International Journal of Food Microbiology 166 (2013) 249–255
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International Journal of Food Microbiology
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Comparative efficacy of Zataria multiflora Boiss., Origanum compactumand Eugenia caryophyllus essential oils against E. coli O157:H7, felinecalicivirus 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, Iranb Departamento de Microbiología y Ecología, Universitat de València. Av. Dr. Moliner, 50. 46100, Burjassot. Valencia, Spainc Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA, CSIC). Av. Agustín Escardino, 7. 46980 Paterna, Valencia, Spaind Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain
⁎ Corresponding author at: Departamento de BiotecnolTecnología de Alimentos. Avda. Agustín Escardino, 7.3900022; fax: +34 96 3939301.
E-mail address: [email protected] (R. Aznar).1 These authors contributed equally to this work and ar
Ready-to-eat salads using baby-leaf and multi-leaf mixes are one of the most promising developments in thefresh-cut food industry. There is great interest in developing novel decontamination treatments, which areboth safe for consumers and more efficient against foodborne pathogens. In this study, emulsions of essentialoils (EOs) from Origanum compactum (oregano), Eugenia caryophyllus (clove), and Zataria multiflora Boiss(zataria)were applied by spray (0.8 ml) after the sanitizingwashing step. The aimwas to investigate their abilityto control the growth of potentially cross-contaminating pathogens and endogenous microbiota in commercialbaby leaves, processed in a fresh-cut produce company. Zataria EO emulsions of 3%, 5% and 10% reducedEscherichia coliO157: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. coliO157:H7 counts remained the samewhen clovewas applied at concentrations of 5% and 10%(2.5 log cfu/g reduction). Oregano (10%) reduced inoculated E. coliO157:H7 counts in baby-leaf salads by amax-imum of 0.5 log cfu/g after 5 days of storage. Zataria showed strong antimicrobial efficacy against E. coliO157:H7and also against the endogenous microbiota of baby-leaf salads stored for 9 days. Feline calicivirus (FCV), anorovirus surrogate, survived on inoculated baby-leaf salads during refrigerated storage (9 days at 7 °C) regard-less of treatment. Refrigeration temperatures completely annulled the effectiveness of the EOs against FCVinoculated 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.
Lettuce is one of the most important ready-to-use products andranks highly among vegetables both in production and economicvalue. The world production of salad and especially the production oflettuce and chicory (the two plants being combined by the FAO forreporting purposes) was 23,622,366 metric tons (FAO, 2012) for calen-dar year 2010. Although prepared salads consist mainly of iceberglettuce, other types of lettuce, with attractive colors and shapes, areused in salad mixes called “mesclun” in France or “spring mix” in theU.S. (Martinez-Sanchez et al., 2012). As consumers are looking for softertextures, baby-sized leaves using baby-leaf at immature stage andmulti-leaf at mature stage have proven one of the most promising
ogía. Instituto de Agroquímica yPaterna, Spain. Tel.: +34 96
e considered joint first authors.
ights reserved.
fresh-cut developments (Martinez-Sanchez et al., 2012). These newsalads represent a new challenge to the food industry, because differ-ences in the morphology and maturity stages of the numerous typesof leafy vegetables that compose them affect sanitizing efficacy. Theincrease in fresh produce consumption has led to a higher incidenceof foodborne illnesses since these vegetables are eaten raw (Warrineret al., 2009). Among the foodborne pathogens related to fresh producesuch as spinach, lettuce, alfalfa sprouts and mixed salads, Escherichiacoli O157 has been identified as the source of around the 21% of out-breaks (Olaimat and Holley, 2012; Rangel et al., 2005). Enteric viruses,and particularly norovirus, form another group of pathogens that areclosely related to fresh produce (Sivapalasingamet al., 2004).Moreover,noroviruses are the most common cause of foodborne illness and havebeen listed in the top 5 pathogens in a cost-related ranking of foodborneillness in the United States (Scharff, 2012). Foods may be contaminatedby contact with human fecal samples in the field or by unhygienicmanipulation by a food handler infected by virus.
