Postharvest Biology and Technology 77 (2013) 87–93

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Postharvest Biology and Technology

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Some conventional and latent anti-listerial effects of essential oils, herbs, carrotand cabbage in fresh-cut vegetable systems

Johann Scollard !, Gillian A. Francis, David O’BeirneFood Science Research Centre, Department of Life Sciences, University of Limerick, Limerick, Ireland

a r t i c l e i n f o

Article history:Received 5 July 2012Accepted 25 November 2012

Keywords:Essential oilsAnti-listerial effectsMacerationSynergySensory appearance

a b s t r a c t

The anti-listerial effectiveness of selected essential oils (EOs) and shredded fresh herbs (thyme, oreganoand rosemary) was examined on a range of modified atmosphere packaged fresh-cut vegetables (lettuce,carrot discs, cabbage and dry coleslaw mix). Anti-listerial effects were in the order: thyme EO > oreganoEO > rosemary herb > rosemary EO. While thyme EO demonstrated the best anti-listerial effect, directapplication of all the EOs damaged product appearance. Shredded fresh rosemary herb appeared to havea major anti-listerial effect, but shredded fresh thyme and oregano showed no anti-listerial effects. How-ever, fresh rosemary herb was only effective in fresh-cut products when it was stomached with theproduct prior to microbial analysis. The effectiveness of these antimicrobials varied depending on theproduct type. Greater anti-listerial effects were recorded on carrot discs and shredded cabbage thanon shredded lettuce. Adding shredded carrot to packages enhanced the apparent anti-listerial effects,suggesting a synergistic effect between carrot and rosemary.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Increasing consumer concerns about the potential harmfuleffects of synthetic antimicrobials has created growth opportuni-ties in the market for natural antimicrobials. The addition of naturalplant antimicrobials such as plant essential oils (EOs) may representan alternative/complementary strategy to reduce contaminationand growth of pathogens during storage of foods. EOs are aromaticoily liquids obtained from plant material (flowers, buds, seeds,leaves, twigs, bark, herbs, wood, fruits and roots). They can beobtained by expression, fermentation or extraction but steam dis-tillation is most commonly used for commercial production (Burt,2004).

Techniques such as antimicrobial dipping, modified atmo-sphere packaging, vacuum packaging and refrigeration, which areemployed in the fresh-cut produce industry, are not sufficientlyeffective at either eliminating undesirable pathogens or delayingmicrobial spoilage (O’Beirne and Zagory, 2010; Tajkarimi et al.,2010). However, if these techniques were combined with the useof naturally occurring antimicrobials such as plant derived EOs,this could provide an added hurdle to pathogen growth and extendshelf-life.

One such pathogen of concern in fresh-cut vegetables is Lis-teria monocytogenes. Since L. monocytogenes occurs widely in soiland in the agricultural environment generally, it may contaminate

! Corresponding author. Tel.: +353 61 213192; fax: +353 61 331490.E-mail addresses: johann.scollard@ul.ie, johannscollard@gmail.com (J. Scollard).

vegetables (O’Beirne and Zagory, 2010). Many studies haveindicated that fresh-cut packaged vegetables may contain L. mono-cytogenes and therefore could represent a health risk to theconsumers. In recent studies, L. monocytogenes was found in freshmixed salad, cabbage samples and on other fresh produce andready-to-eat salad vegetables (Abadias et al., 2008; Johannessenet al., 2002; Johnston et al., 2006; Little et al., 2007). Francis andO’Beirne (2006) recovered 21 L. monocytogenes isolates (2.9% ofsamples) from a range of fresh vegetable products, including drycoleslaw mix (80% shredded cabbage and 20% shredded carrot),bean sprouts, and leafy vegetables such as iceberg, romaine, andradicchio lettuce and mixed salad leaves (curly endive, escarole, andradicchio leaves). L. monocytogenes is facultatively anaerobic, capa-ble of survival and growth under the low O2 and slightly elevatedCO2 concentrations found in MA packages of vegetables.

As a psychrotroph, it can survive and grow at the low tempera-tures. Temperature abuse is a common occurrence in the cold chain,and as a result, this pathogen is of major concern to the fresh-cutproduce industry. Studies by Francis and O’Beirne (1997) foundthat when two operations commonly used in the production ofminimally processed vegetables (modified atmosphere packaging(MAP) and antimicrobial dipping) were combined with storage at8 "C, both enhanced the survival and growth of Listeria populationson shredded lettuce.

