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Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. Coli O157:H7, feline calicivirus and endogenous microbiota... ARTICLE in INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY · JULY 2013 Impact Factor: 3.08 · DOI: 10.1016/j.ijfoodmicro.2013.07.020 · Source: PubMed
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Author's personal copy International Journal of Food Microbiology 166 (2013) 249–255
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Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads Maryam Azizkhani a,1, Patricia Elizaquível b,1, Gloria Sánchez c, María Victoria Selma d, Rosa Aznar b,c,⁎ a
Department of Food Hygiene, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran Departamento de Microbiología y Ecología, Universitat de València. Av. Dr. Moliner, 50. 46100, Burjassot. Valencia, Spain c Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA, CSIC). Av. Agustín Escardino, 7. 46980 Paterna, Valencia, Spain d Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain b
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 16 July 2013 Accepted 19 July 2013 Available online 27 July 2013 Keywords: Origanum compactum Eugenia caryophyllus Zataria multiflora Feline calicivirus E. coli O157:H7 Fresh-cut vegetables
a b s t r a c t Ready-to-eat salads using baby-leaf and multi-leaf mixes are one of the most promising developments in the fresh-cut food industry. There is great interest in developing novel decontamination treatments, which are both safe for consumers and more efficient against foodborne pathogens. In this study, emulsions of essential oils (EOs) from Origanum compactum (oregano), Eugenia caryophyllus (clove), and Zataria multiflora Boiss (zataria) were applied by spray (0.8 ml) after the sanitizing washing step. The aim was to investigate their ability to control the growth of potentially cross-contaminating pathogens and endogenous microbiota in commercial baby leaves, processed in a fresh-cut produce company. Zataria EO emulsions of 3%, 5% and 10% reduced Escherichia coli O157:H7 by 1.7, 2.2 and 3.5 log cfu/g in baby-leaf salads after 5 days of storage at 7 °C. By contrast, reductions in E. coli O157:H7 counts remained the same when clove was applied at concentrations of 5% and 10% (2.5 log cfu/g reduction). Oregano (10%) reduced inoculated E. coli O157:H7 counts in baby-leaf salads by a maximum of 0.5 log cfu/g after 5 days of storage. Zataria showed strong antimicrobial efficacy against E. coli O157:H7 and also against the endogenous microbiota of baby-leaf salads stored for 9 days. Feline calicivirus (FCV), a norovirus surrogate, survived on inoculated baby-leaf salads during refrigerated storage (9 days at 7 °C) regardless of treatment. Refrigeration temperatures completely annulled the effectiveness of the EOs against FCV inoculated in baby-leaf salads as occurred in FCV cultures. This study shows that EOs, and zataria in particular, have great potential use as an additional barrier to reduce contamination-related risks in baby-leaf salads. However, further research should be done into foodborne viruses in order to improve food safety. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lettuce is one of the most important ready-to-use products and ranks highly among vegetables both in production and economic value. The world production of salad and especially the production of lettuce and chicory (the two plants being combined by the FAO for reporting purposes) was 23,622,366 metric tons (FAO, 2012) for calendar year 2010. Although prepared salads consist mainly of iceberg lettuce, other types of lettuce, with attractive colors and shapes, are used in salad mixes called “mesclun” in France or “spring mix” in the U.S. (Martinez-Sanchez et al., 2012). As consumers are looking for softer textures, baby-sized leaves using baby-leaf at immature stage and multi-leaf at mature stage have proven one of the most promising
⁎ Corresponding author at: Departamento de Biotecnología. Instituto de Agroquímica y Tecnología de Alimentos. Avda. Agustín Escardino, 7. Paterna, Spain. Tel.: +34 96 3900022; fax: +34 96 3939301. E-mail address:
[email protected] (R. Aznar). 1 These authors contributed equally to this work and are considered joint first authors. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.07.020