Most of the literature available on the decontamination of freshvegetables has concluded that sanitizing washing processes reduce the
250 M. Azizkhani et al. / International Journal of Food Microbiology 166 (2013) 249–255
endogenousmicrobial populations on the surface of the produce by only2 to 3 log cfu/g (Gil et al., 2009). Furthermore, despite initial differencesin bacterial load, after storage total counts are similar whether theproduce is washed with tap water or a sanitizing solution. Therefore,sanitizing agents are useful to maintain water quality and preventcross-contamination during washing; however, they have scarce or noefficacy in preventing microbial growth during product storage asthey do not have a residual effect (Gil et al., 2009). This fact prompts aninterest in the use of natural and safe compounds with an antimicrobialeffect to be added to processed fruits and vegetables during storage.Essential oils (EOs) have long been applied as flavoring agents to foodssuch as meat and have shown a wide spectrum of antimicrobial activityon several foodborne pathogens and spoilage bacteria, both in vitro andin food matrices (Burt, 2004; Ponce et al., 2011). Composition and activ-ity of some EOs such as oregano, clove, cinnamon, garlic, coriander, rose-mary, 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) anddemonstrate they exert antimicrobial effects against different foodbornepathogens. The antibacterial effect of some EOs on lettuce at maturestages, mainly iceberg and romaine varieties, has also been investigated(Karagözlü et al., 2011; Ponce et al., 2011; Yossa et al., 2013). All thesestudies examined endogenous or pathogenic bacteria reduction in freshproduce when EOs were used as sanitizing agents during the washingstep, but only one of them studied other technological applications ofthe EOs (Ponce et al., 2011). Ponce et al. (2011) assayed spray, immersionand lactose capsule applications of tea tree, clove and rosemary EOs onprocessed romaine lettuce. Only EO spray application maintained anacceptable quality of the product throughout the entire storage period.In addition, there are other EOs which are traditionally used in someparts of the world, whose antimicrobial activity in foods has not beenstudied in detail. Zataria multiflora Boiss (Z. multiflora), belonging to thefamily Laminacae, is native to Iran, Pakistan and Afghanistan. This plantis traditionally used in foods, especially in yoghurt flavoring, as a stimu-lant, condiment, carminative and for treatment of pre-mature laborpains and rupture (Ali et al., 2000; Hosseinzadeh et al., 2000). This EOexhibits beneficial properties against respiratory tract infections andirritable bowel syndrome (Ali et al., 2000). Moreover, Z. multiflora anti-microbial activity has been demonstrated by in vitro experimentsagainst fungi (Khosravi et al., 2012) and bacterial pathogens such asStaphylococcus aureus, Salmonella enterica or Listeria monocytogenes(Basti et al., 2007; Moradi et al., 2011) as well as norovirus surrogatessuch as feline calicivirus (FCV) (Elizaquivel et al., 2013a). However, theapplication and antimicrobial effect of Z. multiflora on fresh vegetablesremains unexplored to date.
In the present study, we have investigated the potential of the EOsfrom Origanum compactum (oregano), Eugenia caryophyllus (clove) andZ. multiflora Boiss. as antimicrobial biopreservatives when applied byspraying on commercial baby-leaf salads processed in a fresh-cut pro-duce company. Their antimicrobial effect, 15 min after application andduring the shelf-life, has been tested on the endogenous microbiotaof baby-leaf salads, as well as on E. coli O157:H7 and FCV, a norovirussurrogate, artificially inoculated in the same product.
2. Materials and methods
2.1. Essential oils
Commercially available EOs from oregano and clove, supplied byPronarôm International (Ghislenghien, Belgium), and zataria EO (pro-duced in-house) were used in this study. The main antimicrobial com-pounds present in each of these EOs are shown in Table 1. Oregano andclove EOs were diluted in 70% ethanol according to the manufacturer’sinstructions and stored at 4 °C before use. Zataria EO was obtainedfrom Z. multiflora Boiss. collected in the Fars province (Iran). Air-driedaerial parts of the plant were subjected to steam distillation for
2 hours, using Clevenger-type apparatus as previously described(Basti et al., 2007). The obtained EO composition was determined byGC and GC-MS at the Institute of Medicinal Plants, Medical Universityof Tehran, Iran (Table 1) (Azizkhani et al., 2013). Themain antimicrobialcompound it contains is carvacrol (71.1%). Zataria EO was stored inairtight glass vials covered with aluminum foil at 4 °C, then prior to itsapplication, 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 theSpanish Type Culture Collection (CECT) was used in this study. Thisstrain is recommended for food safety control assays since gene stx2(virulence factor) has been replaced with gene cat. A nalidixic acid-resistant (NalR) E. coli O157:H7 strain was obtained by consecutive24-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. ThenNalR E. coliO157:H7 colonies were consec-utively subcultured twice in 5 ml of BHI supplemented with nalidixicacid (NalR, 100 mg/ml) at 37 °C for 20 h. This NalR E. coliO157:H7 strainwas 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) underthe same incubation conditions.
