The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter jejuni, Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in food systems
C.A. Phillips, School of Health, The University of Northampton, Boughton Green Road, Northampton, NN2 7AL, UK.
Aims: To investigate the effectiveness of oils and vapours of lemon (Citrus limon), sweet orange (Citrus sinensis) and bergamot (Citrus bergamia) and their components against a number of common foodborne pathogens.
Methods and Results: The disc diffusion method was used to screen the oils and vapours against Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli O157 and Campylobacter jejuni. The survival of each species, demonstrated to be susceptible in the in vitro studies, was tested on cabbage leaf for 60 s by direct contact and on chicken skin for 10 min by direct contact and 24 h by vapour. The results indicate that bergamot was the most inhibitory essential oil (EO) and citral and linalool mimicked its effect (P > 0·001). Citral and linalool vapours produced 6 log reductions in L. monocytogenes, Staph. aureus and B. cereus populations on cabbage leaf after 8–10 h exposure but bergamot vapour exposure, while producing a similar reduction in L. monocytogenes and B. cereus populations, had no effect on Staph. aureus.
Conclusions: Bergamot was the most effective of the oils tested and linalool the most effective anti-bacterial component. Gram-positive bacteria were more susceptible than Gram-negative bacteria in vitro, although Camp. jejuni and E. coli O157 were inhibited by bergamot and linalool oils and by linalool vapour. All bacteria tested were less susceptible in food systems than in vitro. Of the Gram-positive bacteria tested Staph. aureus was the least susceptible to both the oils and the components tested.
Significance and Impact of the Study: Results suggest the possibility that citrus EOs, particularly bergamot, could be used as a way of combating the growth of common causes of food poisoning.
Cases of food poisoning are rising in the UK. Notifications have risen from 14 253 in 1982 to 70 895 in 2003 and this does not seem to have decreased significantly in 2004 with 70 311 notified cases reported (Health Protection Agency 2005). A growing problem seems to be in areas of great expansion like those of the organic food sector (Long et al. 2002). This increasing problem is only emphasized by changes in legislation and consumer trends that request preservative free, safe, but mildly processed, foods (Brul and Coote 1999).
A possible natural alternative to chemical-based bactericides might be the use of essential oils (EOs) as the use of plants and their products are thought to be effective for food safety and preservation generally and because they have a wide range of volatile components (Lanciotti et al. 2003). In vitro studies have shown EOs to have antibacterial properties against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Bacillus cereus and Staphylococcus aureus at between 0·2 and 10 μl ml−1 (Cosentino et al. 1999). However, higher concentrations are required to produce a similar effect when used in food systems (Burt 2004). Out of 11 EOs tested on B. cereus in carrot broth, cinnamon, sage, rosemary and sassafras showed greatest inhibitory effects, with cinnamon inhibiting a strain that was able to grow at low temperatures without the presence of the EO (Valero and Salmerón 2003). Clove has also been shown to exert a significant bactericidal and bacteriostatic action on a variety of strains of E. coli (Moreira et al. 2005). Little research has been carried out on Campylobacter spp. in terms of the effects of EOs on growth and survival, but a study investigating a number of plant EOs in culture medium indicated that a number of these, including the oils of gardenia and jasmine, were effective at inhibiting Camp. jejuni growth (Friedman et al. 2002). Also, it has been shown that oregano has inhibitory effects on Helicobacter pylori, which is a related organism (Chun et al. 2004). Cranberry extracts have also been shown to have inhibitory effects on H. pylori but this is thought to be due to the high phenolic levels present rather than volatile oil components (Vattem et al. 2004). It has been shown that antimicrobial properties of EOs prolong the lag phase and reduce the exponential growth rate of bacteria as well as the final population of the cultures (Valero and Giner 2006).
