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Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate
ARTICLE IN PRESS
LWT 41 (2008) 119–127 www.elsevier.com/locate/lwt
Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate P. Kotzekidou, P. Giannakidis, A. Boulamatsis Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Box 250, 54124 Thessaloniki, Greece Received 6 July 2006; received in revised form 24 January 2007; accepted 24 January 2007
Abstract The efficacy of commercially available plant extracts and essential oils used extensively as flavour ingredients in confectionery products were used as antimicrobials in laboratory media against the following microorganisms: Escherichia coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Using the disc diffusion method, inhibition zones in diameter 420 mm were observed by adding 10 ml of each antimicrobial substance on the following microorganisms: lemon flavour applied on E. coli O157:H7, lemongrass essences against S. aureus, plum using a B. cereus strain and strawberry flavour using a L. monocytogenes strain. E. coli O157:H7 strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Lemon flavour, lemongrass essences, pineapple and strawberry flavour inhibited the foodborne pathogens at the lowest concentration (5 ml/100 ml). Plant extracts and essential oils with potent antimicrobial activities were tested in chocolate held at different temperatures (7 and 20 1C) in dry or humidified environment, which resulted in different aw values of the product (i.e. 0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated pathogens. The most inhibitory action was observed by lemon flavour applied on chocolate inoculated with E. coli cocktail culture after storage at 20 1C for 9 days. Plant extracts tested on chocolate show an enhanced inhibitory effect during storage at 20 1C indicating that their application may provide protection in case of storage at the above temperature or even higher. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Antimicrobials; Chocolate; Essential oils; Foodborne pathogens; Plant extracts
1. Introduction Many naturally occurring compounds found in plants, herbs, and spices have been shown to possess antimicrobial functions and serve as a source of antimicrobial agents against foodborne pathogens (Deans & Ritchie, 1987). As essential oils and their constituents are extensively used as flavour ingredients in a wide variety of foods, beverages, and confectionery products, their application in controlling pathogens could reduce the risk of foodborne outbreaks and assure consumers safe food products (Burt, 2004). Consumer demand for less use of synthetic preservatives Corresponding author. Fax: +30 2310 991632.
E-mail address: [email protected] (P. Kotzekidou).
has led to research and use of ‘‘naturally derived’’ antimicrobials. Certain plants and their extracts used as flavouring agents are known to possess antimicrobial activity offering a potential alternative to synthetic preservatives (Gould, 1996). In modern food industries mild processes are applied in order to obtain safe products which have a natural or ‘‘green’’ image (Burt, 2004). Under these conditions the antimicrobial effects of plant extracts and essential oils intend to reduce the proliferation of foodborne pathogens. The antimicrobial activity of plant extracts used as flavouring agents in foods is due to their essential oil fraction (Conner, 1993). Plant essential oils and their components have broad-spectrum activity against both Gram-negative and Gram-positive foodborne pathogens
0023-6438/$30.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.01.016
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(Jay & Rivers, 1984). However, most studies on antimicrobial action of plant extracts have been conducted in vitro and little information exists regarding the antimicrobial activity of commercially available plant extracts and essential oils used as flavouring agents in confectionery products. The most interesting area of application for plant extracts and essential oils is the inhibition of growth and reduction in numbers of the more serious foodborne pathogens such as Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes (Burt, 2004). Salmonellosis is a growing concern to the chocolate industry as Salmonella Typhimurium infections, caused by contaminated chocolate products, have been reported (Kapperud et al., 1990). L. monocytogenes, a pathogen of concern, causing foodborne disease grows rapidly in chocolate milk at 13 1C (Pearson & Marth, 1990). To date chocolate has not been implicated in cases of infection caused by verocytotoxin-producing E. coli (Baylis et al., 2004). The detection of the above mentioned foodborne pathogens in chocolate and cocoa products as well as the increasing consumer demand for effective, safe, and natural products underlines the importance of quantitative data on plant oils and extracts which can be applied in chocolate confectionery. As the composition of plant oils and extracts varies according to local climatic and environmental conditions (Sivropoulou, Kokkini, Lanaras, & Arsenakis, 1995) the application of commercially available plant extracts and essential oils is of importance. The aim of this study was to determine the efficacy of some commercially available plant extracts and essential oils as antimicrobials in laboratory media against E. coli O157:H7, Salmonella Enteritidis, S. Typhimurium, Staphylococcus aureus, L. monocytogenes, and Bacillus cereus strains. Plant extracts and essential oils showing potent antimicrobial activities were then tested in chocolate with different aw values and held at different temperatures (7 and 20 1C) to determine their efficacy on the fate of the inoculated pathogens. The purpose of this was to create comparable, antimicrobial data between in vitro studies and a real food system. 2. Materials and methods 2.1. Test microorganisms Microorganisms used for the experiment are from the collection of our laboratory: E. coli O157:H7 EDL-932, E. coli O157:H7 EDL-933, E. coli O157:H7 St 1, E. coli O157:H7 St 2, S. aureus, S. aureus S-6, L. monocytogenes Scott A, L. monocytogenes 1, L. monocytogenes 2, B. cereus (the above strains were kindly provided by Professor K. Genigeorgis, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki). The strains of S. Enteritidis and S. Typhimurium were obtained from the Institute of Food Hygiene, Thessaloniki, Greece.