Cultures for inoculation experiments were prepared by transferring100 μl of the overnight culture to 10 ml of TSB with 100 μg/ml nalidixicacid, and incubated at 37 °C for 4 hours (ca. 108 cfu/ml). Thereafter,cultures were serially diluted in phosphate buffered saline (PBS) toobtain 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 propagatedand assayed in CRFK cells. Semi-purified stocks were subsequently pro-duced from the same cells by centrifugation of infected cell lysates at660 ×g for 30 min.
2.4. Baby-leaf salad preparation
Mixed salad containing baby red and green Batavia, Lollo Rossolettuce, spinach and arugula at a ratio of 1:1:1:1:0.3 were processed ina fresh-cut vegetable processing company (Verdifresh, Valencia, Spain).Leaves with defects, such as bruising or discoloration, were handremoved before washing. They were well mixed until homogeneousand thenwashed by immersion for 30–60 s in 100 mg/ml chlorine solu-tion (NaOCl) adjusted to pH 6.5 with phosphoric acid, and using a redoxpotential of 650–750 mV. Leaves were drained for 30 s and then rinsedwith a tapwater shower for 30–60 s. Excesswater was removed by cen-trifugation. Samples of 100 g were mechanically non-vacuum packed
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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 andZataria multiflora EOs compared to water spray as a control treatment. Bars are themean (n = 6) ± standard deviation. Different letters indicate significant differences.
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and transported within 15 min under refrigerated conditions to thelaboratory for EO studies.
2.5. Baby-leaf salad inoculation, EO application, packaging and productquality evaluation
For artificial inoculation experiments, 25 g portions of mixed baby-leaf salad processed in the fresh-vegetable processing company wereplaced in sterile bags and four series were prepared as follows: A(inoculated with E. coliO157:H7 or FCV, sprayedwith tap water insteadof EO emulsion), B (inoculated with E. coli O157:H7 or FCV and sprayedwith EO emulsion), C (control: non-inoculated, sprayed with tap waterinstead of EO emulsion), D (control: non-inoculated, sprayed with EOemulsion). 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 leafsurfaces of A and B sample series by spotting with a micropipette andmaintained 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 preparedin tap water, subjected to shaking at 200 rpm on a rotary shaker for30 min at 4 °C and immediately sprayed on the vegetable leaves. Baby-leaf samples (25 g) were sprayed with water (samples A and C) or EOemulsion (samples B and D) using a spray gun. Water or EO emulsionswere always sprayed on the leaf surface in drops of 5 μl approximately,covering different leaf types and delivering a dose of 0.8 ml EOemulsion/25 g salad. Therefore, 0.024, 0.040 and 0.080 ml of EOs wereapplied to 25 g salads, respectively, when EO emulsions at 3%, 5% and10% were used. After the vegetables had been sprayed, each bag waspacked in a 30 μm thick, oriented polypropylene (OPP) bag, sealedwith a heat sealer (Bifinett, Bochum, Germany) and shaken to facilitateEO dispersion. In order to determine the appropriate EO concentrationfor a commercial baby-leaf salad, the first set of experiments wascarried out by enumeration of inoculated E. coli O157:H7 followingspray application of 0.8 ml of 5% and 10% of clove, 5% and 10% oforegano and 3%, 5% and 10% of zataria, respectively, and stored for5 days at 7 °C. In a second set of experiments, 0.8 ml of 10% of cloveand 10% of zataria emulsions were sprayed on inoculated leaf salads,packed as described above, and stored for 9 days at 7 °C. Passive MAPwas created by the respiration rate of the product and film permeabilitycharacteristics as non-vacuum packages were sealed. The quality of thepackaged salads was evaluated for overall freshness of appearance bysix trained panelists using a 9-point hedonic scale, where 9 = like ex-tremely, 7 = like moderately, 5 = neither like nor dislike, 3 = dislikemoderately, and 1 = dislike extremely (Luo et al., 2009).