Although limited research has been carried out on citrus EOs in terms of their use as antimicrobials in the food industry, it has been shown that they have potential bactericidal properties not only against yeast, moulds and spore forming bacteria but also against food-poisoning bacteria (Deans and Ritchie 1987). Limonene is one of the major components of citrus oils (Moufida and Marzouk 2003) with concentrations from 88% to 95% of lemon or orange oil, although levels in bergamot are lower with concentrations ranging from 32% to 45% (Merle et al. 2004). Citral has been highlighted as an active compound in citrus fruit oils, especially against decay caused by Penicillium digitatum (Caccioni et al. 1998). Linalool is another terpene that is important in determining intensity of fragrance (Svoboda 2003), is present in sweet orange, lemon and bergamot (Moufida and Marzouk 2003) and may be important in any antimicrobial activity (Caccioni et al. 1998). Citral and linalool have also been identified as active antimicrobial components of Pelargonium EOs which have antimicrobial effects against both B. cereus and Staph. aureus (Lis-Balchin et al. 1998).
Citrus and lemony flavours lend themselves to use in foods and are widely used within the food industry. They, and their major components citral, limonene and linalool, have been generally recognised as safe or GRAS (Food and Drug Administration 2005). How citrus EOs alter the organoleptic properties of foodstuffs is important as too much alteration to the natural properties of the foods could make them undesirable to the consumer. A way of combating this problem may be to use EO vapours. Patchouli, tea tree, geranium and lavender EO vapours have been shown to have antimicrobial properties against Staph. aureus, with potential use in the clinical arena (Edwards-Jones et al. 2004). A factor that should be considered when investigating EOs is that they are made of volatile components; therefore their use as antimicrobials may have limited shelf-life, as they rapidly evaporate. However, although this property has disadvantages in the antimicrobial arena, it does have advantages for the organoleptic properties of the food and therefore both consumer acceptance and the food industry.
This study aims to establish the effectiveness of citrus oils and vapours of lemon (Citrus limon), sweet orange (Citrus sinensis) and bergamot (Citrus bergamia) and three components, citral, limonene and linalool against a number of common foodborne pathogens: L. monocytogenes, Staph. aureus, B. cereus, E. coli O157 and Camp. jejuni both in vitro and on foods.
Materials and methods
Essential oils and oil components
Orange (Citrus sinensis), lemon (Citrus limon) and bergamot (Citrus bergamia) were obtained from AMPHORA, (Bristol, UK). Limonene 97%, linalool 97% and citral 95% were obtained from Sigma-Aldrich Co. Ltd. (Dorset, UK). All other chemicals were obtained from Merck (Eurolab Ltd., Leicester, UK) unless otherwise specified.
Analysis of the oils for the three components being tested in this study was carried out by gas chromatography using a Perkin-Elmer auto-system XL (Boston, MA, USA) using a bonded polyethylene glycol column and a flame ionization detector. The initial temperature of the oven was 50°C, initial time 3 min, ramp rate was 6°C min−1, the injector temperature was 250°C and the final temperature 250°C.
Micro-organisms and culture methods
All media were obtained from Oxoid Ltd (Basingstoke, Hampshire, UK). Test organisms included: E. coli O157:H7 (laboratory-attenuated strain), L. monocytogenes ATCC 7644 (C3970), Staph. aureus ATCC 9144 (C7001), B. cereus ATCC 11778 (C1220) and Camp. jejuni ATCC 33291 (C1400). B. cereus was grown in Brain Heart Infusion (BHI) broth (CM225) at 30°C, Staph. aureus and E. coli in nutrient broth (CM1) at 37°C, L. monocytogenes in Listeria enrichment broth (CM0862) without supplementation at 37°C and Camp. jejuni in Bolton broth (CM0983) without supplementation at 42°C. The following solid media were used to culture the bacteria: BHI agar (CM0375) for B. cereus and Staph. aureus; nutrient agar (CM3) for E. coli O157:H7; Listeria selective agar (CM856) for L. monocytogenes and Campylobacter agar base (CM0689) with 5% laked horse blood (SR0048C) for Camp. jejuni. In the experiments on food, selective supplements were added for L. monocytogenes (SR014E) and Camp. jejuni (SR117) in both agar and broth.
