, each skin sample was transferred in 10 mL of Dey-Engley neutralizing broth (catalogue no. C7371, Hardy diagnostic, Santa Maria) and vortexed at 700 rpm for 15 s. The sample was 10-fold diluted in BPD and plated using the spread plate technique onto Campylobacter Line agar (CLA; Line, 2001) for C. jejuni enumeration and tryptic soy agar (TSA; catalogue no. C7121, Hardy diagnostics, Santa Maria, CA) for aerobic bacterial enumeration. The inoculated CLA or TSA plates were incubated at 42◦ C for 48 h under microaerophilic atmosphere or at 37◦ C for 24 h under aerobic condition, respectively. Bacterial colonies were expressed in log10 cfu/sample.

3

4

SHRESTHA ET AL. Table 1. Effect of carvacrol suspension on (a) Campylobacter jejuni and (b) aerobic bacteria survival on chicken skin.1,2,3 (1a) Campylobacter jejuni counts Trial

Treatments

1

2

5.50 3.40 3.45 2.51 2.28 6.13 4.08 3.18 3.69 2.09

± ± ± ± ± ± ± ± ± ±

8h a

0.11 0.43b 0.63b 0.68b 0.56b 0.02a 0.33b 0.49b,c 0.62b 0.48c

5.34 3.79 3.94 3.83 2.96 5.89 4.02 2.34 2.56 3.05

± ± ± ± ± ± ± ± ± ±

24 h a

0.28 0.40b 0.30b 0.48b 0.49b 0.15a 0.34b 0.14c 0.47c 0.63b,c

4.86 2.78 2.16 2.05 2.13 5.77 3.60 3.78 3.70 3.27

± ± ± ± ± ± ± ± ± ±

0.11a 0.26b 0.10b 0.40b 0.42b 0.16a 0.30b 0.22b 0.28b 0.36b

1 n = 5 replicates per treatment per time point per trial. Values (log10 cfu/sample) presented as mean ± standard error of mean. 2 C. jejuni counts within column in the same trial with no common superscript differ significantly (P < 0.05).

(1b) Aerobic bacteria counts Trial

Treatments

1

Control (BPD) 0.25% CR 0.5% CR 1% CR 2% CR Control (BPD) 0.25% CR 0.5% CR 1% CR 2% CR

2

0h 3.58 2.91 2.66 2.57 2.42 4.07 3.79 3.44 3.32 3.27

± ± ± ± ± ± ± ± ± ±

8h a

0.21 0.14a,b 0.21b 0.36b 0.37b 0.09a 0.20a,b 0.19b 0.16b 0.24b

3.94 3.30 3.28 3.01 3.00 4.25 3.48 3.49 3.37 3.35

± ± ± ± ± ± ± ± ± ±

24 h a

0.25 0.40a,b 0.33a,b 0.14b 0.07b 0.04a 0.18b 0.12b 0.26b 0.27b

4.90 3.24 2.44 3.02 2.34 4.59 3.88 3.53 3.43 2.85

± ± ± ± ± ± ± ± ± ±

0.45a 0.12b 0.22b,c 0.19b,c 0.28c 0.19a 0.26a,b 0.16b,c 0.31b,c 0.33c

1 n = 5 replicates per treatment per time point per trial. Values (log10 cfu/sample) presented as mean ± standard error of mean. 3 Aerobic bacteria counts within column in the same trial with no common superscript differ significantly (P < 0.05).

tile before measurements were recorded. Three readings were taken on the lateral side of each sample.

Statistical Analysis For the microbial analysis, C. jejuni and aerobic bacteria counts (cfu/sample) were transformed to log10 cfu/sample to maintain the homogeneity of variance (Byrd et al., 2001). For the color analysis, data from 2 trials were pooled for each treatment before analysis. The data were analyzed using ANOVA with the PROC MIXED procedure in the SAS statistical software, version 9.3 (SAS Institute Inc., Cary, NC). Means were partitioned by LSMEANS analysis, and a P value of <0.05 was required for statistical significance.

RESULTS Antimicrobial Efficacy of CR Suspension Against C. jejuni and Aerobic Bacteria on Chicken Skin The Effect of CR Suspension on C. jejuni Table 1a shows the effect of CR suspension in reduc-

ing C. jejuni on inoculated chicken skin stored at 4◦ C for 3 different time points (0, 8, and 24 h). The C. jejuni counts present on the control (skin washed with BPD) was ∼5.5 and ∼6 log10 cfu/sample for the trials 1 and 2, respectively. All the tested doses of CR suspension (0.25, 0.5, 1, and 2%) significantly reduced C. jejuni counts (>1.4 log10 cfu/sample) compared to the controls. Two percent CR suspension reduced C. jejuni by 3.22 log10 cfu/sample at 0 h in trial 1 and ∼4.0 log10 cfu/sample at 0 h in trial 2 when compared with the controls. All the doses of CR suspensions (0.25, 0.5, 1, or 2%) were equally effective in reducing C. jejuni counts when compared among each other at all storage time points in trial 1. Similar patterns were observed in trial 2 at 24 h; however, 2% CR suspension was more effective in reducing C. jejuni counts than 0.25 or 1% dose at 0 h. The Effect of CR Suspension on Aerobic Bacteria Table 1b shows the efficacy of CR in reducing aerobic bacterial counts on the chicken skin. The aerobic bacterial counts recovered from the controls ranged from ∼3.5 to 5 or from 4 to 4.5 log10 cfu/sample for trials 1 and 2, respectively. The 1 and 2% CR suspension consistently reduced aerobic bacterial counts

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Control (BPD) 0.25% CR 0.5% CR 1% CR 2% CR Control (BPD) 0.25% CR 0.5% CR 1% CR 2% CR

0h

5

CAVACROL WASH REDUCES C. JEJUNI Table 2. Effect of carvacrol suspension, emulsion, and nanoemulsion on Campylobacter jejuni survival on chicken skin.1,2 Time points Treatments