2.6. Microbiological analysis
Prior to the inoculation trials, vegetables were analyzed by culturemethods to ensure the absence of E. coli O157:H7 and FCV. Baby-leafsalad (25 g) was added to 225 ml of buffered peptone water (BPW) ina sterile plastic bag with lateral filter (BagPage S 400, BagSystem,Interscience, St-Nom-la-Breteche, France) and homogenized for 15 susing a Pulsifier (Microgen Bioproducts, Surrey, UK). The resultingmixture was taken from the filter side and concentrated as previouslydescribed (Sanchez et al., 2012). Briefly, polyethylene glycol wasadded to 90 ml homogenate to obtain a final concentration of 10% andafter gentle shaking for 1 h at 4 °C, samples were centrifuged for30 min at 10,000 ×g. Pellets corresponding to the total homogenizedsample were resuspended in 1 ml of PBS and aliquots were seriallydiluted for virus and E. coli O157:H7 enumeration.
Enumeration of the endogenous bacterial groups investigated (totalmesophiles, total psychrotrophs, total coliforms, lactic acid bacteria,yeast and moulds) and the inoculated pathogens (E. coli O157:H7 andFCV) was carried out after 0, 2, 5, 7 and 9 days of storage at 7 °C. Micro-organisms were enumerated using the corresponding agar media andincubation 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
MacConkey agar (Sharlab, Barcelona, Spain) at 37 °C for 24 h; lacticacid 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 microbio-logical counts were expressed as log cfu/g of tissue. Infectious FCVwas enumerated on CRFK cells by determining the TCID50 with eightwells per dilution and 20 μl of inoculum per well.
2.7. Statistical analysis
The two sets of experiments described above, in which EOs wereapplied to commercial baby-leaf salads, were carried out twice, eachwith triplicate samples per treatment and storage period. Analysis ofvariance (ANOVA) was performed with these data, followed by Tukey'stest 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:H7and FCV in artificiallyinoculated 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% cloveand 3%, 5% and 10% zataria were selected based on the obtained resultsand applied at a dose of 0.8 ml/25 g of vegetables. Therefore, the per-centages of EOs applied to vegetables were 0.096% (v/w), 0.160% and0.320% for 3%, 5% and 10%, respectively.
The first set of inoculation experiments in commercial baby-leafsalads was carried out to determine the EO concentration required toinactivate inoculated E. coli O157:H7 (Fig. 1). Fifteen minutes after EOapplication, E. coli O157:H7 levels were not significantly reduced formost treatments. Only 10% clove and 10% oregano significantly(P b 0.001 and P b 0.01, respectively) decreased E. coli O157:H7 levels15 min after application. On the other hand, after 5 days of storage at7 °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 concen-trations) and zataria (3%, 5% and 10% EO concentrations). Higher E. coliO157:H7 reduction was obtained at higher zataria EO concentrations.
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Fig. 3. Survival of FCV inoculated in commercial baby-leaf salads treated with Eugeniacaryophyllus and Zataria multiflora EOs and stored at 7 °C for 9 days. Values are themean (n = 6) ± standard deviation. Dotted vertical lines mark the commercial shelf-life.
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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 at10% did not show higher E. coli O157:H7 reductions than when appliedat a concentration of 5% (2.5 log cfu/g). Oregano application produced asignificant effect (P b 0.01) on inoculated E. coli O157:H7, with a maxi-mum of 0.5 log reductions after 5 days of storage of baby-leaf saladstreated with 10% EO (Fig. 1). Oregano EO was also tested at higherconcentrations (15% and 20%) on inoculated baby-leaf salads, but noimprovements in E. coli O157:H7 reduction rates were observed withrespect to lower concentrations (data not shown).