fresh-cut developments (Martinez-Sanchez et al., 2012). These new salads represent a new challenge to the food industry, because differences in the morphology and maturity stages of the numerous types of leafy vegetables that compose them affect sanitizing efficacy. The increase in fresh produce consumption has led to a higher incidence of foodborne illnesses since these vegetables are eaten raw (Warriner et al., 2009). Among the foodborne pathogens related to fresh produce such as spinach, lettuce, alfalfa sprouts and mixed salads, Escherichia coli O157 has been identified as the source of around the 21% of outbreaks (Olaimat and Holley, 2012; Rangel et al., 2005). Enteric viruses, and particularly norovirus, form another group of pathogens that are closely related to fresh produce (Sivapalasingam et al., 2004). Moreover, noroviruses are the most common cause of foodborne illness and have been listed in the top 5 pathogens in a cost-related ranking of foodborne illness in the United States (Scharff, 2012). Foods may be contaminated by contact with human fecal samples in the field or by unhygienic manipulation by a food handler infected by virus. Most of the literature available on the decontamination of fresh vegetables has concluded that sanitizing washing processes reduce the
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endogenous microbial populations on the surface of the produce by only 2 to 3 log cfu/g (Gil et al., 2009). Furthermore, despite initial differences in bacterial load, after storage total counts are similar whether the produce is washed with tap water or a sanitizing solution. Therefore, sanitizing agents are useful to maintain water quality and prevent cross-contamination during washing; however, they have scarce or no efficacy in preventing microbial growth during product storage as they do not have a residual effect (Gil et al., 2009). This fact prompts an interest in the use of natural and safe compounds with an antimicrobial effect to be added to processed fruits and vegetables during storage. Essential oils (EOs) have long been applied as flavoring agents to foods such as meat and have shown a wide spectrum of antimicrobial activity on several foodborne pathogens and spoilage bacteria, both in vitro and in food matrices (Burt, 2004; Ponce et al., 2011). Composition and activity of some EOs such as oregano, clove, cinnamon, garlic, coriander, rosemary, mint, basil, and parsley have been reported in studies in vitro (Angioni et al., 2004; Elizaquivel et al., 2012; Karagözlü et al., 2011; Oussalah et al., 2007; Piskernik et al., 2011; Tsigarida et al., 2000) and demonstrate they exert antimicrobial effects against different foodborne pathogens. The antibacterial effect of some EOs on lettuce at mature stages, mainly iceberg and romaine varieties, has also been investigated (Karagözlü et al., 2011; Ponce et al., 2011; Yossa et al., 2013). All these studies examined endogenous or pathogenic bacteria reduction in fresh produce when EOs were used as sanitizing agents during the washing step, but only one of them studied other technological applications of the EOs (Ponce et al., 2011). Ponce et al. (2011) assayed spray, immersion and lactose capsule applications of tea tree, clove and rosemary EOs on processed romaine lettuce. Only EO spray application maintained an acceptable quality of the product throughout the entire storage period. In addition, there are other EOs which are traditionally used in some parts of the world, whose antimicrobial activity in foods has not been studied in detail. Zataria multiflora Boiss (Z. multiflora), belonging to the family Laminacae, is native to Iran, Pakistan and Afghanistan. This plant is traditionally used in foods, especially in yoghurt flavoring, as a stimulant, condiment, carminative and for treatment of pre-mature labor pains and rupture (Ali et al., 2000; Hosseinzadeh et al., 2000). This EO exhibits beneficial properties against respiratory tract infections and irritable bowel syndrome (Ali et al., 2000). Moreover, Z. multiflora antimicrobial activity has been demonstrated by in vitro experiments against fungi (Khosravi et al., 2012) and bacterial pathogens such as Staphylococcus aureus, Salmonella enterica or Listeria monocytogenes (Basti et al., 2007; Moradi et al., 2011) as well as norovirus surrogates such as feline calicivirus (FCV) (Elizaquivel et al., 2013a). However, the application and antimicrobial effect of Z. multiflora on fresh vegetables remains unexplored to date. In the present study, we have investigated the potential of the EOs from Origanum compactum (oregano), Eugenia caryophyllus (clove) and Z. multiflora Boiss. as antimicrobial biopreservatives when applied by spraying on commercial baby-leaf salads processed in a fresh-cut produce company. Their antimicrobial effect, 15 min after application and during the shelf-life, has been tested on the endogenous microbiota of baby-leaf salads, as well as on E. coli O157:H7 and FCV, a norovirus surrogate, artificially inoculated in the same product.