1

Baseline

7.52 ± 0.01a

7.25 ± 0.06a

7.28 ± 0.03a

Controls

BPD 4% Tween-80

6.32 ± 0.11 6.55 ± 0.06b

6.17 ± 0.15 6.07 ± 0.11b

5.79 ± 0.05b,c 5.83 ± 0.04b

Suspension

0.25% 0.50% 1% 2%

5.08 4.86 4.40 3.49

± ± ± ±

0.15e,f 0.12f,g 0.22g 0.54h

4.50 4.88 4.77 3.69

± ± ± ±

0.13g 0.23e,f,g 0.39f,g 0.40h

3.80 3.42 3.16 2.80

± ± ± ±

0.07g,h 0.08h,i 0.27i,j 0.27j

Emulsion

0.25% 0.50% 1% 2%

5.53 5.23 5.07 4.43

± ± ± ±

0.10d,e 0.07e,f 0.08e,f 0.10g

4.93 5.71 5.46 5.45

± ± ± ±

0.12d,e,f,g 0.09b,c 0.22c,d 0.17c,d

4.99 4.68 4.38 3.22

± ± ± ±

0.08d,e 0.09e,f 0.08f 0.33i,j

Nanoemulsion

0.25% 0.5% 1% 2%

5.96 5.24 5.25 4.95 7.10

± ± ± ± ±

0.08c,d 0.08e,f 0.04e,f 0.13f 0.07a

5.29 5.63 5.48 5.42 7.31

± ± ± ± ±

0.12c,d,e,f 0.16b,c 0.19c,d 0.17c,d,e 0.06a

5.36 4.93 3.90 3.91 7.08

± ± ± ± ±

0.17c,d 0.15d,e 0.17g 0.12g 0.02a

Controls

BPD 4% Tween-80

5.91 ± 0.09b 6.03 ± 0.07b

Suspension

0.25% 0.50% 1% 2%

4.98 4.17 4.15 2.46

± ± ± ±

0.09c,d 0.21f 0.18f 0.53g

4.56 3.82 3.57 3.53

± ± ± ±

0.14e 0.25f,g 0.26g 0.09g

4.26 4.18 2.69 3.33

± ± ± ±

0.30e,f 0.24e,f,g 0.28i 0.23h

Emulsion

0.25% 0.50% 1% 2%

5.00 4.70 4.26 4.26

± ± ± ±

0.17c,d 0.18d,e 0.11e,f 0.11f

4.98 4.50 4.00 4.02

± ± ± ±

0.15c,d 0.06e 0.12f 0.21f

4.94 4.26 3.93 3.96

± ± ± ±

0.06c,d 0.18e,f 0.24f,g 0.26f,g

Nanoemulsion

0.25% 0.5% 1% 2%

5.49 5.00 4.79 4.30

± ± ± ±

0.10b,c 0.15c,d 0.07d,e 0.12e,f

5.15 4.54 4.67 3.83

± ± ± ±

0.13c 0.12e 0.06d,e 0.14f,g

5.14 4.39 4.51 3.70

± ± ± ±

0.10c 0.18e,f 0.10d,e 0.08g,h

2

0h

Baseline

8h

b,c

24 h

b

5.86 ± 0.08b 5.76 ± 0.09b

5.74 ± 0.08b 5.70 ± 0.10b

1 n = 5 replicates per treatment per time point per trial. Values (log10 cfu/sample) presented as mean ± standard error of mean. 2 C. jejuni counts within column in the same trial with no common superscript differ significantly (P < 0.05).

(>0.93 log10 cfu/sample) across all time points in trial 1 (P < 0.05). In the trial 2, the consistent reduction across time points was observed with 0.5, 1, and 2% doses. In addition, 0.5, 1, and 2% doses were not significantly different in efficacy among each other in both trials.

Comparative Antimicrobial Efficacy of CR Suspension, Emulsion, and Nanoemulsion Against C. jejuni and Aerobic Bacteria on Chicken Skin Properties and Stability of CR Emulsion and Nanoemulsion The average particle size (Z-average), PDI, and zeta-potential for emulsion were ∼505.6 ± 101 nm, 1 ± 0.12, and –24.9 ± 11.3 mV, respectively, whereas for nanoemulsion the values for average particle size (Z-average), PDI, and zeta-potential were ∼260.3 ± 25 nm, 0.039 ± 0.01, and –18.7 ± 5.54 mV, respectively. The distribution was unimodal. The solutions were stable after centrifugation at 3,500× g for 20 min, and resisted 4 cycle of heating and cooling.

The Effect of CR on C. jejuni The effect of CR suspension, emulsion, and nanoemulsion on the survival of C. jejuni on chicken skin is presented in Table 2. C. jejuni recovered from baseline (skin samples not subjected to treatments) was ∼7 log10 cfu/sample. Washing of the skin samples in either BPD (control for CR suspension) or Tween-80 (control for CR emulsion or nanoemulsion) reduced C. jejuni counts by ∼1 to 1.5 log10 cfu/sample when compared with the baseline across all time points in both trials (P < 0.05). All doses of CR (0.25, 0.5, 1, or 2%) suspension significantly reduced C. jejuni counts when compared with its control (BPD) in both trials. The 2% CR suspension treatment was the most effective and reduced C. jejuni counts by at least ∼3 log10 cfu/sample at 0 h in both trials. Most of the tested doses of CR emulsion and nanoemulsion significantly reduced C. jejuni counts at 0, 8, and 24 h of storage when compared to Tween-80 (control) in both trials with the exception of 0.5% CR emulsion or nanoemulsion in trial 1, and 0.25% CR nanoemulsion in trial 2. When the CR suspension was compared with the respective doses of either emulsion or nanoemulsion, no consistent difference in efficacy among the 3

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Trial

6

SHRESTHA ET AL. Table 3. Effect of carvacrol suspension, emulsion, and nanoemulsion against aerobic bacteria counts on chicken skin.1,2 Time points Treatments