Based on these results, 10% clove and zataria concentrations wereselected for the second round of experiments. These experiments inves-tigated the effect of clove and zataria EOs on E. coli O157:H7, FCV virusand the endogenous microbiota of baby-leaf salads during 9 days ofstorage at 7 °C. Oreganowas not applied further during the long storageperiod experiment, due to its low efficacy (less than 1 log reduction)against E. coli O157:H7. Growth of inoculated E. coli O157:H7 in baby-leaf samples sprayed only with water as control treatment was slowat 7 °C, and a statistically significant increase (1.1 ± 0.3 log cfu/g) wasnot observed until the ninth day of storage (Fig. 2). Conversely, a reduc-tion of E. coli O157:H7 inoculated in baby-leaf salads was observedwhen clove and zataria EOs were applied. Maximum E. coli O157:H7reductions were obtained when baby-leaf salads treated with zataria(3.5 log cfu/g reduction) and clove (2.8 log cfu/g reduction)were storedfor 5 and 7 days, respectively (Fig. 2). After 7 days, E. coli O157:H7started to grow in clove-treated samples, reaching levels 1.7 log cfu/glower than in control samples at the end of storage. In contrast, E. coliO157:H7 levels in zataria-treated samples remained 3 log cfu/g lowerthan in control samples until the end of the storage period (Fig. 2).
Feline calicivirus concentrations of 5.3 log TCID50/gwere recorded inbaby-leaf salads after inoculation (Fig. 3). In water-sprayed samples(control treatment) FCV levels declined by1 log unit after 2 days of stor-age at 7 °C, and levels of 4.3 log TCID50/gweremaintained until day 7 ofstorage. The last 2 days of storage, FCV infectivity was significantlyreduced (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 levelswere obtained in EO-treated samples and controls (Fig. 3).
3.2. The effect of EOs on visual quality and the inactivation of endogenousmicrobiota in baby-leaf salad
On each sampling date of trial 3, the overall visual qualities of pack-aged fresh-cut products treated with 10% clove and zataria emulsionswere assessed during 9 days of storage at 7 °C. Although the quality ofthe packaged salads declined during storage, it remained high for thefirst 7 days at 7 °C. Thus, products treated with 10% EO emulsionsmaintained acceptable quality characteristics throughout the 7-daystorage period. On day 9, the quality of samples stored at 7 °C had
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Fig. 2. Survival of E. coli O157:H7 inoculated in commercial baby-leaf salads treatedwith Eugeni(n = 6) ± standard deviation. Dotted vertical lines mark the commercial shelf-life.
deteriorated below the limit of acceptability; however, no negativeeffect of EO application was observed when compared to controlsamples.
Enumeration of the endogenous microbial groups investigated inbaby-leaf salads (totalmesophilic bacteria, total psychrotrophic bacteria,total coliforms, lactic acid bacteria, yeast and moulds) was carriedout initially and after 2, 5, 7 and 9 days of storage at 7 °C (Fig. 4). Innon-EO-treated samples (control), mesophilic bacteria significantlyincreased during storage. In contrast, mesophiles decreased after zatariaapplication compared to control samples (P b 0.05). After 2 days ofstorage, zataria efficacy in mesophilic bacteria reduction became moreevident (2.5 log cfu/g lower than in control samples). After the first 2days of storage, mesophilic bacteria started to grow in zataria-treatedsamples but differences (P b 0.05) with control samples weremaintained throughout the storage period (Fig. 4). In contrast to zataria,the effect of clove on mesophilic bacteria was only significant at 7 and9 days of storage, when bacterial levels were 1.1 ± 0.2 log cfu/g lowerthan those in the control samples.
As occurredwithmesophiles, psychrotrophic populations decreasedin the presence of zataria during the first two days of storage whereasthey increased in the presence of clove and in control samples (Fig. 4).Accordingly, psychrotrophic counts were 3.2 log cfu/g lower inzataria-treated samples than those in controls. After the first 2 days ofstorage, psychrotrophic populations increased in zataria-treated sam-ples but the level was still significantly different (P b 0.05) to those incontrol samples, except at day 9 of storage. In contrast, clove EO
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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 themean (n = 6) ±standard deviation. Dotted vertical lines mark the commercial shelf-life.
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treatment did not delay psychrotrophic growth, but reduced the maxi-mum microbial density in the last 4 days of storage compared to thecontrol samples. Consequently, psychrotrophs were significantlylower in clove-treated samples than in control samples stored for 7and 9 days. Total coliforms significantly decreased 15 min after zatariaand clove application (1.1 and 0.8 log cfu/g reductions) compared tocontrol samples (P b 0.05). Both EOs maintained significantly lowercoliform levels than control sample levels (P b 0.05) during the storageperiod (Fig. 4). In contrast to the other endogenous microbial groupsinvestigated, lactic acid bacteria counts were not significantly differentin samples treated with either zataria or clove as compared to controlsamples. A maximum lactic acid bacteria density of 4.0 log cfu/g wasrecorded in control baby-leaf salads after 7 days of storage, while itwas 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 platesfrom any of the samples, with or without EO.