Table 1 Main compounds of the selected EOs. Plant species
Common name
Origin
Distilled part
Main compounds (%)
Origanum compactum
Oregano
Morocco
Flowering plant
Eugenia caryophyllus
Clove
Madagascar
Flower bud
Zataria multiflora Boiss
Zataria
Iran
Aerial parts
Carvacrol (46.88) Thymol (15.26) p-cymene (13.10) γ-terpinene (11.61) Eugenol (83.96) Eugenile acetate (10.75) β-caryophyllene (3.25) Carvacrol (71.12) γ-terpinene (7.34) α-pinene (4.26) Eucaliptol (3.37) Globulol (2.32) Others compounds (b1)
2 hours, using Clevenger-type apparatus as previously described (Basti et al., 2007). The obtained EO composition was determined by GC and GC-MS at the Institute of Medicinal Plants, Medical University of Tehran, Iran (Table 1) (Azizkhani et al., 2013). The main antimicrobial compound it contains is carvacrol (71.1%). Zataria EO was stored in airtight glass vials covered with aluminum foil at 4 °C, then prior to its application, zataria EO was resupended 1:10 v/v in 50% ethanol. 2.2. Bacterial strain, culture conditions and inoculum preparation Escherichia coli O157:H7 CECT 5947 (non-toxigenic) supplied by the Spanish Type Culture Collection (CECT) was used in this study. This strain is recommended for food safety control assays since gene stx2 (virulence factor) has been replaced with gene cat. A nalidixic acidresistant (NalR) E. coli O157:H7 strain was obtained by consecutive 24-h transfers of brain heart infusion (BHI, Merck, Darmstadt, Germany) cultures to BHI containing increasing concentrations of nalidixic acid (Nal) up to 100 mg/ml. Then NalR E. coli O157:H7 colonies were consecutively subcultured twice in 5 ml of BHI supplemented with nalidixic acid (NalR, 100 mg/ml) at 37 °C for 20 h. This NalR E. coli O157:H7 strain was routinely grown on tryptic soy broth (TSB, Merck) at 37 °C for 18 h, and enumerated by plate count on tryptic soy agar (TSA, Merck) under the same incubation conditions. Cultures for inoculation experiments were prepared by transferring 100 μl of the overnight culture to 10 ml of TSB with 100 μg/ml nalidixic acid, and incubated at 37 °C for 4 hours (ca. 108 cfu/ml). Thereafter, cultures were serially diluted in phosphate buffered saline (PBS) to obtain a final cell density of 106 cfu/ml. 2.3. Virus strain and cell line The cytopathogenic F9 strain of FCV (ATCC VR-782) was propagated and assayed in CRFK cells. Semi-purified stocks were subsequently produced from the same cells by centrifugation of infected cell lysates at 660 ×g for 30 min.
2. Materials and methods
2.4. Baby-leaf salad preparation
2.1. Essential oils
Mixed salad containing baby red and green Batavia, Lollo Rosso lettuce, spinach and arugula at a ratio of 1:1:1:1:0.3 were processed in a fresh-cut vegetable processing company (Verdifresh, Valencia, Spain). Leaves with defects, such as bruising or discoloration, were hand removed before washing. They were well mixed until homogeneous and then washed by immersion for 30–60 s in 100 mg/ml chlorine solution (NaOCl) adjusted to pH 6.5 with phosphoric acid, and using a redox potential of 650–750 mV. Leaves were drained for 30 s and then rinsed with a tap water shower for 30–60 s. Excess water was removed by centrifugation. Samples of 100 g were mechanically non-vacuum packed
Commercially available EOs from oregano and clove, supplied by Pronarôm International (Ghislenghien, Belgium), and zataria EO (produced in-house) were used in this study. The main antimicrobial compounds present in each of these EOs are shown in Table 1. Oregano and clove EOs were diluted in 70% ethanol according to the manufacturer’s instructions and stored at 4 °C before use. Zataria EO was obtained from Z. multiflora Boiss. collected in the Fars province (Iran). Air-dried aerial parts of the plant were subjected to steam distillation for
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2.7. Statistical analysis The two sets of experiments described above, in which EOs were applied to commercial baby-leaf salads, were carried out twice, each with triplicate samples per treatment and storage period. Analysis of variance (ANOVA) was performed with these data, followed by Tukey's test with a significance level of P ≤ 0.05, using SPSS 14.0 for Windows. 3. Results 3.1. Effect of EOs on the inactivation of E. coli O157:H7 and FCV in artificially inoculated baby-leaf salad Different concentrations of oregano, clove and zataria EOs (0.5%, 1%, 2%, 5%, 10%, 15% and 20%) were assayed in vitro against E. coli O157:H7 (data not shown). Emulsions of 5% and 10% oregano, 5% and 10% clove and 3%, 5% and 10% zataria were selected based on the obtained results and applied at a dose of 0.8 ml/25 g of vegetables. Therefore, the percentages of EOs applied to vegetables were 0.096% (v/w), 0.160% and 0.320% for 3%, 5% and 10%, respectively. The first set of inoculation experiments in commercial baby-leaf salads was carried out to determine the EO concentration required to inactivate inoculated E. coli O157:H7 (Fig. 1). Fifteen minutes after EO application, E. coli O157:H7 levels were not significantly reduced for most treatments. Only 10% clove and 10% oregano significantly (P b 0.001 and P b 0.01, respectively) decreased E. coli O157:H7 levels 15 min after application. On the other hand, after 5 days of storage at 7 °C, the E. coli O157:H7 population was significantly (P b 0.001) reduced in baby-leaf salads treated with clove (5% and 10% EO concentrations) and zataria (3%, 5% and 10% EO concentrations). Higher E. coli O157:H7 reduction was obtained at higher zataria EO concentrations. 7