Cocktail bacterial cultures obtained by mixing the same population of different strains of each microorganism (i.e. four strains of E. coli O157:H7, three strains of L. monocytogenes, and two strains of S. aureus) were used in the experiments concerning the fate of the inoculated pathogens on chocolate. 2.2. Media All survivor experiments were carried out on Trypticase Soy Agar supplemented with yeast extract (TSAYE: 17 g tryptone, 3 g soya peptone, 2.5 g glucose, 5 g NaCl, 2.5 g K2HPO4, 5 g yeast extract, 15 g agar in 1L distilled water, pH 7.3). All ingredients were purchased by Merck (Darmstadt, Germany). The number of viable cells was determined with plate count method after incubation at 37 1C for 24–48 h. 2.3. Plant extracts and essential oils The plant extracts and essential oils used in this study are listed in Table 1. 2.4. Antibacterial assay using the disc diffusion method Screening of the plant extracts and essential oils was studied by three-point inoculations of each antimicrobial substance on TSAYE in a standard Petri dish from a 16–18 h culture grown in Trypticase Soy Broth supplemented with yeast extract incubated at 37 1C. The cells of the inoculum were in the exponential phase. The concentration of bacteria inoculated in TSAYE was 2.4–5 107 CFU/ml. All experiments were performed in duplicate. Sterilized filter paper discs (Whatman type 1, 0.6 cm in diameter) were placed on the surface of TSAYE. Each one of the antimicrobial substances was tested undiluted as well as diluted in ethanol at concentrations of 5, 15, 25, and 50 ml/100 ml, and 10 ml of each dilution was added to each filter paper disc. In the control, 10 ml ethanol was added to the filter paper. The lowest concentration of plant extracts or essential oils tested for which an inhibitory zone was observed at the points of inoculation after incubation at 37 1C for 24–48 h was recorded. The inhibition zone diameter was measured (including the filter paper disc, 6 mm in diameter) using Vernier calipers and expressed in millimetres. 2.5. Application of plant extracts and essential oils in chocolate inoculated with E. coli O157:H7, L. monocytogenes, and S. aureus Freshly produced milk chocolate bars of 1 kg (ALKH, Loutraki, Greece) were used in this experiment. The product was analysed for occurrence of E. coli O157:H7, L. monocytogenes, and S. aureus. Each chocolate bar was melted in a waterbath at 80 1C, allowed to cool to 30 1C, and inoculated with 1 ml of an exponential culture which
ARTICLE IN PRESS P. Kotzekidou et al. / LWT 41 (2008) 119–127 Table 1 Plant extracts and essential oils used in the experiment Plant extracts and essential oils
Manufacturer
Almond (bitter) Almond (bitter) Apricot Beluda flavouring Apricot Banana Bergamot flavouring oil Bergamot Cointreu Gum mastic oil
Frutarom, Israel ((FI) Greek Green Herbs (GGH) Bush Boake Allen, UK (BBA) Greek Green Herbs Frutarom, Israel Bush Boake Allen, UK Greek Green Herbs Bush Boake Allen, UK Chios Gum Mastic Growers Association, Greece Bush Boake Allen, UK TAKASAGO Europe GmbH (TE) Greek Green Herbs Greek Green Herbs Greek Green Herbs Primavera Life, Germany (PL) Bush Boake Allen, UK Greek Green Herbs Greek Green Herbs Neroli Flavouring (NF) TAKASAGO Europe GmbH Greek Green Herbs Renee` Laurent, France (RL)
Hazelnut flavouring Lemon flavour Lemon Lemongrass essences Mandarin essences Orange Pineapple Plum Rosewater Rosewater Strawberry flavour Strawberry Vanilla flavouring
was a cocktail of bacterial cultures obtained by mixing the same population of the different strains of each microorganism (i.e. E. coli O157:H7, L. monocytogenes, and S. aureus), followed by the addition of 1 ml of a plant extract or essential oil and mixed manually. In control samples 1 ml ethanol was added instead of essential oil. The mixture was dispensed in Petri dishes (each containing 20 g of the mixture) and maintained at 7 or 20 1C in dry as well as in humidified environment (obtained in a desiccator chamber containing distilled water instead of desiccant) at the above temperatures until they were analysed. Each sample was prepared in duplicate, and two independent trials were carried out for each experiment. At 0, 3, 6, 9, 12, 24, 48, 72, 96, 120, and in