1

Baseline

5.76 ± 0.07a

5.43 ± 0.19a

5.15 ± 0.06a

Controls

BPD 4% Tween-80

5.78 ± 0.09 5.58 ± 0.09a,b

4.86 ± 0.15 4.67 ± 0.11b,c,d

4.90 ± 0.22a,b 4.87 ± 0.26a,b

Suspension

0.25% 0.50% 1% 2%

4.88 4.66 4.67 3.95

± ± ± ±

0.15c,d,e 0.22e 0.36d,e 0.15f

3.93 3.54 3.52 3.43

± ± ± ±

0.19e,f,g 0.46f,g 0.22f,g 0.41g

3.19 3.66 3.47 4.11

± ± ± ±

0.30e 0.23d,e 0.21e 0.22c,d

Emulsion

0.25% 0.50% 1% 2%

5.61 5.33 4.89 4.57

± ± ± ±

0.11a,b 0.08a,b,c 0.29c,d,e 0.26e

4.04 4.24 4.12 4.87

± ± ± ±

0.3d,e,f,g 0.28b,c,d,e,f 0.32c,d,e,f,g 0.15a,b

4.28 4.85 4.37 4.55

± ± ± ±

0.10c 0.22a,b 0.33b,c 0.09b,c

Nanoemulsion

0.25% 0.5% 1% 2%

5.57 5.76 5.01 5.16

± ± ± ±

0.06a,b 0.07a 0.2c,d,e 0.11b,c,d

4.71 4.40 4.78 4.62

± ± ± ±

0.44a,b,c,d 0.15b,c,d,e 0.22a,b,c 0.19b,c,d,e

5.23 4.19 4.40 4.45

± ± ± ±

0.07a 0.11c,d 0.03b,c 0.15b,c

2

0h

8h

a

24 h

a,b

Baseline

4.23 ± 0.08a,b

4.31 ± 0.06a

4.29 ± 0.11a

Controls

BPD 4% Tween-80

3.89 ± 0.09 3.78 ± 0.08b,c,d,e

3.85 ± 0.11 4.16 ± 0.24a,b

4.14 ± 0.20a,b 4.02 ± 0.08a,b,c

Suspension

0.25% 0.50% 1% 2%

4.10 2.65 2.93 2.18

± ± ± ±

0.20b 0.18g,h 0.29f,g 0.12h

3.14 2.34 2.26 2.46

± ± ± ±

0.19d,e 0.16f 0.26f 0.14f

3.71 2.91 2.67 2.31

± ± ± ±

0.24a,b,c 0.29d,e 0.24e,f 0.2f

Emulsion

0.25% 0.50% 1% 2%

3.93 3.50 3.40 4.24

± ± ± ±

0.22b,c,d 0.17d,e 0.07e,f 0.34a,b

3.54 3.00 3.97 3.98

± ± ± ±

0.11c,d 0.14e 0.17a,b,c 0.19a,b,c

3.52 2.91 3.61 3.44

± ± ± ±

0.12c 0.26d,e 0.06b,c 0.30c,d

Nanoemulsion

0.25% 0.5% 1% 2%

4.05 3.73 3.56 4.71

± ± ± ±

0.23b,c 0.10b,c,d,e 0.02c,d,e 0.23a

4.03 3.67 3.92 3.86

± ± ± ±

0.27a,b,c 0.08b,c 0.22a,b,c 0.15a,b,c

4.16 3.76 3.92 3.88

± ± ± ±

0.16a,b 0.09a,b,c 0.25a,b,c 0.28a,b,c

b,c,d,e

a,b,c

1 n = 5 replicates per treatment per time point per trial. Values (log10 cfu/sample) presented as mean ± standard error of mean. 2 Aerobic bacteria counts within column in the same trial with no common superscript differ significantly (P < 0.05).

different forms of CR was observed (except 1% CR suspension vs. 1% CR nanoemulsion). When the CR emulsion vs. nanoemulsion were compared within time points and doses, the 0.25 and 0.5% CR emulsions produced efficacy similar to the respective doses of nanoemulsion in both trials. The Effect of CR on Aerobic Bacteria The total aerobic bacterial counts (Table 3) recovered from baseline was ∼5.5 and 4.3 log10 cfu/sample in trials 1 and 2, respectively. Washing of skin sample with either BPD or 4% Tween-80 did not reduce aerobic counts in either trial when compared to the baseline (P > 0.05). Most of the CR suspension treatments significantly reduced aerobic counts as compared to the BPD controls in both trials (except for CR 0.25% at 0 and 24 h in trial 2). The 1% CR emulsion or 2% CR nanoemulsion in trial 1 and only 0.5% emulsion in trial 2 significantly reduced aerobic counts as compared to the baseline (P < 0.05); however, when compared with the 4% Tween-80, none of the treatments of either emulsion or nanoemulsion consistently reduced total aerobic counts in both trials (P > 0.05). When the CR suspension was compared with the respective doses of

either emulsion or nanoemulsion, there was no consistent difference in efficacy among the 3 different forms of CR. The efficacy of CR emulsion and nanoemulsion were similar in majority of time points in both trials.

Survival of C. jejuni in CR Wash Treatments The number of C. jejuni surviving in BPD wash treatments ranged from ∼5.75 to 6.35 log10 cfu/mL in trial 1 and from ∼6.13 to 6.50 log10 cfu/mL in trial 2 (data not shown). In case of the 4% Tween-80 solution, the number of C. jejuni was similar when compared with BPD (P > 0.05) (data not shown). However, C. jejuni counts were reduced to below detection limit (0.6 log10 cfu/mL) in all CR wash treatments (data not shown).

Effect of CR Treatments on the Color of Skin Table 3 shows the effect of CR suspension, emulsion, or nanoemulsion on the color of skin stored at 4◦ C for 0

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Trial

CAVACROL WASH REDUCES C. JEJUNI

and 24 h. Samples washed with control (BPD) showed lightness (L∗ ), redness (a∗ ), and yellowness (b∗ ) values of 74.80 ± 1.54, 2.27 ± 0.57, and 10.80 ± 0.70, respectively for 0 h and 77.8 ± 0.76, 1.68 ± 0.49, and 10.08 ± 0.50, respectively for 24 h. None of the suspensions, emulsions, or nanoemulsions of CR significantly changed the L∗ , a∗ , and b∗ of samples at 0 h or at 24 h of storage.