Screening of essential oils and oil components
Aliquots (0·1 ml) of each of the oils, citral, limonene or linalool were spotted onto 2 cm diameter filter paper discs. Three discs impregnated with the same oil were then placed onto the surface of an agar plate previously spread with either L. monocytogenes, Staph. aureus, B. cereus, E. coli O157 or Camp. jejuni. The experiment was carried out in duplicate on two separate occasions (n = 12).
In order to test the effect of the vapour of lemon/orange/bergamot/citral/limonene/linalool, spread-plated bacterial cultures were exposed to each vapour by placing one disc impregnated with one oil onto the lid of the Petri dish approximately 8 mm from the bacteria (Edwards-Jones et al. 2004). Three Petri dishes were used for each vapour. This was repeated on four separate occasions (n = 12).
All plates were then incubated at the appropriate temperatures for 24 h: E. coli, L. monocytogenes and Staph. aureus at 37°C, B. cereus at 30°C and Camp. jejuni at 42°C. Zones of inhibition were measured (diameter in mm) using Vernier callipers. Controls were plates that had not been exposed to any oils, components or vapour.
Determination of minimum inhibitory concentrations (MIC)
MICs for the EOs and their components were established using an agar dilution method (Banes-Marshall et al. 2001). Before the addition of the oils, 0·5% (v/v) of Tween 20 was first added to the agar. Use of Tween 20, an emulsifier, enhances the solubility of oils in agar up to a final concentration of 4% (v/v) oil, although at concentrations higher than this the oil will not solubilize even with Tween 20 incorporated into the agar. Dilutions of the antimicrobials were added to the appropriate agar plates at concentrations of 0·03%, 0·06%, 0·12%, 0·5%, 1%, 2% and 4% (v/v). Initial experiments demonstrated that the incorporation of Tween 20 only into the agar had no effect on the growth of the bacteria under test. Therefore controls had no oil nor Tween incorporated in the agar. Plates were then spread with 0·1 ml of Staph. aureus or L. monocytogenes or E. coli O157 or Camp. jejuni or B. cereus cultures containing approximately 107 ml−1 and incubated for 24 h. The MIC was defined as the lowest concentration of the oil inhibiting visible growth. The experiment was carried out in duplicate on two separate occasions (n = 4).
On-food model using cabbage leaf and chicken skin
In order to remove competing microflora, cabbage leaves (Sweetheart) were rinsed first with 70% ethanol, then with sterile deionized water and used immediately. The chicken skin was removed from the muscle, separated from any visible fat layers and 2 × 2 cm pieces were placed separately in McCartney bottles and heated to 121°C for 2 min (Long and Phillips 2002). The samples were then stored for a maximum of 4 weeks at −20°C and, before use, thawed and all excess liquid removed. The resulting texture of the chicken skin was similar to that of untreated skin.
Aliquots (0·1 ml) of exponential phase B. cereus, Camp. jejuni, E. coli O157, Staph. aureus, L. monocytogenenes cultures containing approximately 109 cells ml−1 were added to 2 × 2 cm squares of either cabbage or chicken skin and allowed to dry for 5 min to allow maximum adhesion to the food matrix. Each sample of either cabbage leaf or chicken skin was then subjected to a solution of either Tween 20 and the oil or citral or limonene or linalool for 15, 30, 45 or 60 s at the MIC established. The food was then placed in phosphate buffered saline (PBS) solution (BR14, Oxoid) and stomached (IUL Instruments stomacher, Biotrace International Plc, UK) for 2 min. The resulting suspension was spiral plated (Don-Whitley, West Yorks, UK) onto the appropriate agar and incubated at the appropriate temperatures for 24 h: E. coli, L. monocytogenes and Staph. aureus at 37°C, B. cereus at 30°C and Camp. jejuni at 42°C. The control was a food sample that had only been exposed to sterile deionized water. Each experiment was carried out in duplicate on two separate occasions.