Essential oils have been shown to possess antibacterial proper-ties against a range of pathogenic and spoilage microorganisms.In vitro studies have demonstrated antibacterial activity of EOsagainst L. monocytogenes, Salmonella Typhimurium, Escherichia coli,Bacillus cereus and Staphylococcus aureus at levels between 0.2

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88 J. Scollard et al. / Postharvest Biology and Technology 77 (2013) 87–93

and 10 !L/mL (Burt, 2004). Thyme, basil and marjoram EOs wereeffective against up to 20 different strains of L. monocytogenes (Lis-Balchin and Deans, 1997). Oussalah et al. (2006) found that oreganoand thyme oils showed a strong antimicrobial activity againstL. monocytogenes and that L. monocytogenes was destroyed com-pletely by twenty EOs at concentrations ! 0.8% (vol/vol). AlthoughEOs perform well in antibacterial assays in vitro, there are still rel-atively few studies in real food systems. Work has been carried outin some food, namely in meat, fish, dairy products, fruit and somevegetables (Burt, 2004).

Some vegetable tissues have naturally occurring antimicrobialsthat provide varying levels of protection against pathogens. Manystudies have shown carrot and carrot juice to have a lethal effect onListeria (Nguyen-the and Lund, 1991; Beuchat et al., 1994; Beuchatand Doyle, 1995). Most authors have attributed this anti-listerialactivity to antimicrobials or phytoalexins in cellular and vascularfluids released as a result of rupturing carrot cells. Cabbage and cab-bage juice have also been recognised as possessing antimicrobialactivity (Yildiz and Westhoff, 1981; Kyung and Fleming, 1994a,b).The antimicrobial activity of cabbage is believed to be due tothiosulfinates enzymatically generated from S-alk(en)yl-l-cysteinesulfoxides when the tissue is ruptured. The antimicrobial action ofthiosulfinates and their derivatives (namely methyl methanethio-sulfinate in the case of cabbage) are effective due to the S(O) Sgroup in the molecules, which readily react with SH group ofessential proteins of microorganisms (Kyung and Lee, 2001).

In the current study the effectiveness of natural plant antimi-crobials (EOs and herbs) against L. monocytogenes on fresh-cutvegetables was examined. Interactions with product type werestudied in order to examine whether components of produce mayenhance the effectiveness of these natural plant antimicrobials.

2. Materials and methods

2.1. Preparation of the vegetable products

2.1.1. LettuceHeads of Iceberg lettuce were purchased from a local fruit and

vegetable supplier (Richardson’s Foods, Limerick) on the day oftesting. The outer and damaged leaves as well as the core of theheads were removed and discarded. Inner leaves were sliced man-ually using a sharp stainless steel knife to approximately 10 mmstrips.

2.1.2. Carrot discsLocally grown Irish carrots were used for the production of car-

rot discs (Richardson’s Foods, Limerick). Carrots were topped andtailed using a sharp knife and the end slices were discarded. Car-rots were then peeled using a hand-held peeler and were sliced into6 mm thick slices, using a sharp knife.

2.1.3. Dry coleslaw mixThe dry coleslaw mix (80% shredded white cabbage, 20% shred-

ded carrot) was prepared by the local processor (Richardson’sFoods, Limerick).

2.1.4. CabbageHeads of white cabbage were purchased from a local fruit and

vegetable supplier (Richardson’s Foods, Limerick). On the day oftesting, the outer and damaged leaves as well as the core of theheads were removed and discarded. Inner leaves were sliced man-ually using a sharp stainless steel knife to approximately 10 mmstrips.

2.1.5. Packaging of vegetable samplesVegetable portions (25 g) were transferred into bags

(18 cm " 10 cm), composed of 35 !m oriented polypropylenepackaging film (Cannings Packaging Ltd., Dublin, Ireland), whichwere later heat-sealed. According to the manufacturer, this filmhad a permeability to O2 of 1200 mL/m2/day/atm and to CO2 of4000 mL/m2/day/atm.