With increasing consumer demand for natural, antibiotic-free, and minimally processed foods, the use of GRAS status plant-derived compounds is gaining more attention as safe and effective antimicrobials for decontamination of poultry carcass. The rough surface of chicken skin facilitates attachment and survival of C. jejuni during the multiple stages of processing (Wempe et al., 1983; Corry and Atabay, 2001; Stern et al., 2001; Tan et al., 2014). Therefore, we used chicken skin as a model to represent the whole carcass for testing the efficacy of CR against C. jejuni. Currently, peracetic acid and chlorine are the most frequently used chemicals for carcass decontamination by poultry processors (USDA FSIS, 2017), however, the aforementioned chemicals results in minimal reduction of C. jejuni (Bauermeister et al., 2008; Nagel et al., 2013). In our preliminary trial, chlorine (50 ppm) and peracetic acid (1,000 ppm) wash treatments reduced C. jejuni on chicken skin by ∼0.34 and 1.75 log10 cfu/sample, respectively (data not shown). In the present study, we observed that CR as an antimicrobial wash treatment significantly reduced C. jejuni counts on chicken skin. The reduction was at least by ∼2.4 log10 cfu/sample for 2% CR suspension compared with the samples washed in BPD controls (Table 1a). Similar results were reported previously against various pathogens on other foods. Ultee and coworkers (2000) reported that CR showed a dosedependent inhibition of Bacillus cereus in artificially inoculated cooked rice. Antibiotic-resistant Salmonella enterica was significanlty reduced on celery (below detection limit; detection limit < 1 log10 ) on day 0 and oysters (∼5 log10 ) on day 3 washed in 1% CR for 10 min or 1 h (Ravishankar et al., 2010). Upadhyay and coworkers (2014) showed that dipping (for 1 and 3 min) of cantaloupes rind plugs in 2% CR alone or in combination with 2% hydrogen peroxide significantly reduced Listeria monocytogenes on cantaloupes by > 2.0 to 2.5 log10 . The group also observed that CR with hydrogen peroxide reduced L. monocytogenes to undectable level (detection limit < 1 log10 /cm2 ) with the 10 min dipping time at 25◦ C. Recently, a study demonstrated that 3 common foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Tyhphimurium) were significantly reduced on beef when immersed in 0.3 or 0.5% CR with teriyaki sauce after 1, 3, and 7 D stored at 4◦ C (Moon et al., 2017). The

reduction of C. jejuni on inoculated chicken skin by CR shows its potential to be used as a natural safe chemical decontamination of chicken carcasses in processing plants. Since meat and meat products are very perishable foods and require protection from microbial spoilage, the meat industry is constantly looking for intervention strategies to increase the shelf-life and safety of meat products. The use of a GRAS compound that reduces both foodborne pathogens and spoilage organisms could be a good option to improve safety and quality of food products. Many studies have been conducted using oregano oil to control spoilage microorgansims thereby increasing shelf-life of perishable foods (Chouliara et al., 2007; Mexis et al., 2009; Karabagias et al., 2011). Since most of the meat spoilage bacteria are either aerobic or facultative anerobic (Gill and Greer, 1993), we evalutated the efficacy of CR against total aerobic bacteria. Data from our study showed that 1 and 2% CR suspension significantly reduced (P < 0.05) aerobic bacterial counts when compared to the controls (Table 1b). The maximum reduction (∼2.5 log10 cfu/sample) was obtained with 2% CR. Thus, CR could be used to inhibit the growth of meat spoilage organisms and thereby increase the shelf-life of poultry products. Although the majority of the essential oils are categorized as GRAS (Kabara, 1991), their use in the food products are greatly limited due to their flavor consideration and limited solubility in water. Recently, several studies have shown that formulation of emulsion and nanoemulsion significantly improved the water solubility and altered the antimicrobial efficacy of essential oils against various microorganisms (Dons`ı et al., 2011, 2012; Ghosh et al., 2013; Bhargava et al., 2015; Speranza et al., 2015). The oil in water nanoemulsion consists of a fine dispersion of ultra-small oil particles with diameter smaller than 400 nm (Arbor 2008), whereas emulsion (macroemulsions) is characterized by particle size of 0.5 to 100 μm (Windhab et al., 2005). The decrease in oil droplet size in a colloidal solution leads to an increase in the total surface area for interaction between essential oils and bacteria, thereby potentially enhancing the antimicrobial efficacy of essential oils (Solans et al., 2005; Weiss et al., 2009). Previous studies have explored the antimicrobial efficacy of CR emulsion and/or nanoemulsion against Salmonella Enteritidis, Escherichia coli O157:H7 (Landry et al., 2014, 2015) and Lactobacillus plantarum (Char et al., 2016). However, studies investigating CR emulsion/nanoemulsion efficacy against C. jejuni have not been conducted. Stable formulation of CR emulsion [surfactant-to-oil ratio (SOR)=1.25] and nanoemulsion (SOR = 2) were selected from various SOR based on our priliminary experiments (data not shown). The droplet size in CR nanoemulsion was more uniform than emulsion based on PDI (0.039 ± 0.01 vs. 1 ± 0.12). However, the emulsion or nanoemulsion formulations did not

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

DISCUSSION

7

8

SHRESTHA ET AL. Table 4. Color values of chicken skin samples treated with carvacrol.1 Sampling time

Treatments

0h

Baseline Controls Suspension

Nanoemulsion

24 h

Baseline Controls Suspension

Emulsion

Nanoemulsion

BPD 4% Tween-80 0.25% 0.50% 1% 2% 0.25% 0.50% 1% 2% 0.25% 0.50% 1% 2% BPD 4% Tween-80 0.25% 0.50% 1% 2% 0.25% 0.50% 1% 2% 0.25% 0.50% 1% 2%

74.78 74.80 75.22 76.10 76.86 74.28 73.81 75.06 75.13 73.99 73.73 75.40 74.95 74.95 75.16 76.80 77.80 76.76 78.93 79.14 78.83 78.89 77.68 77.51 77.65 76.60 76.05 76.96 77.48 76.02

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a∗ 0.60 1.54 0.64 0.46 0.81 0.51 0.82 1.10 1.94 1.55 1.40 1.30 1.40 0.98 0.96 0.39 0.76 0.73 0.67 0.85 0.50 0.38 1.37 2.57 0.98 0.56 1.12 1.98 1.31 0.86

2.06 2.27 2.02 2.44 2.43 2.74 2.09 1.83 1.81 1.88 2.24 1.70 2.34 1.76 1.72 1.69 1.68 1.78 2.43 2.36 1.36 1.33 1.26 1.47 1.75 1.79 1.41 1.90 1.57 1.48