some experiments in addition at 168, and 216 h the content of the plate was mixed with sterile peptone water (0.1 g/100 ml) in a ratio 1:9 and homogenized in a stomacher for 2 min. The samples were plated on CT-SMAC medium, PALCAM agar, and BairdParker agar (Merck, Darmstadt, Germany) for enumeration of inoculated E. coli O157:H7, L. monocytogenes, and S. aureus counts, respectively. At the end of the experiments the pH and water activity (aw) values of the control samples were measured. The pH of the melted chocolate was measured by a pH-meter (WTW, Weilheim, Germany). The mean pH value of the control samples was 5.3. The aw was measured with a Rotronic-Hygroscop DT (Rotronic, Bassersdorf, Switzerland). The equilibrium aw values of chocolate samples held at 7 and 20 1C were 0.450 and 0.340, respectively, whereas those held at the above temperatures in humidified environment were 0.822.
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Each experiment was repeated on two separate occasions, in duplicate. 3. Results The 22 plant extracts and essential oils tested showed various degrees of inhibition against the 12 bacterial strains using the disc diffusion method as presented in Table 2. Plant extracts with an enhanced inhibitory effect in decreasing order were: strawberry (GGH) which inhibited all strains; strawberry flavour and almond (GGH) (inhibition of 11 strains); apricot Beluda flavouring, apricot, bergamot, plum, rosewater (GGH), and pineapple (inhibition of nine strains); lemon flavour; almond bitter (FI) and banana (inhibition of seven strains); hazelnut flavouring and rosewater (NF) (inhibition of six strains); cointreu (inhibition of five strains); lemon (GGH) and lemongrass essences (inhibition of four strains); bergamot flavouring oil, and gum mastic oil (inhibition of three strains); mandarin essences (inhibition of two strains); orange and vanilla flavouring which inhibited one strain. Inhibition zones in diameter 420 mm were observed by adding 10 ml of each antimicrobial substance using inoculum of 2.4–5 107 CFU/ml of the following microorganisms: lemon flavour against a strain of E. coli O157:H7 (zone diameter 21.8 mm), lemongrass essences against S. aureus (zone diameter 25.1 mm), plum using a B. cereus strain (zone diameter 20.2 mm) and strawberry flavour using a L. monocytogenes strain (zone diameter 28.2 mm). E. coli strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Plant extracts and essential oils which were the most inhibitory against the bacterial strains tested as well as those producing a clear zone of inhibition 420 mm (in diameter) were selected for determining their inhibitory action diluted in ethanol obtaining a concentration of 5, 15, 25, 50 ml/100 ml, respectively. Table 3 shows the lowest concentration of 11 plant extracts and oils against the foodborne pathogens tested by the disc diffusion assay. Lemon flavour, pineapple, and rosewater (GGH) with the minimum inhibitory concentration of 5 ml/100 ml had an inhibitory action against E. coli O157:H7 EDL-933. S. Typhimurium was inhibited by 5 ml/100 ml of lemon flavour and lemongrass essences. Lemon flavour and lemongrass essences at concentrations of 15 ml/100 ml inhibited strains of S. aureus. Strains of L. monocytogenes and S. Enteritidis were inhibited by 15 ml/100 ml of lemon flavour, whereas B. cereus was inhibited by 15 ml/100 ml of plum. The results show that the zone of inhibition is a practical approach for screening different concentrations of potential antimicrobial substances. Based on the disc diffusion studies apricot, lemon, lemongrass, plum, and strawberry were selected for further studies in chocolate. Since in disc diffusion experiment the different bacterial strains of E. coli O157:H7, S. aureus, and L. monocytogenes responded differently to the action of the
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Table 2 Zone of inhibition of growth of foodborne pathogensa by plant extracts and essential oils Plant extracts and essential oils
E. coli EDL-933
E. coli O157:H7 St 1
E. coli O157:H7 St 2
S. Enteritidis
S. Typhimurium
S. auureus
S. aureus S-6
L. monocytogenes Scott A
L. monocytogenes 1
L. monocytogenes 2
B. cereus
7.070.40c
n.i.d
9.270.3
7.570.9
n.i.