On-food vapour model using cabbage leaf
Food samples were prepared as in previous experiments. The cabbage was then suspended above either citral or linalool or bergamot oil in a sterile 1 l beaker. The amount of oil (1·85 ml) used was calculated to be directly proportional to that in the vapour disc diffusion method. The beakers were double foiled, taped and incubated for each organism as in previous experiments for various time intervals up to 24 h. After 24 h physical changes of the food were apparent and therefore 24 h was considered the maximum time of exposure. Food samples were then removed from the beaker, placed in sterile PBS and stomached for 2 min. The resulting suspension was spiral plated onto the appropriate agar and incubated as previously for 24 h. The control was a food sample which had only been exposed to sterile air. Each experiment was carried out in duplicate on two separate occasions.
Independent t-tests for unpaired data or Mann–Whitney U-tests for data that was not normally distributed were carried out using SPSS Version 11.5 for Windows. Significance was set at P = 0·05.
Gas chromatographic analysis of the oils for three components indicated that limonene was more abundant than citral or linalool in the oils tested, while bergamot contained 15% linalool and orange 3% citral (Table 1). Using the disc diffusion method, lemon and orange oils were not as effective at inhibiting bacterial growth as bergamot, citral and linalool (Table 2) while limonene had no effect on any of the bacteria tested (results not shown). As orange, lemon and limonene oils were not as effective as the other oils or components tested in vitro and many oils or components have generally been previously shown to be less effective in terms of inhibitory effect on foods than in vitro, orange, lemon and limonene were not tested in the on-food investigations. Overall the EOs and the components tested were more effective against the Gram-positive bacteria tested, when grouped together, than the Gram-negative (P < 0·001). Overall, Staph. aureus was the least affected by the oils tested compared with either L. monocytogenes or B. cereus. Camp. jejuni was not inhibited by orange or citral whereas E. coli O157 was not inhibited by citral at the concentrations tested.
Table 1. Analysis of oils by gas chromatography
Table 2. Mean (±SEM) diameter (mm) of area of inhibition of E. coli O157, Camp. jejuni, L. monocytogenes, B. cereus and Staph. aureus when exposed to citrus EOs and vapour or citral or linalool oil and vapour (n = 12)
|E. coli O157||24 ± 0·3|| 0||18 ± 2||0||21 ± 0·3||0||0|| 0||70 ± 5||>90|
|Camp. jejuni||23 ± 0·3|| 0|| 0||0||18 ± 3||0||0|| 0||>90||43 ± 1|
|L. monocytogenes||>90||54 ± 2||27 ± 2||0||41 ± 2||0||>90||79 ± 0·9||>90||62 ± 0·7|
|B. cereus||36 ± 1||28 ± 1||19 ± 2||0||29 ± 1||0||>90||>90||>90||35 ± 1·3|
|Staph. aureus||46 ± 2||26 ± 3||14 ± 3||0||23 ± 0·6||0|| 6 ± 2||47 ± 2||63 ± 4·7||>90|
Orange, lemon and limonene vapours were not inhibitory against any of the bacteria tested in vitro (Table 2). Bergamot, citral and linalool vapours were more effective on the Gram-positive bacteria tested than on either E. coli O157 or Camp. jejuni so only bergamot, citral and linalool were tested against L. monocytogenes, B. cereus and Staph. aureus in the on-food model. As linalool also inhibited Camp. jejuni and E. coli O157, this was tested against Camp. jejuni and E. coli O157 in the on-food system. When comparing the effect of bergamot oil with bergamot vapour, the oil was significantly more effective against both Gram-positive and Gram-negative bacteria (P < 0·001). Linalool oil was also significantly more effective (inhibitory) than its vapour against Gram-positive bacteria (P = 0·004). When comparing the effectiveness of linalool oil or vapour there was no significant difference between the inhibition produced by either on the Gram-negative bacteria tested, although the oil was significantly more effective than the vapour on the Gram-positive bacteria tested. There was no significant difference between the inhibition by citral oil as compared with vapour against any of the bacteria.