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b∗ 0.16 0.57 0.14 0.41 0.30 0.26 0.33 0.09 0.46 0.21 0.34 0.31 0.82 0.19 0.23 0.11 0.49 0.16 0.35 0.59 0.50 0.84 0.50 0.39 0.39 0.17 0.30 0.41 0.42 0.46

10.18 10.80 10.40 10.18 9.85 10.93 10.70 9.70 9.17 9.61 10.43 9.69 9.88 9.35 11.00 9.30 10.08 9.46 10.31 10.48 9.66 10.68 9.45 9.00 9.88 10.44 9.23 8.93 9.97 9.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.40 0.70 0.31 1.03 0.92 0.71 0.54 1.03 0.79 0.77 0.97 1.03 0.49 0.61 0.78 0.39 0.50 0.30 0.50 1.15 0.31 0.39 0.90 1.87 0.93 0.65 1.04 0.66 0.98 1.04

1 n = 5 replicates per treatment per time. Values (means ± standard error of the mean). Within the same column at the same time, none of the treatments were significantly different with the controls (BPD or 4% Tween-80) or baseline (P > 0.05).

enhance the antimicrobial efficacy of CR against C. jejuni on chicken skin samples vs. the suspension alone (Table 2). In fact, for several CR suspension treatments, the anti-Campylobacter efficacy was found to be better in suspension than emulsion or nanoemulsion formulations. One potential possiblity is the formation of transient, small droplets of CR (with increased surface area and efficacy) in the CR suspension while the treatments are vigrously shaken at washing step. This could increase anti-Campylobacter efficacy of CR suspension. C. jejuni was not detected in the CR wash treatements indicating that the wash treatments would not lead to environmental contamination with C. jejuni. The type of microorganisms that colonize fresh meat products depend highly on the characteristics of meat, processing methods and storage conditions (Huis in’t Veld, 1996). In addtion to the better antiCampylobacter activity of CR suspension over emulsion or nanoemulsion, we observed the effects of CR suspension is promising against aerobic bacteria when compared with other 2 forms of CR (Table 3). The color of poultry carcasses and poultry meat products is one of the important attributes of product quality because it is directly related to the purchasing decision of consumers. The lightness value, L∗ ,

represents the darkest black at L∗ = 0, and the brightest white at L∗ = 100. The –a∗ to +a∗ and –b∗ to +b∗ represent green-red and blue-yellow, respectively (Pathare et al., 2013). We did not observe a significant change (P > 0.05) in the color values (L∗ , a∗ , and b∗ ) either at 0 or 24 h after washing with CR suspension, emulsion, or nanoemulsion treatments when compared with the controls (BPD or Tween-80) or baseline (Table 4). These results indicate that CR treatments could be used as a potential wash treatment to reduce the load of C. jejuni and meat spoilage bacteria without changing chicken skin color. In conclusion, CR suspension, emulsion, and nanoemulsion as a wash treatment produced a consistent reduction in C. jejuni counts on chicken skin. The reduction (by ∼2.4 to 4 log10 cfu/sample) by the highest dose of CR suspension is promising since a 2-log10 reduction of Campylobacter on the poultry carcass could reduce the risk of human campylobacteriosis by up to 30-fold (Rosenquist et al., 2003). In addition, CR suspension reduced aerobic bacteria counts without changing the color of chicken skin. Thus, CR treatments could be a good alternative of conventional chemicals to reduce the Campylobacter and meat spoilage bacteria on the chicken carcasses; however, follow-up investigations testing the effects of CR

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Emulsion

L∗

CAVACROL WASH REDUCES C. JEJUNI

should be conducted in an industrial setting prior to implementing these treatments in the poultry processing plant.

ACKNOWLEDGMENTS

REFERENCES Abd-Elsalam, K. A., and A. R. Khokhlov. 2015. Eugenol oil nanoemulsion: antifungal activity against Fusarium oxysporum f. sp. vasinfectum and phytotoxicity on cottonseeds. Appl. Nanosci. 5:255–265. Aeschbach, R., J. L¨ oliger, B. Scott, A. Murcia, J. Butler, B. Halliwell, and O. Aruoma. 1994. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem. Toxicol. 32:31–36. Alonso-Hernando, A., J. Guevara-Franco, C. Alonso-Calleja, and R. Capita. 2013. Effect of the temperature of the dipping solution on the antimicrobial effectiveness of various chemical decontaminants against pathogenic and spoilage bacteria on poultry. J. Food Prot. 76:833–842. Arbor, A. 2008. Nanoemulsion vaccines show increasing promise. Oilbased nasal vaccine technique produces immunity against smallpox, HIV. AIDS Res. Hum. Retroviruses 24:1–9. Arrebola, M. L., M. C. Navarro, J. Jim´enez, and F. A. Oca˜ na. 1994. Yield and composition of the essential oil of Thymus serpylloides subsp. serpylloides. Phytochemistry 36:67–72. Arsi, K., A. Donoghue, K. Venkitanarayanan, A. Kollanoor-Johny, A. Fanatico, P. Blore, and D. Donoghue. 2014. The efficacy of the natural plant extracts, thymol and carvacrol against Campylobacter colonization in broiler chickens. J. Food Saf. 34:321–325. Arunasree, K. 2010. Anti-proliferative effects of carvacrol on a human metastatic breast cancer cell line, MDA-MB 231. Phytomedicine. 17:581–588. Bauermeister, L. J., J. W. Bowers, J. C. Townsend, and S. R. McKee. 2008. Validating the efficacy of peracetic acid mixture as an antimicrobial in poultry chillers. J. Food Prot. 71:1119–1122. Bhargava, K., D. S. Conti, S. R. P. da Rocha, and Y. Zhang. 2015. Application of an oregano oil nanoemulsion to the control of foodborne bacteria on fresh lettuce. Food Microbiol. 47:69–73. Bilgili, S., D. Conner, J. Pinion, and K. Tamblyn. 1998. Broiler skin color as affected by organic acids: influence of concentration and method of application. Poult. Sci. 77:752–757. Birk, T., A. C. Grønlund, B. B. Christensen, S. Knøchel, K. Lohse, and H. Rosenquist. 2010. Effect of organic acids and marination ingredients on the survival of Campylobacter jejuni on meat. J. Food Prot. 73:258–265. Blaser, M. J., D. N. Taylor, and R. A. Feldman. 1983. Epidemiology of Campylobacter jejuni infections. Epidemiol. Rev. 5:157–176. Byrd, J. A., B. M. Hargis, D. J. Caldwell, R. H. Bailey, K. L. Herron, J. L. McReynolds, R. L. Brewer, R. C. Anderson, K. M. Bischoff, T. R. Callaway, and L. F. Kubena. 2001. Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter Contamination of Broilers. Poult. Sci. 80:278–283. Centers for Disease Control and Prevention (CDC). 2017. Campylobacter. Questions and Answers. Available at https://www.cdc. gov/campylobacter/faq.html. Accessed 6 Nov. 2017. Chang, Y., L. McLandsborough, and D. J. McClements. 2013. Physicochemical properties and antimicrobial efficacy of carvacrol nanoemulsions formed by spontaneous emulsification. J. Agric. Food Chem. 61:8906–8913. Char, C., L. Cisternas, F. P´erez, and S. Guerrero. 2016. Effect of emulsification on the antimicrobial activity of carvacrol. CyTA 14:186–192.