6.170.6
7.570.6
n.i.
n.i.
10.171.1
7.270.6
n.i.
8.370.3
8.470.4
9.170.7
9.871.0
9.270.5
8.270.7
9.870.9
10.271.1
7.670.8
10.270.9
n.i.
7.270.7
11.270.2
7.370.4
10.471.2
11.371.2
9.570.8
6.370.5
10.171.0
11.371.4
n.i.
11.371.3
n.i.
n.i.
10.570.5 13.270.2 5.170.1
9.570.3 8.470.6 n.i.
11.671.1 n.i. n.i.
9.670.8 10.271.1 n.i.
8.470.6 7.770.8 n.i.
11.470.8 9.571.1 6.670.7
7.370.6 n.i. 14.271.3
n.i. n.i. n.i.
n.i. n.i. n.i.
12.470.9 9.270.8 n.i.
n.i. n.i. n.i.
8.170.9 7.270.8 n.i.
9.670.6 7.770.7 n.i. 5.170.1
12.370.5 13.770.6 n.i. n.i.
13.570.9 14.171.3 n.i. 6.770.9
10.270.6 10.771.2 n.i. 7.370.8
10.271.2 12.371.2 n.i. 6.270.6
12.271.3 n.i. 6.770.8 n.i.
n.i. n.i. 13.571.4 n.i.
15.271.5 n.i. 12.471.3 n.i.
n.i. n.i. n.i. n.i.
11.270.9 n.i. n.i. 7.270.8
n.i. n.i. n.i. 14.171.2
8.170.9 n.i. n.i. n.i.
n.i. n.i. n.i.
13.470.6 21.871.2 n.i.
n.i. 10.371.2 n.i.
n.i. 11.470.7 n.i.
n.i. 11.170.9 n.i.
6.470.6 9.671.2 8.570.7
13.271.1 7.470.8 15.271.3
n.i. n.i. 25.171.8
n.i. n.i. 15.271.3
n.i. 9.370.7 n.i.
15.371.4 n.i. n.i.
n.i. n.i. n.i.
n.i.
n.i.
n.i.
n.i.
6.570.5
n.i.
n.i.
12.271.1
n.i.
n.i.
n.i.
n.i.
n.i. 6.27 0.1 5.270.2 11.870.8 11.670.6 10.47 0.4
n.i. 9.470.6 10.771.1 10.970.6 8.370.4 11.271.5
n.i. 11.270.9 11.370.7 11.571.1 10.770.8 9.671.0
n.i. 8.570.8 11.771.2 10.370.9 8.470.7 9.370.8
n.i. 9.370.7 8.670.7 11.271.3 8.770.8 9.570.6
n.i. 11.271.1 6.270.4 8.670.9 7.570.6 6.470.5
n.i. 5.370.4 n.i. n.i. n.i. 10.271.2
12.371.2 n.i. 9.470.9 11.370.8 n.i. 10.570.7
n.i. n.i. n.i. n.i. n.i. 15.171.6
n.i. n.i. 12.371.2 12.271.3 n.i. 9.570.9
n.i. 6.470.7 n.i. n.i. n.i. 9.570.8
n.i. 6.370.5 20.271.9 7.270.8 n.i. 10.271.3
12.770.7
13.771.1
13.471.3
10.671.3
n.i.