Each of the bacteria-oil combinations shown to be effective in the initial screening by the disc diffusion method was tested to establish the MIC of the oil against the organism (Table 3). When the MIC was >4%, for example, lemon and bergamot EO against Camp. jejuni, orange EO against B. cereus and lemon EO against Staph. aureus, these combinations were not carried forward to the on-food investigation as there would be too great an effect on the organoleptic properties of the foodstuff for these levels to be applicable in an industrial setting.
Table 3. Mean (±SD) minimum inhibitory concentrations (% v/v) of orange, lemon and bergamot EOs, citral and linalool against E. coli O157, Camp. jejuni, L. monocytogenes, B. cereus and Staph. aureus using the agar dilution method (n = 4)
|E. coli O157||0·5||1||1||n/a||0·25|
On cabbage leaf the EOs/components became effective at approximately 15 s and continued to produce an inhibitory effect over 60 s (Fig. 1). This time was chosen to minimize the changes in organoleptic properties of the cabbage leaf. The greatest reduction was that of linalool oil acting against B. cereus (Fig. 1c) within 45 s, after which no viable cells were isolated. Staph. aureus was the least susceptible in terms of linalool oil only reducing by 2–3 logs over 60 s. However, generally on chicken skin the initial reduction was not so marked except in the case of bergamot on L. monocytogenes and B. cereus (Fig. 2a).
Over 60 s exposure the EOs and the components tested had a greater inhibitory effect on the bacteria when tested on cabbage leaf (Fig. 1) than on chicken skin (Fig. 2). In the case of bergamot there were approximately log 3 survivors (Fig. 1a) after this time but on chicken skin there were 4–5 log survivors, except for Staph. aureus, which showed no log reduction (Fig. 2a). For citral there were 2–4 log survivors on cabbage (Fig. 1b) as compared with 5–6 log survivors on chicken skin, with Staph. aureus again being the exception (Fig. 2b). For linalool there were <2 log survivors on cabbage (Fig. 1c) compared with 5–6 log survivors on chicken skin (Fig. 2c), however in the case of Staph. aureus there was no significant difference between the counts at 60 s on cabbage leaf when compared with chicken skin. The time of exposure on chicken skin was extended to determine whether the oils would produce greater log reductions but, except for B. cereus when exposed to citral oil, when there was approximately a further 2 log reduction, no further significant reduction in viable count was seen over a total exposure time of 10 min (results not shown). Ten minutes was considered the longest time that would be practicable in an industrial situation. For this reason the vapour experiments were only carried out on cabbage leaf, as vapours generally have less of an effect (Table 2).
Survival of L. monocytogenes and B. cereus in the presence of bergamot vapour (Fig. 3a) showed an initial decrease in numbers, no further decrease for 10 h and finally resulted in a 6 log reduction over 24 h. Bergamot had no effect on Staph. aureus. Citral vapour exposure produced a similar log reduction over 24 h, but the decrease in survival occurred between 1 and 10 h (Fig. 3b). In the presence of linalool vapour the final reduction was similar to both exposure to bergamot or citral vapour (Fig. 3c). Although at 5 h exposure L. monocytogenes appeared to be the least susceptible to linalool vapour, after 7 h a 5–6 log reduction occurred. A similar log reduction in numbers of Staph. aureus occurred over 24 h. Although there were differences in time scale between the effectiveness of oils and vapours, in that the oils were at their most effective at 60 s (Fig. 1) and the vapours at 18 h (Fig. 3), when comparing the log reductions at these two time points, there was no significant difference between the performances, in terms of inhibitory activity, of bergamot, citral or linalool oil as compared with their vapours on cabbage leaf.