Chavan, P. S., and S. G. Tupe. 2014. Antifungal activity and mechanism of action of carvacrol and thymol against vineyard and wine spoilage yeasts. Food Control 46:115–120. Chouliara, E., A. Karatapanis, I. Savvaidis, and M. Kontominas. 2007. Combined effect of oregano essential oil and modified atmosphere packaging on shelf-life extension of fresh chicken breast meat, stored at 4◦ C. Food Microbiol. 24:607–617. Cole, K., M. B. Farnell, A. M. Donoghue, N. J. Stern, E. A. Svetoch, B. N. Eruslanov, L. I. Volodina, Y. N. Kovalev, V. V. Perelygin, E. V. Mitsevich, and I. P. Mitsevich. 2006. Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults. Poult. Sci. 85:1570–1575. Corry, J. E. L., and H. I. Atabay. 2001. Poultry as a source of Campylobacter and related organisms. J. Appl. Microbiol. 90:96S–114S. Corry, J. E., S. J. James, G. Purnell, C. S. Barbedo-Pinto, Y. Chochois, M. Howell, and C. James. 2007. Surface pasteurisation of chicken carcasses using hot water. J. Food Eng. 79:913–919. Cox, N., A. Mercuri, J. Thomson, and D. Gregory. 1974. Quality of broiler carcasses as affected by hot water treatments. Poult. Sci. 53:1566–1571. Cui, S., B. Ge, J. Zheng, and J. Meng. 2005. Prevalence and antimicrobial resistance of Campylobacter spp. and Salmonella serovars in organic chickens from Maryland retail stores. Appl. Environ. Microbiol. 71:4108–4111. Dawson, L., R. Walker, M. Zabik, D. Bigbee, and W. Mallmann. 1963. Influence of surface pasteurization and chlortetracycline on bacterial incidence on fyers. Food Technol. 17:218. Dons`ı, F., M. Annunziata, M. Sessa, and G. Ferrari. 2011. Nanoencapsulation of essential oils to enhance their antimicrobial activity in foods. LWT. 44:1908–1914. Dons`ı, F., M. Annunziata, M. Vincensi, and G. Ferrari. 2012. Design of nanoemulsion-based delivery systems of natural antimicrobials: effect of the emulsifier. J. Biotechnol. 159:342–350. Elvers, K. T., V. K. Morris, D. G. Newell, and V. M. Allen. 2011. Molecular tracking, through processing, of Campylobacter strains colonizing broiler flocks. Appl. Environ. Microbiol. 77:5722–5729. Evans, S. J. 1991. Introduction and spread of thermophilic Campylobacters in broiler flocks. Vet. Rec. 131:574–576. Ghosh, V., A. Mukherjee, and N. Chandrasekaran. 2013. Ultrasonic emulsification of food-grade nanoemulsion formulation and evaluation of its bactericidal activity. Ultrason. Sonochem. 20:338–344. Gill, C. O., and G. G. Greer, 1993. Enumeration and Identification of Meat Spoilage Bacteria. Technical Bulletin 1993–8E. Research Branch, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada. Gradel, K. O., H. L. Nielsen, H. C. Schønheyder, T. Ejlertsen, B. Kristensen, and H. Nielsen. 2009. Increased short-and long-term risk of inflammatory bowel disease after Salmonella or Campylobacter gastroenteritis. Gastroenterology. 137:495–501. Guyard-Nicodeme, M., A. Keita, S. Queens, M. Amelot, T. Poezevara, B. Le Berre, J. S´ anchez, P. Vesseur, A. Mart´ın, P. Medel, and M. Chemaly. 2016. Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period. Poult. Sci. 95:298–305. Hoffmann, S. A., B. Maculloch, and M. Batz. 2015. Economic burden of major foodborne illnesses acquired in the United States (No. 205081). United States Department of Agriculture. https://www.ers.usda.gov/webdocs/publications/43984/52807 eib140.pdf. Accessed 31 Oct. 2018. Huis in’t Veld, J. H. 1996. Microbial and biochemical spoilage of foods: an overview. Int. J. Food Microbiol. 33:1–18. Huneau-Sala¨ un, A., M. Guyard-Nicod`eme, G. Benzoni, X. Gautier, S. Quesne, T. Po¨ezevara, and M. Chemaly. 2018. Randomized control trial to test the effect of a feed additive on Campylobacter contamination in commercial broiler flocks up to slaughter. Zoonoses Public Health. 65:404–411. James, C., S. J. James, N. Hannay, G. Purnell, C. Barbedo-Pinto, H. Yaman, M. Araujo, M. L. Gonzalez, J. Calvo, and M. Howell. 2007. Decontamination of poultry carcasses using steam or hot water in combination with rapid cooling, chilling or freezing of carcass surfaces. Int. J. Food Microbiol. 114:195–203. Kabara, J. 1991. Phenols and chelators. Pages 200–214 in Food Preservatives. N. J. Russell, and G. W. Gould, eds. Blackie, London.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

This research was funded in part by the US Department of Agriculture, National Institue of Food and Agriculture, Organic Agriculture Research and Extension Initiative, 2017-51300-26815.