2.6. Microbiological analysis
Inoculum 15 min after EO application After 5 day storage at 7 ºC
6 aa
a a a
a b
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a
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a a b
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Prior to the inoculation trials, vegetables were analyzed by culture methods to ensure the absence of E. coli O157:H7 and FCV. Baby-leaf salad (25 g) was added to 225 ml of buffered peptone water (BPW) in a sterile plastic bag with lateral filter (BagPage S 400, BagSystem, Interscience, St-Nom-la-Breteche, France) and homogenized for 15 s using a Pulsifier (Microgen Bioproducts, Surrey, UK). The resulting mixture was taken from the filter side and concentrated as previously described (Sanchez et al., 2012). Briefly, polyethylene glycol was added to 90 ml homogenate to obtain a final concentration of 10% and after gentle shaking for 1 h at 4 °C, samples were centrifuged for 30 min at 10,000 ×g. Pellets corresponding to the total homogenized sample were resuspended in 1 ml of PBS and aliquots were serially diluted for virus and E. coli O157:H7 enumeration. Enumeration of the endogenous bacterial groups investigated (total mesophiles, total psychrotrophs, total coliforms, lactic acid bacteria, yeast and moulds) and the inoculated pathogens (E. coli O157:H7 and FCV) was carried out after 0, 2, 5, 7 and 9 days of storage at 7 °C. Microorganisms were enumerated using the corresponding agar media and incubation temperatures: mesophiles in TSA at 28 °C for 24–48 h; psychrotrophes in TSA at 7 °C for 10 to 12 days; total coliforms in
%
For artificial inoculation experiments, 25 g portions of mixed babyleaf salad processed in the fresh-vegetable processing company were placed in sterile bags and four series were prepared as follows: A (inoculated with E. coli O157:H7 or FCV, sprayed with tap water instead of EO emulsion), B (inoculated with E. coli O157:H7 or FCV and sprayed with EO emulsion), C (control: non-inoculated, sprayed with tap water instead of EO emulsion), D (control: non-inoculated, sprayed with EO emulsion). Aliquots of 250 μl of E. coli O157:H7 NalR (ca. 1 × 105 cfu) and 100 μl of FCV (ca. 2 × 107 TCID50) were inoculated on the leaf surfaces of A and B sample series by spotting with a micropipette and maintained in a flow-chamber for 1 h. The EO emulsions (5% and 10% (v/v) oregano, 5% and 10% clove, 3%, 5% and 10% zataria) were prepared in tap water, subjected to shaking at 200 rpm on a rotary shaker for 30 min at 4 °C and immediately sprayed on the vegetable leaves. Babyleaf samples (25 g) were sprayed with water (samples A and C) or EO emulsion (samples B and D) using a spray gun. Water or EO emulsions were always sprayed on the leaf surface in drops of 5 μl approximately, covering different leaf types and delivering a dose of 0.8 ml EO emulsion/25 g salad. Therefore, 0.024, 0.040 and 0.080 ml of EOs were applied to 25 g salads, respectively, when EO emulsions at 3%, 5% and 10% were used. After the vegetables had been sprayed, each bag was packed in a 30 μm thick, oriented polypropylene (OPP) bag, sealed with a heat sealer (Bifinett, Bochum, Germany) and shaken to facilitate EO dispersion. In order to determine the appropriate EO concentration for a commercial baby-leaf salad, the first set of experiments was carried out by enumeration of inoculated E. coli O157:H7 following spray application of 0.8 ml of 5% and 10% of clove, 5% and 10% of oregano and 3%, 5% and 10% of zataria, respectively, and stored for 5 days at 7 °C. In a second set of experiments, 0.8 ml of 10% of clove and 10% of zataria emulsions were sprayed on inoculated leaf salads, packed as described above, and stored for 9 days at 7 °C. Passive MAP was created by the respiration rate of the product and film permeability characteristics as non-vacuum packages were sealed. The quality of the packaged salads was evaluated for overall freshness of appearance by six trained panelists using a 9-point hedonic scale, where 9 = like extremely, 7 = like moderately, 5 = neither like nor dislike, 3 = dislike moderately, and 1 = dislike extremely (Luo et al., 2009).