9.770.8
7.370.7
12.470.8
28.271.9
12.271.4
18.271.5
8.670.9
5.370.3
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
Obtained by adding 10 ml of each undiluted antimicrobial substance. The diameter of the filter paper discs (6 mm) is included. Inoculum level: 2.4–5 107 CFU/ml. c Values are mean7standard deviation. d No zone of inhibition observed. b
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a
E. coli EDL-932
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Almond (bitter) FI Almond (bitter) GGH Apricot Beluda flavouring Apricot GGH Banana Bergamot flavouring oil Bergamot GGH Cointreu Gum mastic oil Hazelnut flavouring Lemon GGH Lemon flavour Lemongrass essences Mandarin essences Orange Pineapple Plum Rosewater GGH Rosewater (NF) Strawberry GGH Strawberry flavour Vanilla flavouring
Inhibition zone diameter (mm)b
Table 3 Zones of inhibition of growth of foodborne pathogens obtained by the lowest concentration (i.e. 5, 15, 25, or 50 ml/100 ml) of plant extract and essential oil Plant extracts and essential oils
Inhibition zone diameter (mm)a Con. ml/100 ml
Control Almond (bitter) GGH
Apricot Beluda flavouring
Apricot GGH
Lemongrass essences
Pineapple
Plum
Rosewater GGH
Strawberry GGH
Strawberry flavour
a
E. coli O157:H7 St 1
E. coli O157:H7 St 2
S. Enteritidis
S. Typhimurium
S. aureus
S. aureus S6
L. monocytogenes Scott A
L. monocytogenes 1
L. monocytogenes 2
B. cereus
n.i.b n.i. n.i. 12.371.2 14.171.4 n.i. n.i. n.i. 17.271.5 n.i. n.i. n.i. n.i. n.i. n.i. 11.271.3 15.470.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.371.0 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.571.9
n.i. n.i. n.i. n.i. 13.271.0 n.i. n.i. n.i. 12.470.9 n.i. n.i. 10.671.1 12.371.3 n.i. n.i. n.i. 12.571.4 18.471.9 22.371.8 23.271.7 25.471.6 n.i. n.i. n.i. n.i. 11.271.3 11.171.1 12.271.0 13.471.5 n.i. 14.371.6 14.270.9 15.171.2 11.270.8 13.670.7 12.670.8 14.271.2 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.371.3
n.i. n.i. n.i. 10.270.7 13.271.4 n.i. n.i. 9.370.8 10.171.2 n.i. 8.470.9 11.270.8 15.171.2 n.i. n.i. n.i. 10.670.9 n.i. n.i. n.i. 10.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.571.4 n.i. n.i. n.i. 13.271.1 n.i. n.i. n.i. 15.371.2 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 16.471.6
n.i. n.i. n.i. n.i. 12.270.9 n.i. n.i. 11.370.7 14.270.6 n.i. n.i. n.i. 12.170.5 n.i. n.i. n.i. 10.370.9 n.i. n.i. n.i. 16.571.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.470.8 n.i. n.i. n.i. 16.570.9 n.i. n.i. n.i. n.i. n.i. n.i. 12.270.9 12.371.1 12.171.2 13.671.0 14.771.3 15.470.9
n.i. n.i. n.i. n.i. 12.270.8 n.i. n.i. n.i. 13.470.8 n.i. n.i. 11.370.9 12.171.1 n.i. n.i. 10.270.9 13.471.1 n.i. 12.371.2 13.471.1 12.570.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.270.9 n.i. 10.270.8 11.470.9 12.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.471.0 n.i. n.i. n.i. n.i.
n.i. n.i. n.i. 8.370.6 10.170.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.170.9 9.270.7 10.370.8 11.271.0 10.371.2 11.271.1 12.471.5 13.670.9 n.i. n.i. n.i. 11.270.8 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.370.9
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.070.5 9.270.6 10.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.270.8 n.i. n.i. n.i. n.i. n.i. 19.370.9 21.271.8 23.471.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 15.170.9 16.271.0 18.371.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.270.9 9.470.8 11.271.0 15.47 1.5 22.571.8
n.i. n.i. n.i. n.i. 10.370.5 n.i. n.i. 11.570.7 13.270.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.370.8 n.i. 11.570.9 9.370.6 8.470.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.270.8 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.370.7 n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.170.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 9.470.7 11.270.6 13.570.9 20.271.4
n.i. n.i. n.i. n.i. 8.570.6 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.470.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 7.270.3 n.i. 10.170.4 12.470.6 17.370.6 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 7.570.5 n.i. n.i. n.i. n.i.