2.2. Preparation of inocula

For experiments on lettuce and carrot discs Listeria innocua wasused as a model for L. monocytogenes (Francis and O’Beirne, 1997).For the work carried out on the coleslaw mix, L. monocytogenesNCTC 11994 was used. For work carried out on cabbage, a mixtureof Listeria strains were used, where the five strains listed belowwere mixed together and then used to inoculate the vegetables.

The Listeria strains used were as follows: two L. monocytogenesreference strains (i.e. NCTC 11994 and NCTC 7973), two L. monocyto-genes strains which had been previously isolated from packagedvegetables (L. monocytogenes IL323 and CW 329) and one L. innocuastrain (i.e. NCTC 11288). All cultures were maintained at #20 $Cin Tryptone soya broth (TSB) supplemented with 15% (v/v) glyc-erol, and were grown in Tryptone soya broth (10 mL TSB, OxoidCM129, Fannin Healthcare, Cork, Ireland) at 35 $C for 24 h. Cultureswere then centrifuged (5000 " g, 15 min), resuspended, mixed anddiluted in phosphate buffered saline (PBS, Oxoid BR014, FanninHealthcare) to allow for contamination of vegetables at initial levelsof approximately 105–106 CFU g#1 of vegetable.

2.3. Inoculation of vegetables

After appropriate dilutions, 100 !L aliquots of the cell suspen-sion were distributed uniformly over the vegetables containedwithin each of the packages. Following inoculation, the antimi-crobial treatments were applied to the vegetables and the bagswere heat-sealed using an impulse bench-top heat sealer (Relco,UK Ltd., England). After sealing, packs were gently shaken to assistdistribution of the inoculum.

2.4. Effects of antimicrobial treatments and storage

The effectiveness of the plant antimicrobial treatments on thesurvival and growth of Listeria were examined as described below.

2.4.1. Essential oilsThyme, oregano and rosemary essential oils were obtained from

Guinness Chemicals Ltd. Portlaoise Ireland and were CO2 supercriti-cal fluid extracts. The oil (0.5 g/25 g) was sprayed over the vegetableproducts in a fine mist using a spray bottle.

2.4.2. Fresh herbsFresh thyme, oregano and rosemary herbs were obtained from a

local supplier. The fresh herbs were cut into approx 30 mm portionsusing a sterile sharp knife and 5 g quantities were added to samplesof packaged vegetables and mixed.

2.4.3. Fresh shredded carrotTo evaluate possible additional benefits from the addition of

freshly shredded carrot, 5 g of 10 mm (in length) shreds were addedto samples of packaged vegetables and mixed.

Immediately after application of antimicrobial treatments andsealing of packages, vegetable samples were transferred to tem-peratures of 4 $C and 8 $C in order to test the effects of storagetemperature on the anti-listerial effectiveness of treatments.

J. Scollard et al. / Postharvest Biology and Technology 77 (2013) 87–93 89

2.5. Microbiological analyses

Microbiological analyses were carried out on day 0 (day of inocu-lation) and at regular intervals throughout the storage period (days2, 6, 9). At each sampling duplicate packs from the same experimentwere analysed for Listeria populations and total viable cell counts.The 25 g sample of each package was transferred into a stomacherbag and samples were homogenised for 2 min at high speed with225 mL of phosphate buffer saline (PBS, Oxoid BR0014) using aSeward laboratory stomacher (Stomacher Model 400, AGB Scien-tific, Ireland). Serial dilutions of each homogenised sample weremade in PBS and were surface spread (100 !L/plate) in duplicateon to appropriate media.

Numbers of Listeria were determined on Listeria selective agar(LSA, Oxoid CM 856) containing a modified Listeria selective sup-plement (Oxoid SR0206) after 48 h at 35 !C. Total bacterial countswere determined on tryptone soya agar (TSA, Oxoid CM131) afterincubation at 35 !C for 48 h. Following incubation, colonies werecounted and results were expressed as colony forming units pergram of sample.

2.6. Evaluation of sensory quality

Sensory evaluation was performed on sampling days using a 5member sensory panel (members of laboratory with experiencein sensory evaluation of fresh-cut vegetables). The panellists wereasked to evaluate the appearance of samples using a scale for‘appearance score’ ranging from 10 to 0 (10 = excellent, 0 = verypoor quality). The samples were coded and randomly offered topanellists. Sensory analyses were carried out in daylight at 18 to20 !C.