9

10

SHRESTHA ET AL. Park, H., Y. C. Hung, and R. E. Brackett. 2002. Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing. Int. J. Food Microbiol. 72:77–83. Pathare, P. B., U. L. Opara, and F. A. J. Al-Said. 2013. Colour measurement and analysis in fresh and processed foods: a review. Food Bioprocess Tech. 6:36–60. Patterson, M. 1995. Sensitivity of Campylobacter spp. to irradiation in poultry meat. Lett. Appl. Microbiol. 20:338–340. Ramos, M., A. Beltr´ an, M. Peltzer, A. J. Valente, and M. del Carmen Garrig´ os. 2014. Release and antioxidant activity of carvacrol and thymol from polypropylene active packaging films. LWT 58:470– 477. Ravishankar, S., L. Zhu, J. Reyna-Granados, B. Law, L. Joens, and M. Friedman. 2010. Carvacrol and cinnamaldehyde inactivate antibiotic-resistant Salmonella enterica in buffer and on celery and oysters. J. Food Prot. 73:234–240. Riedel, C. T., L. Brøndsted, H. Rosenquist, S. N. Haxgart, and B. B. Christensen. 2009. Chemical decontamination of Campylobacter jejuni on chicken skin and meat. J. Food Prot. 72:1173– 1180. Rosenquist, H., N. L. Nielsen, H. M. Sommer, B. Nørrung, and B. B. Christensen. 2003. Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens. Int. J. Food Microbiol. 83:87–103. Shafiq, S., and F. Shakeel. 2010. Stability and self-nanoemulsification efficiency of ramipril nanoemulsion containing labrasol and plurol oleique. Clin. Res. Regul. Affairs. 27:7–12. Sharma, H., S. K. Mendiratta, R. K. Agrawal, K. Gurunathan, S. Kumar, and T. P. Singh. 2017. Use of various essential oils as bio preservatives and their effect on the quality of vacuum packaged fresh chicken sausages under frozen conditions. LWT 81:118– 127. Shrestha, S., K. Arsi, B. R. Wagle, A. M. Donoghue, and D. J. Donoghue. 2017. Ability of select probiotics to reduce enteric Campylobacter colonization in broiler chickens. Int. J. Poult. Sci. 16:37–42. Smith-Palmer, A., J. Stewart, and L. Fyfe. 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26:118– 122. Solans, C., P. Izquierdo, J. Nolla, N. Azemar, and M. J. GarciaCelma. 2005. Nano-emulsions. Curr. Opin. Colloid Interface Sci. 10:102–110. Speranza, P., A. P. B. Ribeiro, R. L. Cunha, J. A. Macedo, and G. A. Macedo. 2015. Influence of emulsion droplet size on antimicrobial activity of interesterified Amazonian oils. LWT 60:207–212. Spiller, R. C. 2007. Role of infection in irritable bowel syndrome. J. Gastroenterol. 42:41–47. Stern, N. J., P. Fedorka-Cray, J. S. Bailey, N. A. Cox, S. E. Craven, K. L. Hiett, M. T. Musgrove, S. Ladely, D. Cosby, and G. C. Mead. 2001. Distribution of Campylobacter spp. in selected US poultry production and processing operations. J. Food Prot. 64:1705–1710. Tan, S. M., S. M. Lee, and G. A. Dykes. 2014. Fat contributes to the buffering capacity of chicken skin and meat but enhances the vulnerability of attached Salmonella cells to acetic acid treatment. Food Res. Int. 66:417–423. Thormar, H., H. Hilmarsson, J. Thrainsson, F. Georgsson, E. Gunnarsson, and S. Dadadottir. 2011. Treatment of fresh poultry carcases with emulsions of glycerol monocaprate (monocaprin) to reduce contamination with Campylobacter and psychrotrophic bacteria. Br. Poult. Sci. 52:11–19. Ultee, A., R. Slump, G. Steging, and E. Smid. 2000. Antimicrobial activity of carvacrol toward Bacillus cereus on rice. J. Food Prot. 63:620–624. Upadhyay, A., I. Upadhyaya, S. Mooyottu, A. Kollanoor-Johny, and K. Venkitanarayanan. 2014. Efficacy of plant-derived compounds combined with hydrogen peroxide as antimicrobial wash and coating treatment for reducing Listeria monocytogenes on cantaloupes. Food Microbiol. 44:47–53. USDA and Foreign Agricultural Service (USDA-FAS). 2018. Livestock and poultry: world markets and trade. Available at https://apps.fas.usda.gov/psdonline/circulars/livestock poultry. pdf. Accessed 31 Oct. 2018.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