No zone of inhibition observed. Values are mean7standard deviation.
b
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Lemon flavour
E. coli EDL-933
P. Kotzekidou et al. / LWT 41 (2008) 119–127
Bergamot GGH
5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50
E. coli EDL-932
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above plant extracts, a cocktail of bacterial cultures obtained by mixing the same population of the different strains of each microorganism was used in the experiments concerning the fate of the inoculated pathogens in chocolate. For practical application of plant extracts or essential oils in chocolate, it is important to know if very low concentrations (i.e. 0.1 ml/100 g) have any antimicrobial effects. Plant extracts differed significantly in their ability to inhibit the growth of E. coli pooled inoculum in chocolate with aw 0.450 held at 7 1C. Among the four extracts (i.e. apricot, lemon, plum, and strawberry) added at concentration of 0.1 ml/100 g, lemon was the most inhibitory to E. coli O157:H7. At low temperature storage (7 1C), a nearly 1.7-log reduction in E. coli O157:H7 was observed in samples inoculated with ca. 105 CFU/g and treated with lemon. A quick repression of the inoculum was observed within 2 days (Fig. 1). The results showed that strawberry is able to suppress E. coli O157:H7 by 1-log during 7 days of storage at 7 1C. In contrast, plum completely inhibited the growth of the inoculated E. coli O157:H7 showing bacteriostatic results and a reduction of 0.4-log after 9 days of storage. Apricot resulted in no clear decline of the population of E. coli O157:H7. In control samples of chocolate a slight increase of the inoculated population was observed during storage at 7 1C. In chocolate samples held at 20 1C with aw 0.340, lemon added at concentration of 0.1 ml/100 g suppressed quickly the inoculated population of E. coli O157:H7 (ca. 105 CFU/g) within 24 h by about 1.8-log cycles. The most inhibitory/bactericidal action was observed after 9 days of storage causing a 3.3-log reduction of the inoculum (Fig. 1). Strawberry and plum possessed the ability to suppress E. coli O157:H7. However, the reduction in populations was not as much as with lemon treatment. Chocolate samples treated with apricot extract suppressed growth of E. coli O157:H7 and reduced the inoculum (ca. 105 CFU/g) after 9 days of storage by about 1-log
cycle. During the first 24 h after inoculation of the control chocolate samples a slight increase of the inoculated bacterial population was observed (by about 0.6-log), followed by reduction of E. coli O157:H7 population after prolonged storage of chocolate at 20 1C, due to the low aw (0.340) of the product. As shown in Fig. 1 increased storage temperature enhanced the inhibitory effect of the plant extracts tested on chocolate. Especially, application of lemon flavour on chocolate resulted in 3.3-log decrease of the inoculated E. coli O157:H7 after prolonged storage at 20 1C. According to this observation, plant extracts may be more effective in case of abused storage temperatures. The high initial population of L. monocytogenes (inoculum ca. 105 CFU/g) reduced by about 0.6 to 1.2-log cycles in control samples as well as in chocolate containing strawberry flavour (0.1 ml/100 g) during the first 9 h of storage (Fig. 2). The most drastic effect (i.e. a reduction of 1.5-log) was observed in chocolate containing strawberry flavour held at 20 1C after 5 days of storage. The effect of strawberry flavour was bactericidal and the reduced L. monocytogenes population remained at low levels during prolonged storage in dry or humidified environment at 7 or 20 1C. In control samples the inoculated L. monocytogenes was reduced in the first 24 h, but later during storage at 7 1C the population of L. monocytogenes was slightly increased in dry as well as in humidified environment, whereas this effect was less pronounced at 20 1C. In samples where 0.1 ml/100 g strawberry extract was added a reduction of 1.5- and 1.2-log cycles occurred in dry and humidified environment, respectively. Lemongrass essences added in chocolate held in humidified or dry environment at 7 as well as at 20 1C (Fig. 3) had a slight bactericidal effect on inoculated S. aureus (inoculum level ca. 105 CFU/g) but there was no great difference between samples where lemongrass essences were added and control samples. The reduced population of inoculated pathogen fluctuated during prolonged storage at the different environmental conditions. The
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Fig. 1. Inhibition of E. coli O157:H7 pooled inoculum by plant extracts added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C and aw 0.450 and (b) at 20 1C and aw 0.340. Symbols: (x) control experiment without plant extract added; (n) lemon flavour; (&) plum; (J) strawberry flavour; (B) apricot Beluda flavouring. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