10
2.5. Baby-leaf salad inoculation, EO application, packaging and product quality evaluation
MacConkey agar (Sharlab, Barcelona, Spain) at 37 °C for 24 h; lactic acid bacteria in MRS agar (Conda, Madrid, Spain) at 28 °C for 48 h; yeast and moulds in Yeast Glucose Broth (YGB) at 28 °C for 7 days. Escherichia coli O157:H7 was enumerated on CT-SMAC (Scharlab, Barcelona, Spain) supplemented with Nalidixic acid (Nal) (100 μg/ml) at 37 °C for 24 h. Replicates were analyzed in duplicate and microbiological counts were expressed as log cfu/g of tissue. Infectious FCV was enumerated on CRFK cells by determining the TCID50 with eight wells per dilution and 20 μl of inoculum per well.
or eg
and transported within 15 min under refrigerated conditions to the laboratory for EO studies.
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Fig. 1. Reduction of E. coli O157:H7, artificially inoculated on commercial baby-leaf salads, by decontamination with suspensions of Origanum compactum, Eugenia caryophyllus and Zataria multiflora EOs compared to water spray as a control treatment. Bars are the mean (n = 6) ± standard deviation. Different letters indicate significant differences.
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3.2. The effect of EOs on visual quality and the inactivation of endogenous microbiota in baby-leaf salad
Log cfu/g
On each sampling date of trial 3, the overall visual qualities of packaged fresh-cut products treated with 10% clove and zataria emulsions were assessed during 9 days of storage at 7 °C. Although the quality of the packaged salads declined during storage, it remained high for the first 7 days at 7 °C. Thus, products treated with 10% EO emulsions maintained acceptable quality characteristics throughout the 7-day storage period. On day 9, the quality of samples stored at 7 °C had
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deteriorated below the limit of acceptability; however, no negative effect of EO application was observed when compared to control samples. Enumeration of the endogenous microbial groups investigated in baby-leaf salads (total mesophilic bacteria, total psychrotrophic bacteria, total coliforms, lactic acid bacteria, yeast and moulds) was carried out initially and after 2, 5, 7 and 9 days of storage at 7 °C (Fig. 4). In non-EO-treated samples (control), mesophilic bacteria significantly increased during storage. In contrast, mesophiles decreased after zataria application compared to control samples (P b 0.05). After 2 days of storage, zataria efficacy in mesophilic bacteria reduction became more evident (2.5 log cfu/g lower than in control samples). After the first 2 days of storage, mesophilic bacteria started to grow in zataria-treated samples but differences (P b 0.05) with control samples were maintained throughout the storage period (Fig. 4). In contrast to zataria, the effect of clove on mesophilic bacteria was only significant at 7 and 9 days of storage, when bacterial levels were 1.1 ± 0.2 log cfu/g lower than those in the control samples. As occurred with mesophiles, psychrotrophic populations decreased in the presence of zataria during the first two days of storage whereas they increased in the presence of clove and in control samples (Fig. 4). Accordingly, psychrotrophic counts were 3.2 log cfu/g lower in zataria-treated samples than those in controls. After the first 2 days of storage, psychrotrophic populations increased in zataria-treated samples but the level was still significantly different (P b 0.05) to those in control samples, except at day 9 of storage. In contrast, clove EO
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Thus, zataria EO concentrations of 3%, 5% and 10% reduced E. coli O157: H7 levels by 1.7, 2.2 and 3.5 log cfu/g, respectively. In contrast, clove at 10% did not show higher E. coli O157:H7 reductions than when applied at a concentration of 5% (2.5 log cfu/g). Oregano application produced a significant effect (P b 0.01) on inoculated E. coli O157:H7, with a maximum of 0.5 log reductions after 5 days of storage of baby-leaf salads treated with 10% EO (Fig. 1). Oregano EO was also tested at higher concentrations (15% and 20%) on inoculated baby-leaf salads, but no improvements in E. coli O157:H7 reduction rates were observed with respect to lower concentrations (data not shown). Based on these results, 10% clove and zataria concentrations were selected for the second round of experiments. These experiments investigated the effect of clove and zataria EOs on E. coli O157:H7, FCV virus and the endogenous microbiota of baby-leaf salads during 9 days of storage at 