2.7. Statistical analyses

All experiments were carried out in duplicate (two weeks apart,on different vegetable material using fresh cultures) and replicatedtwice. Duplicate packs were serially diluted and plated in duplicateat each analysis time. Reported populations therefore represent themeans of four values ± standard deviation. Means were comparedby analysis of variance (ANOVA) followed by least difference testingat P < 0.05. Changes in appearance score were followed with paired-sample t tests (P < 0.05).

3. Results

3.1. Effects of EOs and fresh herbs on L. innocua in fresh-cutlettuce

Fresh-cut MAP lettuce was inoculated with L. innocua and theeffectiveness of antimicrobial treatments against Listeria examinedduring storage. Thyme EO significantly reduced Listeria populations(P < 0.001) by 6 log counts with no viable cells being detected fromday 0 to day 6 (Fig. 1a). The addition of oregano EO (Fig. 1c) resultedin a 2 log reduction in growth by day 2, however this effect waslost by the end of storage. No anti-listerial effects were seen withthe addition of rosemary EO (Fig. 1b). By contrast, adding freshrosemary herb appeared to significantly (P < 0.001) reduce Listeriacounts by 3 log cycles on lettuce from day 0, and reductions of thisorder persisted up to day 9. However, these effects were only evi-dent after stomaching the product with the herb, in preparation formicrobial enumeration. When the rosemary was removed prior tostomaching there were no anti-listerial effects. Thyme and oreganoherb had no inhibitory effects on Listeria survival and growth. Sim-ilar trends were found for all treatments when stored at 4 !C (seesupplementary data).

0 2 4 6 8 100

2

4

6

8

10ControlThyme HerbThyme EO

a)

Time (Days)

Log

CFU

/g

0 2 4 6 8 100

2

4

6

8

10ControlRosemary HerbRosemary EO

b)

Time (Days)Lo

g C

FU/g

0 2 4 6 8 100

2

4

6

8

10ControlOregano HerbOregano EO

c)

Time (Days)

Log

CFU

/g

Fig. 1. Effects of (a) thyme, (b) rosemary and (c) oregano (essential oils and shreddedfresh herb) on survival and growth of Listeria innocua on MAP fresh-cut lettuce storedat 8 !C. Reported populations are means of 4 values. Error bars show s.d.

3.2. Effects of removing the herb prior to maceration instomacher on L. innocua survival and growth

Powerful anti-listerial effects of rosemary herb were observedwhen rosemary was stomached in preparation for plate count-ing (Fig. 2). Listerial populations were reduced by 4 logs on day 0(P < 0.001) and to a slightly lesser extent throughout storage for 9days. However, presence of the rosemary herb in fresh-cut pack-ages is not sufficient to have an effect, as anti-listerial effects wereonly seen when the herb was macerated during stomaching. Whenrosemary herb was removed prior to maceration in the stomacher,the anti-listerial effects were absent (P > 0.05).

0 2 4 6 8 100

2

4

6

8

10ControlRosemary herbRosemary herb (removed)

Time (Days)

Log

CFU

/g

Fig. 2. Effects of removing rosemary herb before maceration on survival and growthof Listeria innocua on fresh-cut MAP cabbage stored at 8 !C. Reported populationsare means of 4 values. Error bars show s.d.

90 J. Scollard et al. / Postharvest Biology and Technology 77 (2013) 87–93

0 2 4 6 8 100

2

4

6

8

10ControlThyme HerbThyme EO

a)

Time (Days)

Log

CFU/

g

0 2 4 6 8 100

3

5

8

10Con trolRosemary HerbRosemary EO

b)

Time (Days)

Log

CFU/

g

Fig. 3. Effects of (a) thyme and (b) rosemary (EO and fresh shredded herb) on sur-vival and growth of Listeria innocua on MAP carrot discs stored at 8 !C. Reportedpopulations are means of 4 values. Error bars show s.d.