ARTICLE IN PRESS P. Kotzekidou et al. / LWT 41 (2008) 119–127
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Fig. 2. Inhibition of L. monocytogenes pooled inoculum by strawberry flavour added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C at aw 0.450 (), and aw 0.822 (’); (b) at 20 1C at aw 0.340 (m), and aw 0.822 (’). Open symbols: control experiment without essential oil added. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
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Fig. 3. Inhibition of S. aureus pooled inoculum by lemongrass essences added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C at aw 0.450 (), and aw 0.822 (’); (b) at 20 1C at aw 0.340 (m), and aw 0.822 (’). Open symbols: control experiment without essential oil added. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
strongest inhibitory effect (reduction of ca. 1.4-log) was observed in chocolate samples containing lemongrass essences and held at 20 1C in humidified environment for 5 days. In some cases the inhibitory effect observed within the first 12 h could be overcome by inoculated microorganisms during prolonged storage time. Therefore, lemongrass alone cannot provide complete protection against S. aureus in chocolate. 4. Discussion Extracts from plants are widely used in the food industry and are considered GRAS. They usually contain more than a single compound with antimicrobial activity. Hence, using extracts has, as a consequence, to take advantage of all active compounds present in extracts as reported by Hao, Brackett, and Doyle (1998). Plant-derived essential oils due to their content of antimicrobial compounds
possess potential as natural agents for food preservation. Their antimicrobial activity is assigned to a number of small terpenoid and phenolic compounds which, due to their lipophilic character, accumulate in bacterial membranes causing energy depletion (Conner, 1993). Our results show that Gram-positive and Gram-negative organisms were affected by the plant extracts and essential oils tested. S. Typhimurium (Gram-negative) was the most sensitive strain as it was inhibited by 17 of the compounds tested, while L. monocytogenes Scott A (Gram-positive) was the most resistant strain, as it was inhibited by four of the compounds tested. Our results agree with the observation of Dorman and Deans (2000) that the susceptibility of bacteria to plant volatile oils and the Gram reaction appears to have little influence on growth inhibition. Similar observations were reported by Hao et al. (1998) that the Gram-negative Aeromonas hydrophila is more sensitive to plant extracts and essential oils than the
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Gram-positive L. monocytogenes. Other researchers reported that essential oils are slightly more active against Gram-positive than Gram-negative bacteria as Gramnegative organisms are less susceptible to the action of antibacterials (Shelef, Jyothi, & Bulgarelli, 1984; Vaara, 1992). Gram-negative bacteria possess a hydrophilic outer membrane, due to the presence of lipopolysaccharide molecules (Nikaido, 1996). Small hydrophilic solutes are able to pass the outer membrane through abundant porin proteins providing hydrophilic trans-membrane channels, whereas the outer membrane serves as a penetration barrier towards macromolecules and to hydrophobic compounds (Nikaido, 1996). But the outer membrane is not totally impermeable to hydrophobic molecules, some of which can slowly traverse through porins. Bypassing the outer membrane is a prerequisite for any solute to exert bactericidal activity. Thus, it is worthwhile to evaluate antimicrobial compounds in a real food system, because low molecular lipophilic compounds, despite their limited solubility in water, are able to penetrate Gram-negative bacteria and influence their proliferation in food-related environments (Helander et al., 1998). Plant extracts and essential oils tested may exhibit different modes of action against the bacterial strains, such as interference with the phospholipid bilayer of the cell membrane which has as a consequence a permeability increase and loss of cellular constituents, damage of the enzymes involved in the production of cellular energy and synthesis of structural components, and destruction or inactivation of genetic material (Kim, Marshall, & Wei, 1995). In general, the mechanism of action is considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents (Davidson, 1997; Sikkema, de Bont, & Poolman, 1995). Comparison of published data is complicated, as the outcome of a test is affected by factors such as the volume of inoculum, growth phase, culture