Karabagias, I., A. Badeka, and M. Kontominas. 2011. Shelf life extension of lamb meat using thyme or oregano essential oils and modified atmosphere packaging. Meat Sci. 88:109–116. Kollanoor Johny, A., M. Darre, A. Donoghue, D. Donoghue, and K. Venkitanarayanan. 2010. Antibacterial effect of transcinnamaldehyde, eugenol, carvacrol, and thymol on Salmonella Enteritidis and Campylobacter jejuni in chicken cecal contents in vitro. J. Appl. Poult. Res. 19:237–244. Kramer, J. M., J. A. Frost, F. J. Bolton, and D. R. Wareing. 2000. Campylobacter contamination of raw meat and poultry at retail sale: identification of multiple types and comparison with isolates from human infection. J. Food Prot. 63:1654– 1659. Landry, K. S., S. Micheli, D. J. McClements, and L. McLandsborough. 2015. Effectiveness of a spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli ˆ contaminated broccoli and radish seeds. Food MiO157:H7 onA crobiol. 51:10–17. Landry, K. S., Y. Chang, D. J. McClements, and L. McLandsborough. 2014. Effectiveness of a novel spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli O157:H7 on contaminated mung bean and alfalfa seeds. Int. J. Food Microbiol. 187:15–21. Lima, I. O., F. D. O. Pereira, W. A. D. Oliveira, E. D. O. Lima, E. A. Menezes, F. A. Cunha, and M. D. F. F. M. Diniz. 2013. Antifungal activity and mode of action of carvacrol against Candida albicans strains. J. Essent. Oil Res. 25:138–142. Line, J. E. 2001. Development of a selective differential agar for isolation and enumeration of Campylobacter spp. J. Food Prot. 64:1711–1715. Loretz, M., R. Stephan, and C. Zweifel. 2010. Antimicrobial activity of decontamination treatments for poultry carcasses: a literature survey. Food Control 21:791–804. Luo, Y., J. Y. Wu, M. H. Lu, Z. Shi, N. Na, and J. M. Di. 2016. Carvacrol alleviates prostate cancer cell proliferation, migration, and invasion through regulation of PI3K/Akt and MAPK signaling pathways. Oxid. Med. Cell. Longev. 2016:1–11 Marder, E. P., P. R. Cieslak, A. B. Cronquist, J. Dunn, S. Lathrop, T. Rabatsky-Ehr, P. Ryan, K. Smith, M. Tobin-D’Angelo, D. J. Vugia, and S. Zansky. 2017. Incidence and trends of infections with pathogens transmitted commonly through food and the effect of increasing use of culture-independent diagnostic tests on surveillance-foodborne diseases active surveillance network, 10 US Sites, 2013–2016. MMWR Morb. Mortal. Wkly. Rep. 66:397– 403. Mehdi, S. J., A. Ahmad, M. Irshad, N. Manzoor, and M. M. A. Rizvi. 2011. Cytotoxic effect of carvacrol on human cervical cancer cells. Biol. Med. 3:307–312. Mexis, S., E. Chouliara, and M. Kontominas. 2009. Combined effect of an oxygen absorber and oregano essential oil on shelf life extension of rainbow trout fillets stored at 4◦ C. Food Microbiol. 26:598–605. Moon, H., N. H. Kim, S. H. Kim, Y. Kim, J. H. Ryu, and M. S. Rhee. 2017. Teriyaki sauce with carvacrol or thymol effectively controls Escherichia coli O157: H7, Listeria monocytogenes, Salmonella Typhimurium, and indigenous flora in marinated beef and marinade. Meat Sci. 129:147–152. Musavian, H. S., N. H. Krebs, U. Nonboe, J. E. Corry, and G. Purnell. 2014. Combined steam and ultrasound treatment of broilers at slaughter: A promising intervention to significantly reduce numbers of naturally occurring Campylobacters on carcasses. Int. J. Food Microbiol. 176:23–28. Nagel, G. M., L. J. Bauermeister, C. L. Bratcher, M. Singh, and S. R. McKee. 2013. Salmonella and Campylobacter reduction and quality characteristics of poultry carcasses treated with various antimicrobials in a post-chill immersion tank. Int. J. Food Microbiol. 165:281–286. Nauta, M. J., W. F. Jacobs-Reitsma, and A. H. Havelaar. 2007. A risk assessment model for Campylobacter in broiler meat. Risk Anal. 27:845–861. Ostertag, F., J. Weiss, and D. J. McClements. 2012. Low-energy formation of edible nanoemulsions: factors influencing droplet size produced by emulsion phase inversion. J. Colloid Interface Sci. 388:95–102.

CAVACROL WASH REDUCES C. JEJUNI

Whyte, P., K. McGill, and J. Collins. 2003. An assessment of steam pasteurization and hot water immersion treatments for the microbiological decontamination of broiler carcasses. Food Microbiol. 20:111–117. Williams, A., and O. A. Oyarzabal. 2012. Prevalence of Campylobacter spp. in skinless, boneless retail broiler meat from 2005 through 2011 in Alabama, USA. BMC Microbiol. 12:P184. Windhab, E. J., M. Dressler, K. Feigl, P. Fischer, and D. MegiasAlguacil. 2005. Emulsion processing—from single-drop deformation to design of complex processes and products. Chem. Eng. Sci. 60:2101–2113. Xu, J., F. Zhou, B. P. Ji, R. S. Pei, and N. Xu. 2008. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Lett. Appl. Microbiol. 47:174–179. Yadav, S., V. Sharma, A. Prasad, S. Ganapathy, and S. Vij. 2014. Comparative evaluation of antimicrobial effects of a new root canal irrigant carvacrol with sodium hypochlorite. Indian J. Health Sci. Care 1:13–17. Zainol, S., M. Basri, H. B. Basri, A. F. Shamsuddin, S. S. AbdulGani, R. A. Karjiban, and E. Abdul-Malek. 2012. Formulation optimization of a palm-based nanoemulsion system containing levodopa. IJMS 13:13049–13064 Zhao, T., and M. P. Doyle. 2006. Reduction of Campylobacter jejuni on chicken wings by chemical treatments. J. Food Prot. 69:762– 767.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez198/5471362 by guest on 23 April 2019

USDA FSIS. 2017. Table of safe and suitable ingredients: antimicrobial update. Available at https://www.fsis.usda.gov/ wps/wcm/connect/24346cbd-ad28-4223-8db1-55f067ce3879/ 7120.1-Antimicrobials.pdf?MOD=AJPERES. Accessed 31 Oct. 2018. Wagenaar, J. A., D. G. Newell, R. S. Kalupahana, and L. MughiniGras. 2015. Campylobacter: animal reservoirs, human infections, and options for control. Pages 159–177 in Zoonoses— Infections Affecting Humans and Animals: Focus on Public Health Aspects. A., and Sing, ed. Springer, Dordrecht, The Netherlands. Wagle, B. R., A. Upadhyay, K. Arsi, S. Shrestha, K. Venkitanarayanan, A. M. Donoghue, and D. J. Donoghue. 2017. Application of β -resorcylic acid as potential antimicrobial feed additive to reduce Campylobacter colonization in broiler chickens. Front. Microbiol. 8:599. Weiss, J., S. Gaysinsky, M. Davidson, and J. McClements. 2009. Nanostructured encapsulation systems: food antimicrobials. Pages 425–479 in Global Issues in Food Science and Technology. Academic Press, Burlington, MA, USA. Wempe, J. M., C. A. Genigeorgis, T. B. Farver, and H. I. Yusufu. 1983. Prevalence of Campylobacter jejuni in two California chicken processing plants. Appl. Environ. Microbiol. 45:355–359. WHO. 2017. Campylobacter. Available at http://www.who.int/ mediacentre/factsheets/fs255/en/. Accessed 6 Nov. 2017.

11