3.3. Effects of EOs and fresh herbs on L. innocua in fresh-cutcarrot discs

Thyme EO had a major anti-listerial effect, with no viable cellsbeing detected from day 0 of storage onwards. As in the case oflettuce, thyme herb had no effect (Fig. 3a).

By contrast, rosemary EO had no effect but rosemary herbappeared to significantly reduce Listeria populations with no viablecells being detected on day 0 (P < 0.001). Listeria populationsincreased during storage and by day 9, populations were only0.5 log cycles lower than the untreated carrot discs (Fig. 3b). Similarresults were found at 4 !C (see supplementary data).

3.4. Effects of EOs and fresh herbs on L. monocytogenes infresh-cut coleslaw mix

Thyme and oregano EOs (Fig. 4a, c) resulted in no viable cellsbeing detected from day 0 and throughout storage (P < 0.001).By contrast, thyme and oregano herb had no anti-listerial effect,with listerial populations behaving similarly to those on untreatedcoleslaw.

Rosemary EO reduced counts by 1 log cycle throughout storagecompared to the control values (P < 0.05). Rosemary herb, however,appeared to have a greater effect (Fig. 4b), reducing log countsby 3 log cycles (day 0), and maintaining a 4 log reduction by theend of storage (P < 0.001). Similar results were recorded at 4 !C (seesupplementary data).

3.5. Effects of rosemary EO, fresh herb and shredded carrot on L.monocytogenes in fresh-cut shredded cabbage

The effects of different rosemary and shredded carrot combi-nation treatments on Listeria survival and growth during storageon shredded cabbage at 8 !C are presented in Fig. 5. Rosemary EOreduced counts by 2 log cycles (day 0) and up to 3 log cycle by day9. Rosemary EO combined with shredded carrot slightly enhancedthis effect. Rosemary herb appeared to have a major anti-listerialeffect, with a 6 log reduction on day 0. When shredded carrot was

0 2 4 6 8 100

2

4

6

8

10ControlThyme herbThyme EO

a)

Time (Days)

Log

CFU

/g0 2 4 6 8 10

0

2

4

6

8

10ControlRosemary HerbRosemary EO

b)

Time (Days)Lo

g C

FU/g

0 2 4 6 8 100

2

4

6

8

10ControlOregano EOOregano herb

c)

Time (Days)

Log

CFU

/g

Fig. 4. Effects of (a) thyme, (b) rosemary and (c) oregano (EO and fresh shred-ded herb) on survival and growth of Listeria monocytogenes (NCTC 11994) on MAPcoleslaw mix stored at 8 !C. Reported populations are means of 4 values. Error barsshow s.d.

added, populations were reduced further by approximately a 1 logcycle, eliminating Listeria initially. While the effects of rosemaryherb alone were lost by the end of storage, populations were still3 log cycles lower than that of the control when rosemary and carrotwere combined.

The effects of all the antimicrobial treatments are summarisedin Table 1.

3.6. Effects of EOs and fresh herbs on appearance of fresh-cutvegetables

The addition of rosemary essential oil to lettuce, coleslawmix and shredded cabbage (Fig. 6) reduced appearance scores

0 2 4 6 8 100

2

4

6

8

10ControlCarrotRosemary herbCarrot + rosemary herbRosemary EOCarrot + rosemary EO

Time (Days)

Log

CFU

/g

Fig. 5. Effects of rosemary (EO and shredded fresh herb), carrot and rosemary andcarrot combinations on survival and growth of Listeria monocytogenes (mixed strain)on fresh-cut MAP cabbage stored at 8 !C. Reported populations are means of 4 values.Error bars show s.d.

J. Scollard et al. / Postharvest Biology and Technology 77 (2013) 87–93 91

Table 1Summary of the overall maximum reduction in counts for all the antimicrobial treatments tested on lettuce, carrot discs, coleslaw mix and shredded cabbage at 8 !C(TBCs = total bacterial counts, ND = not determined).