4. Discussion
Outbreaks of E. coli O157:H7 have been increasingly associated withthe consumption of lettuce (Soderstrom et al., 2008) or spinach (Grantet al., 2008; Wendel et al., 2009). Observations on the ability of patho-genic bacteria to grow on refrigerated, fresh-cut salads are not new,with reports of E. coli O157:H7 growing on shredded lettuce and babyspinach stored at 8 °C (Luo et al., 2009; Posada-Izquierdo et al., 2013).The results of the present study on mixed salad (containing baby redand green Batavia, Lollo Rosso lettuce, spinach and arugula) haveshown that E. coliO157:H7 is one of several pathogenic bacteria capableof growing on commercial baby-leaf salads stored at refrigeration tem-perature, i.e., 7 °C.
Plant EOs are a potentially useful source of antimicrobial compoundsfor fresh produce preservation because a considerable number of EOcomponents are GRAS. Results reported in other studies are difficult tocompare, mainly due to differences in foods, technological applicationmethods, bacterial strains and sources of antimicrobial compounds
(Jerkovic et al., 2001). Furthermore, in the fresh-cut industry, EO anti-microbial effects on novel fresh-cut developments, such as baby-leafsalads, as well as EO technological applications require further investi-gation. Our results on Zataria multiflora EO reveal it exerts a strongantibacterial activity against E. coli O157:H7 in culture medium(Elizaquivel et al., 2013b) and in baby-leaf salads. According to thechemical 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 havereported its high efficiency against both Gram-positive and Gram-negative 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 bacteri-cidal effect of low concentrations of Eugenia caryophillus (clove) EO,containing 83.96% of eugenol and 10.75% eugenile acetate (Leuschnerand Lelsch, 2003; Oussalah et al., 2007). Similarly, in the presentstudy, clove EO proved effective against E. coli O157:H7 and endoge-nous microbiota of baby-leaf salads. Origanum compactum (oregano)with 46.88% carvacrol and 15.26% thymol showed a strong bactericidalactivity when applied to pure culture. However, oregano had a signifi-cant (P b 0.05) but low antimicrobial activity against E. coli O157:H7(maximumof 0.5 log reductions) when applied to artificially inoculatedbaby-leaf salads. Although it is statistically significant, a reduction of 0.5log cfu/g is generally not considered a significant reduction in foodmicrobiology. Therefore, oregano EO did not prove effective in termsof practical application against E. coli O157:H7 in baby-leaf salads.
It appears that the difference in antibacterial activities of EOsmay berelated to the concentration and nature of contents, to the respectivecomposition, volatility, the functional groups, the structural configura-tion of the components and their possible synergistic interaction. Differ-ences between EOs are due to ecological and plant growth factors(Chang et al., 2001). The major chemical constituents of these oils areessentially monoterpene phenolics and monoterpene hydrocarbons.Eugenol, carvacrol and thymol are phenolic compounds that haveproven to be highly active against pathogenic bacteria. Other authors
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(Kim et al., 1995) observed that carvacrol was the most active constitu-ent of 11 EOs tested, and the zataria EO extract used in the present studycontained high concentrations of this constituent.
The antiviral effect of EOs was previously determined by our teamusing 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,withno effect at 4 °C. The effects of clove and zataria EOon FCV incubateda 4 °C showed similar trends in titer reductions to those obtained withoregano EO, achieving the maximum titer reduction when FCV suspen-sion was treated at 37 °C with 0.1% of zataria EO. Results of the presentstudy showed that FCV can survive on baby-leaf salads during refriger-ated storage (9 days at 7 °C).Moreover, as occurred in FCV suspensions,in FCV-inoculated baby-leaf salads, refrigeration temperature totallyannulled the antiviral efficacy of EOs. This could be because the lowtemperature produced a conformational change in the capsid, reducingthe accessibility of EOs to some viral regions. Several studies haveevaluated the effectiveness of decontamination treatments againstfoodborne viruses in vegetable samples. Most of them have reportedthat 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 washingtreatments have been evaluated mainly on norovirus surrogates. Sofar, an extent of antiviral activity on norovirus surrogate suspensionshas been observed using EOs of oregano, zataria, and clove (Elizaquivelet al., 2013a); chitosan (Su et al., 2009); cranberry juice and cranberryproanthocyanidins (Su et al., 2010a); pomegranate juice and pomegran-ate 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 onvegetable samples. This study reported that after 1 min of washingtreatment, 0.25–1 mg/ml GSE caused a reduction of 2–3 log for FCVtiters (Su and D'Souza, 2013). These results highlight the potential useof the combination of natural compounds and other treatments toreduce the presence of enteric viruses in food produce.