7 °C. Oregano was not applied further during the long storage period experiment, due to its low efficacy (less than 1 log reduction) against E. coli O157:H7. Growth of inoculated E. coli O157:H7 in babyleaf samples sprayed only with water as control treatment was slow at 7 °C, and a statistically significant increase (1.1 ± 0.3 log cfu/g) was not observed until the ninth day of storage (Fig. 2). Conversely, a reduction of E. coli O157:H7 inoculated in baby-leaf salads was observed when clove and zataria EOs were applied. Maximum E. coli O157:H7 reductions were obtained when baby-leaf salads treated with zataria (3.5 log cfu/g reduction) and clove (2.8 log cfu/g reduction) were stored for 5 and 7 days, respectively (Fig. 2). After 7 days, E. coli O157:H7 started to grow in clove-treated samples, reaching levels 1.7 log cfu/g lower than in control samples at the end of storage. In contrast, E. coli O157:H7 levels in zataria-treated samples remained 3 log cfu/g lower than in control samples until the end of the storage period (Fig. 2). Feline calicivirus concentrations of 5.3 log TCID50/g were recorded in baby-leaf salads after inoculation (Fig. 3). In water-sprayed samples (control treatment) FCV levels declined by 1 log unit after 2 days of storage at 7 °C, and levels of 4.3 log TCID50/g were maintained until day 7 of storage. The last 2 days of storage, FCV infectivity was significantly reduced (P b 0.01) 0.3 log cfu/g. The two tested EOs, clove and zataria, did not have a significant effect on FCV infectivity, as similar FCV levels were obtained in EO-treated samples and controls (Fig. 3).
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Fig. 2. Survival of E. coli O157:H7 inoculated in commercial baby-leaf salads treated with Eugenia caryophyllus and Zataria multiflora EOs and stored at 7 °C for 9 days. Values are the mean (n = 6) ± standard deviation. Dotted vertical lines mark the commercial shelf-life.
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Days of storage
Fig. 4. Effect of Eugenia caryophyllus and Zataria multiflora EOs on different bacterial groups in commercial baby-leaf salads during storage at 7 °C for 9 days. Values are the mean (n = 6) ± standard deviation. Dotted vertical lines mark the commercial shelf-life.
treatment did not delay psychrotrophic growth, but reduced the maximum microbial density in the last 4 days of storage compared to the control samples. Consequently, psychrotrophs were significantly lower in clove-treated samples than in control samples stored for 7 and 9 days. Total coliforms significantly decreased 15 min after zataria and clove application (1.1 and 0.8 log cfu/g reductions) compared to control samples (P b 0.05). Both EOs maintained significantly lower coliform levels than control sample levels (P b 0.05) during the storage period (Fig. 4). In contrast to the other endogenous microbial groups investigated, lactic acid bacteria counts were not significantly different in samples treated with either zataria or clove as compared to control samples. A maximum lactic acid bacteria density of 4.0 log cfu/g was recorded in control baby-leaf salads after 7 days of storage, while it was 4.2 and 4.7 log cfu/g in samples treated with clove and zataria, respectively. No growth of yeast or moulds was observed on YGB plates from any of the samples, with or without EO. 4. Discussion Outbreaks of E. coli O157:H7 have been increasingly associated with the consumption of lettuce (Soderstrom et al., 2008) or spinach (Grant et al., 2008; Wendel et al., 2009). Observations on the ability of pathogenic bacteria to grow on refrigerated, fresh-cut salads are not new, with reports of E. coli O157:H7 growing on shredded lettuce and baby spinach stored at 8 °C (Luo et al., 2009; Posada-Izquierdo et al., 2013). The results of the present study on mixed salad (containing baby red and green Batavia, Lollo Rosso lettuce, spinach and arugula) have shown that E. coli O157:H7 is one of several pathogenic bacteria capable of growing on commercial baby-leaf salads stored at refrigeration temperature, i.e., 7 °C. Plant EOs are a potentially useful source of antimicrobial compounds for fresh produce preservation because a considerable number of EO components are GRAS. Results reported in other studies are difficult to compare, mainly due to differences in foods, technological application methods, bacterial strains and sources of antimicrobial compounds