Reduction in counts (log CFU/g) Thyme Rosemary Oregano

EO Herb EO Herb EO Herb

Lettuce Listeria counts 6 0 0 3 2 0TBCs 2 0 0 0 0 0

Carrot discs Listeria counts 6 0 0 6 ND NDTBCs 2 0 0 0 ND ND

Coleslaw mix Listeria counts 6 0 1 4 6 0TBCs 1 0 0 0 ND ND

Shredded cabbage Listeria counts 6 0 1 6 6 NDTBCs 5 0 0 0 5 ND

0 2 4 6 8 100

2

4

6

8

10

12ControlRosemary HerbRosemary EO

(a) Lettuce

Time (Days)

Appe

aran

ce s

core

0 2 4 6 8 100

2

4

6

8

10

12ControlRosemary HerbRosemary EO

(b) Coleslaw mix

Time (Days)

Appe

aran

ce s

core

0 2 4 6 8 100

2

4

6

8

10

12Control

Rosemary herb

Rosemary EO

(c) Cabbage

Time (Days)

Appe

aran

ce s

core

Fig. 6. Effects of rosemary (essential oil and shredded fresh herb on appearancescores of MAP (a) lettuce, (b) coleslaw and (c) cabbage stored at 8 !C. Reported scoresare means of 4 values. Error bars show s.d.

throughout storage to unacceptable levels. The addition of freshrosemary herb had no effect on the appearance scores. Sim-ilar results were found for all the oils and herbs tested (seesupplementary data).

4. Discussion

There are powerful anti-listerial effects available from nat-ural antimicrobials; however, direct use in fresh-cut systems isgenerally impossible due to phytotoxic effects. The separationof anti-listerial and phytotoxic effects, and exploitation of these

anti-listerial effects is a major challenge. A deeper understandingof both conventional, latent and unexpected anti-listerial effectsmay contribute to more successful practical exploitation. Thispaper presents both conventional effects and novel effects andinteractions.

The work demonstrates that the antimicrobial effects of EOsvaried depending on which EO was used and the type of fresh-cut vegetable involved. Both anti-listerial and general anti-bacterialeffects were observed for thyme and oregano EOs. Thyme EO wasfound to be the most effective treatment against Listeria. Previousstudies found thyme EO to be effective against a number of differ-ent strains of L. monocytogenes and to be the oil with the widestspectrum of antimicrobial activity (Lis-Balchin and Deans, 1997;Smith-Palmer et al., 1998; Dorman and Deans, 2000; Viuda-Martoset al., 2008).

Oregano EO was also found to have strong anti-listerial effects,but not as strong as those of thyme EO. Burt (2004) reportedoregano EO to be effective against bacteria such as Listeria, S. aureusand E. coli. Gutierrez et al. (2008) found that oregano and thymeEOs had the highest activity against all the tested bacteria includ-ing Listeria spp. Govaris et al. (2010) found that both oregano andthyme EO exhibited equal activity against L. monocytogenes. Chemi-cal analysis of EOs, have found carvacrol and thymol to be present inhigh quantities in both oregano and thyme EOs. Carvacrol and thy-mol possess antimicrobial activity in vitro against a broad spectrumof Gram-positive bacteria including L. monocytogenes (Lis-Balchinand Deans, 1997; Davidson, 2000; Lambert et al., 2001).

Rosemary EO showed no anti-listerial effects except in thepresence of shredded cabbage, and these effects were considerablysmaller than those of the other EOs. By contrast, strong anti-listerialeffects were evident from rosemary herb. However, these wereonly observed after stomaching. Removing the rosemary herbprior to stomaching resulted in the loss of the anti-listerial effects;it appears that the herb is only effective when it is completely mac-erated with the vegetable sample in the stomacher. This effect maybe due to the stomaching releasing particular antimicrobial com-ponents from the herb which are effective against Listeria. Studiesby Smallfield et al. (2000) found that coriander subjected to heavycrushing had an increased oil yield due to all vittae being ruptured.Crushing garlic, was found to transform the alliin present into thebiologically active allicin molecule, and it was this allicin moleculethat was responsible for it’s remarkable antibacterial activity (Ankriand Mirelman, 1999). Rosemary herb has been shown to exhibitinhibitory effects against a number of bacteria. Yano et al. (2006)found rosemary herb effective against E. coli at both 5 and 30 !C.Shelef et al. (1980) found that a concentration of 0.3% rosemaryin the media inhibited the growth of 21 Gram positive organisms.However, of the fresh herbs tested, only rosemary had these effects,as no reductions in Listeria populations were evident with oreganoor thyme herb. Pandit and Shelef (1994) also found that thymeherb did not exhibit any anti-listerial effects. Reports by other

92 J. Scollard et al. / Postharvest Biology and Technology 77 (2013) 87–93

investigators found that camphor, verbenone and borneolaccounted for the main composition of rosemary EO (Pintoreet al., 2002; Okoh et al., 2010). The anti-listerial effects of rosemarywere lost by the end of storage, possibly due to the higher levelsof microorganisms consuming the antimicrobials produced duringstomaching.