Baby-leaf salads include a diverse microbiota dominated by variousGram-negative and also Gram-positive bacteria. Similar microbialcounts obtained in TSA incubated at 7 and 30 °C from baby-leaf samplesstored at 7 °C, indicated that mesophilic bacteria can be consideredpsychrotrophs. Zataria showed the highest inhibition of mesophilicpopulation levels; meanwhile levels were the same for zataria andclove after 7 and 9 days storage. The phenolic compounds accountingfor EO antimicrobial activity are lipophilic, and this quality confers astrong antibacterial capacity as it enables them to partition out of thewater phase and be dissolved in the hydrophobic domain of the cyto-plasmic membrane. This causes an increase in membrane permeability,which in turn dissipates the proton motive force, resulting in lethaldamage to the bacterial cell (Burt, 2004).
Refrigeration is often the main and, frequently, the only factor con-trolling the growth of foodborne pathogens in fresh produce. Fresh-cutproduce processors often recommend that fresh-cut products be storedat 1 to 3 °C to maintain food quality. However, in fact, temperatureabuse frequently occurs during fresh-cut product distribution and retaildisplay (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 efficientdecontamination treatments against pathogens. In this study, EOswere applied to commercial baby leaves by spray after the sanitizingwashing step, and subsequently investigated for their ability to controlthe growth of potentially cross-contaminating pathogens and endoge-nous microbiota. Our results have shown that EOs, especially zataria,have a great potential to be used as an additional barrier to reducecontamination-related risks in fresh produce. This study has shownthat clove and zataria EOs exert antibacterial activity against E. coliO157:H7 inoculated in baby-leaf salads while different concentrationsof zataria control bacterial growth. Also, the present study generateddirectly comparable, quantitative, antimicrobial data for EOs,
underpinning their potential as food preservatives. When spices andherbs are used as integral ingredients in prepared foods or added asflavoring agents, the active ingredients they contain are present in insuf-ficient quantities to exert significant antimicrobial effects. By contrast,volatile oils, which often contain the principal aromatic and flavoringcomponents of herbs and spices, can reduce bacterial contamination,even when added in small quantities to foodstuffs, without affectingorganoleptic properties. The use of zataria for the preservation of fishhas recently been reported (Choobkar et al., 2010). In a study carriedout by Ekhtiarzadeh et al. (2012) fish fillets were treated with zatariaand subsequent organoleptic evaluation showed that they were highlyacceptable when zataria was applied at concentrations of 0.135% and0.405% whereas an undesirable sensory effect was observed at 0.810%.Our study is the first in which zataria has been applied to vegetablesand total concentrations of EO in produce (v/w %) were 0.096, 0.160and 0.320% without negative effects on sensory properties. Therefore,further studies should be undertaken to determine the effective concen-tration of EOs against microorganisms in other foodstuffs, as well as theorganoleptic impact of these concentrations. In addition to spoilagedelay, improvement of sensory attributes of fresh-cut fruit and vegeta-bles may also be interesting from a commercial point of view. In thisrespect, EOs not previously associated with a herby or spicy flavor,could be used in fresh-cut produce, looking for synergies betweenantibacterial effect and flavor. When good hygienic practices arefollowed throughout the production chain of fresh-cut vegetables, theuse of EOs as preservatives can contribute to the improvement of foodsafety and food quality.
Acknowledgments
This studywas supported by grantAGL2009-08603 from the SpanishMinistry of Science and Innovation, ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. G. Sánchez was the recipientof a JAE doctor grant from the “Consejo Superior de InvestigacionesCientíficas” (CSIC) and M. Azizkhani was supported by a grant fromthe Ministry of Sciences, Research and Technology of Iran. We thankthe Institute of Medicinal Plants, Medical University of Tehran (Iran)for supplying the Z. multiflora essential oil. English text revised byF. Barraclough.
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