This study has shown that, using the disc diffusion method, citrus EOs and particularly their components, citral and linalool, have antibacterial properties both in direct oil and vapour form (Table 2). Previous studies using other EOs have shown similar results (Valero and Salmerón 2003; Burt 2004), although little research has been carried out on citrus EOs. Overall citrus oils are more effective on Gram-positive bacteria than on Gram-negative in vitro which is similar for other EOs/components (Smith-Palmer et al. 1998; Delaquis et al. 2002; Burt 2004). Although the theories for this occurrence vary, results from this investigation suggest that Gram-negative bacteria are in fact inhibited but it depends on both the compound and the bacteria tested. In this study on cabbage leaf, there was no significant difference at 60 s exposure between the effect of bergamot on L. monocytogenes, B. cereus, E. coli O157 or Staph. aureus, whereas, citral has no effect on either E. coli O157 or Camp. jejuni and linalool inhibited all the bacteria tested.
Lemon and orange oils had little inhibitory effect on the bacteria tested in the disc diffusion method although bergamot oil was effective (Table 2). Similar results have been reported previously, also using the disc diffusion method (Deans and Ritchie 1987) when orange and lemon were shown to have no inhibitory effect on E. coli while bergamot produced an inhibitory diameter of 4·5 cm. When testing the three components, the results mimicked that of the oil composition in that lemon and orange both have high limonene content (Table 1) with orange, lemon nor limonene having any inhibitory effect on the bacteria tested (Table 2). Bergamot contained the highest percentage of linalool, which was also demonstrated to be the most effective component. All the essential oils contain a low percentage of citral ranging from 0·1% to 3%; this may account for the little inhibitory effect that lemon and orange EOs have demonstrated in this, and other, studies. Citral and linalool were effective at inhibiting in vitro growth of the three Gram-positive bacteria tested which corresponds to the effect of Pelargonium EOs previously reported on Staph. aureus and B. cereus (Lis-Balchin et al. 1998). Another point that should be considered, which has been suggested by other studies (Caccioni et al. 1998; Burt 2004), is a synergistic effect of the components of essential oils. This has not been tested in this study but research is on-going.
The mechanisms by which EOs bring about their antibacterial effect is incompletely understood but there are a number of proposed mechanisms (Holley and Patel 2005). Certainly there are morphological changes that are apparent. The outer membrane of both E. coli and Salmonella typhimurium disintegrates following exposure to carvacrol and thymol (Helander et al. 1998) and major thickening and disruption of the cell wall, together with increased roughness and lack of cytoplasm has recently been reported in L. monocytogenes on treatment with thyme essential oils (Rasooli et al. 2006). In other studies Gram-positive bacteria have been found to be more sensitive to essential oils than Gram-negative bacteria, which, it has been suggested, may be due to the relatively impermeable outer membrane that surrounds Gram-negative bacteria (Smith-Palmer et al. 1998). Although this explanation has been generally well accepted (Delaquis et al. 2002; Burt 2004), there have also been studies that suggest there is only a time delay in the growth of Gram-negative bacteria. Therefore, over a longer time period the essential oils would have the same effect on both Gram-negative and Gram-positive bacteria (Deans and Ritchie 1987; Tassou and Nychas 1996). This differential sensitivity was seen in this study in vitro (Table 3) but not in the on food studies when Staph. aureus was the least sensitive to the oils or components tested.
Previous studies carried out on the inhibitory effects of essential oil vapours are limited, although the fact that the oils comprise a number of volatile components makes them ideal for this type of research. In this study, although there was a significant difference between the effectiveness of bergamot oil when compared with the vapour on Gram-positive and Gram-negative bacteria individually, overall when all the bacteria were grouped together there was no significant difference between the inhibitory effect of bergamot oil as compared with vapour in vitro. When applied to food (Fig. 3), there was also no statistical significant difference in the performances of the oils when compared with vapours (t = −2·153, d.f. = 15 P = 0·05), although the vapour did take longer to be effective. The oil/components all inhibit bacterial survival in the same way in that there was an initial decrease in viable count followed by a further reduction in number during the next several hours until no further effect was seen. The results of this study suggest that the use of bergamot, citral or linalool vapour may be an effective method for reducing contamination by Gram-positive bacteria on food systems but not Gram-negative contamination. However, exposure time was relatively long which may change the organoleptic properties of the food, thus having implications for costs in the food processing industry.