There appeared to be significant interactions between someantimicrobial effects of EOs/herbs and the presence of other veg-etable components, notably carrot and cabbage. Rosemary EO wasfound to have little or no anti-listerial effects on lettuce, butshowed stronger anti-listerial effects on cabbage and coleslaw.Thyme EO, rosemary EO and rosemary herb effects were enhancedon shredded cabbage. The effectiveness of rosemary herb wasincreased from 3 log cycles on lettuce to 6 log cycles when appliedto shredded cabbage. Cabbage has been shown to generate methylmethanethiosulfinate (MMTSO), enzymatically when the tissue isdisrupted (Kyung et al., 2002). The antimicrobial activity has beenexplained as a general reaction between thiosulfinates and SHgroups of essential cellular proteins (Kyung and Lee, 2001). Kyungand Fleming (1997) found that MMTSO was inhibitory againstL. monocytogenes. The combination of thyme EO, rosemary EOand rosemary herb with cabbage appeared to create a synergisticenhancement of effectiveness in both coleslaw mix and shreddedcabbage.

This impact of product was again observed in the carrot discs,and was particularly evident when rosemary herb is added to car-rot discs. Addition of rosemary herb to lettuce resulted in a 3 logreduction in Listeria populations on Day 0, whereas the additionof rosemary herb to carrot discs resulted in a 6 log reduction ininitial populations. This heightened anti-listerial effect observedon the carrot discs led to the inclusion of shredded carrot as anantimicrobial treatment in experiments on other products. To ourknowledge, no studies have been carried out on the combinationeffects of carrot and EOs. However, carrot itself is recognised ashaving strong anti-listerial effects and the lethal effect of shreddedcarrot on L. monocytogenes has been previously reported (Beuchatand Brackett, 1990; Beuchat and Doyle, 1995; Noriega et al., 2010).On cabbage at 8 !C, rosemary herb resulted in a 5 log reductionin Listeria, and this was increased to 6 log cycles when combinedwith shredded carrot. The effect seemed to be synergistic, possiblyinvolving a combination of the increased cell permeability causedby the EO (Burt, 2004) and greater access for the anti-listerial com-ponent of carrot.

The application of EOs in an undiluted form to lettuce and othervegetables led to unacceptable damage to appearance. This effectis probably due to the dark colour of the oils and phytotoxic effects.Similar results were seen by De Azeredo et al. (2011) who foundthat the essential oils of rosemary and oregano caused undesirableeffects on the sensory quality of minimally processed vegetables.Uyttendaele et al. (2004) found that the addition of thyme EO tochopped bell peppers led to softening of the tissue and moistureloss, suggesting thyme EO probably has a cytotoxic effect. Somepanellists commented on the unattractive odour associated withthe oil. Undesirable organoleptic effects might be limited by carefulmatching of EO and food type, by diluting EOs, by using combina-tions of EOs, or by using EOs in combination with plant materialsuch as carrot or cabbage shreds.

The anti-listerial properties observed were in the order:thyme EO > oregano EO > rosemary fresh herb > rosemary EO. Freshrosemary herb however, was ineffective in fresh-cut products untilit was stomached with the product prior to microbial analysis. Atthis point it produced strong anti-listerial effects. While no practi-cal anti-listerial treatment using EOs or herb can yet be reported,important sources of potential anti-listerial effects have been iden-tified from rosemary herb and synergies between rosemary herb,some EOs and carrot and cabbage. Greater understanding of the

molecular basis of such effects and the effect of maceration on rose-mary herb will help researchers navigate the difficult but rewardingpath to commercial exploitation of natural antimicrobials.

Acknowledgements

This work was funded by FIRM and the Department of Agricul-ture and Food.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.postharvbio.2012.11.011.

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