(Jerkovic et al., 2001). Furthermore, in the fresh-cut industry, EO antimicrobial effects on novel fresh-cut developments, such as baby-leaf salads, as well as EO technological applications require further investigation. Our results on Zataria multiflora EO reveal it exerts a strong antibacterial activity against E. coli O157:H7 in culture medium (Elizaquivel et al., 2013b) and in baby-leaf salads. According to the chemical analysis of this and another study (Misaghi and Basti, 2007), the main active compounds in zataria EO are carvacrol (71.12%), γ-terpinene (7.34%) and α–terpinene (4.26%). Previous studies have reported its high efficiency against both Gram-positive and Gramnegative bacteria when tested in culture media (Basti et al., 2007; Saei-Dehkordi et al., 2010); and food models (Ekhtiarzadeh et al., 2012; Moradi et al., 2011). There are also studies reporting the bactericidal effect of low concentrations of Eugenia caryophillus (clove) EO, containing 83.96% of eugenol and 10.75% eugenile acetate (Leuschner and Lelsch, 2003; Oussalah et al., 2007). Similarly, in the present study, clove EO proved effective against E. coli O157:H7 and endogenous microbiota of baby-leaf salads. Origanum compactum (oregano) with 46.88% carvacrol and 15.26% thymol showed a strong bactericidal activity when applied to pure culture. However, oregano had a significant (P b 0.05) but low antimicrobial activity against E. coli O157:H7 (maximum of 0.5 log reductions) when applied to artificially inoculated baby-leaf salads. Although it is statistically significant, a reduction of 0.5 log cfu/g is generally not considered a significant reduction in food microbiology. Therefore, oregano EO did not prove effective in terms of practical application against E. coli O157:H7 in baby-leaf salads. It appears that the difference in antibacterial activities of EOs may be related to the concentration and nature of contents, to the respective composition, volatility, the functional groups, the structural configuration of the components and their possible synergistic interaction. Differences between EOs are due to ecological and plant growth factors (Chang et al., 2001). The major chemical constituents of these oils are essentially monoterpene phenolics and monoterpene hydrocarbons. Eugenol, carvacrol and thymol are phenolic compounds that have proven to be highly active against pathogenic bacteria. Other authors
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(Kim et al., 1995) observed that carvacrol was the most active constituent of 11 EOs tested, and the zataria EO extract used in the present study contained high concentrations of this constituent. The antiviral effect of EOs was previously determined by our team using a cell-culture assay at 4 and 37 °C (Elizaquivel et al., 2013a). Oregano EO (2%) decreased FCV titers by 3.75 log TCID50/ml at 37 °C, with no effect at 4 °C. The effects of clove and zataria EO on FCV incubated a 4 °C showed similar trends in titer reductions to those obtained with oregano EO, achieving the maximum titer reduction when FCV suspension was treated at 37 °C with 0.1% of zataria EO. Results of the present study showed that FCV can survive on baby-leaf salads during refrigerated storage (9 days at 7 °C). Moreover, as occurred in FCV suspensions, in FCV-inoculated baby-leaf salads, refrigeration temperature totally annulled the antiviral efficacy of EOs. This could be because the low temperature produced a conformational change in the capsid, reducing the accessibility of EOs to some viral regions. Several studies have evaluated the effectiveness of decontamination treatments against foodborne viruses in vegetable samples. Most of them have reported that these treatments do not guarantee the complete elimination/ inactivation of enteric viruses in food produce (Baert et al., 2009). In the last decade, natural compounds used as biocides in washing treatments have been evaluated mainly on norovirus surrogates. So far, an extent of antiviral activity on norovirus surrogate suspensions has been observed using EOs of oregano, zataria, and clove (Elizaquivel et al., 2013a); chitosan (Su et al., 2009); cranberry juice and cranberry proanthocyanidins (Su et al., 2010a); pomegranate juice and pomegranate polyphenols (Su et al., 2010b, 2011); black raspberry juice (Oh et al., 2012); and Korean red ginseng extract and ginsenosides (Lee et al., 2011). To date, only grape seed extract (GSE) has been evaluated on vegetable samples. This study reported that after 1 min of washing treatment, 0.25–1 mg/ml GSE caused a reduction of 2–3 log for FCV titers (Su and D'Souza, 2013). These results highlight the potential use of the combination of natural compounds and other treatments to reduce the presence of enteric viruses in food produce. Baby-leaf salads