Citrus oils were more effective on bacteria on cabbage leaf than on chicken skin (Figs 1 and 2). Other studies have noted that higher concentrations of essential oils are needed to be effective when being used on food compared with in vitro studies. In one such study, an increase in concentration of 10-fold when used in pork sausages, 50-fold when used in soup and 25 to 100-fold when used in soft cheese was required to produce a similar effect to that reported in vitro (Tassou and Nychas 1996). One suggestion has been that there is a greater availability of nutrients in the food than in culture medium for the bacteria, thus enabling cells to repair themselves more efficiently. It has also been suggested that the increased concentrations required on salad and vegetables is less than that on foodstuffs with higher lipid contents, as the latter are thought to aid in repair of damage to the cell, as essential oils act upon the lipids of the cell wall (Smith-Palmer et al. 1998). Also the oils may have been less effective on the chicken skin because of the rough surface of the skin, which allowed for greater adhesion by the bacteria. Staph. aureus was not inhibited by citral or bergamot when on chicken skin, which reflected the in vitro results (Table 2), since neither of these substances were as effective on Staph. aureus as they were on the other two Gram-positive bacteria.
Using the disc diffusion method, bergamot, lemon and linalool oils were effective against Camp. jejuni (Table 2) but on chicken skin only linalool has any effect. This emphasizes the fact that care must be taken when suggesting the use of EOs based on in vitro studies. Camp. jejuni is an important enteropathogen present on poultry and, based on in vitro studies, it has been suggested that some Thai medicinal plants would be effective in controlling its entry into the human food chain (Wannissorn et al. 2005). However, the results of this present study, together with other studies (Smith-Palmer et al. 1998; Delaquis et al. 2002; Burt 2004), suggest any recommendations must be based on results from on-food studies.
With changes in legislation and consumer trends for more natural alternatives to chemical-based bactericides (Brul and Coote 1999), citrus EOs may provide a solution for both industry and consumers, although when discussing EOs as potential bactericides for food products the effect that they have on the organoleptic properties must be considered. Although components of citrus EOs are GRAS and their scent and taste lend themselves to food use, too much alteration is not acceptable to the consumer. In one recent study, although the growth of B. cereus was inhibited effectively in carrot broth by carvacrol, cinnamaldehyde and thymol, any amount of carvacrol or thymol was unacceptable to a tasting panel, although cinnamaldehyde was acceptable at levels below 6 μl 100 ml−1 (Valero and Giner 2006). However, the application of ‘hurdle technology’ may allow the use of low levels of EOs in combination with other food preservation techniques such as refrigeration and/or modified atmospheres. Low temperature in combination with cinnamaldehyde and thymol is effective in the case of B. cereus (Valero and Frances 2006), and the combination of low temperature, modified atmospheres and oregano EO is effective against S. typhimurium (Skandamis et al. 2002).
The use of EO vapours may be a potential way of combating the organoleptic effect brought about by direct contact between the food and oil. However, longer exposure to the vapour is required to produce a similar inhibitory effect (18 h as against 60 s) which has cost implications for the food industry. Also, there is the possibility that the vapours would be absorbed into the food matrix, also influencing the organoleptic properties of the food. Certainly after 24 h exposure, in this study, there were obvious physical changes to cabbage leaf. As cross-contamination of foodstuffs can occur at many stages during ‘farm to fork’, for example, during growth or at harvest or during transportation, an assessment of when the application of EOs or their vapours would be most effective needs to be established.
The authors would like to thank Belmay Ltd Wellingborough for the gas chromatography analysis, AMPHORA (Bristol, UK) for the oils and technical information and the Society for Applied Microbiology for a Students into Work grant, which helped fund part of this study.