include a diverse microbiota dominated by various Gram-negative and also Gram-positive bacteria. Similar microbial counts obtained in TSA incubated at 7 and 30 °C from baby-leaf samples stored at 7 °C, indicated that mesophilic bacteria can be considered psychrotrophs. Zataria showed the highest inhibition of mesophilic population levels; meanwhile levels were the same for zataria and clove after 7 and 9 days storage. The phenolic compounds accounting for EO antimicrobial activity are lipophilic, and this quality confers a strong antibacterial capacity as it enables them to partition out of the water phase and be dissolved in the hydrophobic domain of the cytoplasmic membrane. This causes an increase in membrane permeability, which in turn dissipates the proton motive force, resulting in lethal damage to the bacterial cell (Burt, 2004). Refrigeration is often the main and, frequently, the only factor controlling the growth of foodborne pathogens in fresh produce. Fresh-cut produce processors often recommend that fresh-cut products be stored at 1 to 3 °C to maintain food quality. However, in fact, temperature abuse frequently occurs during fresh-cut product distribution and retail display (Luo et al., 2009). These abusive refrigeration temperatures, such as 7 °C, allow the growth of some pathogens such as E. coli O157: H7. There is a great interest in alternative safer and more efficient decontamination treatments against pathogens. In this study, EOs were applied to commercial baby leaves by spray after the sanitizing washing step, and subsequently investigated for their ability to control the growth of potentially cross-contaminating pathogens and endogenous microbiota. Our results have shown that EOs, especially zataria, have a great potential to be used as an additional barrier to reduce contamination-related risks in fresh produce. This study has shown that clove and zataria EOs exert antibacterial activity against E. coli O157:H7 inoculated in baby-leaf salads while different concentrations of zataria control bacterial growth. Also, the present study generated directly comparable, quantitative, antimicrobial data for EOs,
underpinning their potential as food preservatives. When spices and herbs are used as integral ingredients in prepared foods or added as flavoring agents, the active ingredients they contain are present in insufficient quantities to exert significant antimicrobial effects. By contrast, volatile oils, which often contain the principal aromatic and flavoring components of herbs and spices, can reduce bacterial contamination, even when added in small quantities to foodstuffs, without affecting organoleptic properties. The use of zataria for the preservation of fish has recently been reported (Choobkar et al., 2010). In a study carried out by Ekhtiarzadeh et al. (2012) fish fillets were treated with zataria and subsequent organoleptic evaluation showed that they were highly acceptable when zataria was applied at concentrations of 0.135% and 0.405% whereas an undesirable sensory effect was observed at 0.810%. Our study is the first in which zataria has been applied to vegetables and total concentrations of EO in produce (v/w %) were 0.096, 0.160 and 0.320% without negative effects on sensory properties. Therefore, further studies should be undertaken to determine the effective concentration of EOs against microorganisms in other foodstuffs, as well as the organoleptic impact of these concentrations. In addition to spoilage delay, improvement of sensory attributes of fresh-cut fruit and vegetables may also be interesting from a commercial point of view. In this respect, EOs not previously associated with a herby or spicy flavor, could be used in fresh-cut produce, looking for synergies between antibacterial effect and flavor. When good hygienic practices are followed throughout the production chain of fresh-cut vegetables, the use of EOs as preservatives can contribute to the improvement of food safety and food quality.
Acknowledgments This study was supported by grant AGL2009-08603 from the Spanish Ministry of Science and Innovation, ACOMP/2010/279 and ACOMP/ 2012/199 from the Generalitat Valenciana. G. Sánchez was the recipient of a JAE doctor grant from the “Consejo Superior de Investigaciones Científicas” (CSIC) and M. Azizkhani was supported by a grant from the Ministry of Sciences, Research and Technology of Iran. We thank the Institute of Medicinal Plants, Medical University of Tehran (Iran) for supplying the Z. multiflora essential oil. English text revised by F. Barraclough.
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