medium used, pH of the media, and incubation time and temperature (Friedman, Henika, & Mandrell, 2002; Rios, Recio, & Villar, 1988). Especially, during their application in foods the lower water content of food compared to laboratory media may hamper the progress of antibacterial agents to the target site in the bacterial cell (Smith-Palmer, Stewart, & Fyfe, 2001). The plant extracts and essential oils added to chocolate not only affected the progress of bacterial growth but also resulted in a greater than 1-log reduction of the initial viable bacterial population. Carbohydrates in foods do not appear to protect bacteria from the action of essential oils as much as fat do (Shelef et al., 1984). In artificially contaminated chocolate verocytotoxin-producing strains E. coli O157:H7, O111:H-, and O26:H11 survived in products stored at 22 1C for up to 90 days and in products stored at 10 1C they could be detected after 366 days of storage (Baylis et al., 2004). Our results revealed that the
plant extracts and essential oils applied to chocolate were bactericidal to E. coli O157:H7, L. monocytogenes, and S. aureus. Application of the above antimicrobial substances resulted in an immediate reduction in population of pathogens. The results indicate that lemon flavour was the most effective extract against E. coli O157:H7, and that strawberry flavour had a more inhibitory effect than plum and apricot extract. Especially in case of S. aureus and B. cereus, it is of importance that essential oils cause inhibition of toxin production, due to lower specific growth rate which has as a consequence that the cells use all the available energy to sustain viability, leaving little left for toxin production, whereas if toxin excretion is an active process, there may be insufficient ATP or proton motive force to export it from the cell (Ultee & Smid, 2001). In conclusion, our results show that plant extracts and essential oils alone cannot provide complete protection against pathogens in chocolate. Their antimicrobial activity diminishes in food systems as they appeared less efficacious when added to chocolate. But, they might prove to be more effective in food products that have a low level of bacterial contamination. Increased storage temperature enhanced the inhibitory effect of all plant extracts tested on chocolate, indicating that the application of the tested plant extracts may provide protection in case of storage at 20 1C or higher temperature. The use of plant extracts and essential oils in consumer goods is expected to increase in the future due to the rise of ‘‘green consumerism’’, which stimulates the use and development of products derived from plants (Tuley de Silva, 1996), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials. References Baylis, C. L., MacPhee, S., Robinson, A. J., Griffiths, R., Lilley, K., & Betts, R. P. (2004). Survival of Escherichia coli O157:H7, O111:H- and O26:H11 in artificially contaminated chocolate and confectionery products. International Journal of Food Microbiology, 96, 35–48. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology, 94, 223–253. Conner, D. E. (1993). Naturally occurring compounds. In P. M. Davidson, & A. L. Branen (Eds.), Antimicrobials in foods (pp. 441–468). New York: Marcel Dekker. Davidson, P. M. (1997). Chemical preservatives and natural antimicrobial compounds. In M. P. Doyle, L. R. Beuchat, & T. J. Montville (Eds.), Food microbiology. Fundamentals and frontiers (pp. 520–556). Washington, DC: ASM Publications. Deans, S. G., & Ritchie, G. A. (1987). Antimicrobial properties of plant essential oils. International Journal of Food Microbiology, 5, 165–180. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308–316. Friedman, M., Henika, P. R., & Mandrell, R. E. (2002). Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella enterica. Journal of Food Protection, 65, 1545–1560. Gould, G.W., 1996. Industry perspectives on the use of natural antimicrobials and inhibitors for food applications. Journal of Food Protection (suppl.), 82–86.
ARTICLE IN PRESS P. Kotzekidou et al. / LWT 41 (2008) 119–127 Hao, Y. Y., Brackett, R. E., & Doyle, M. P. (1998). Efficacy of plant extracts in inhibiting Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked poultry. Food Microbiology, 15, 367–378. Helander, I. M., Alakomi, H.-L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E. J., et al. (1998). Characterization of the action of selected essential oil components on Gram-negative bacteria. Journal of Agricultural and Food Chemistry, 46, 3590–3595. Jay, J. M., & Rivers, G. M. (1984). Antimicrobial activity of some food flavouring compounds. Journal of Food Safety, 6, 129–139. Kapperud, G., Gustavsen, S., Hellesnes, I., Hansen, A. H., Lassen, J., Hirn, J., et al. (1990). Outbreak of Salmonella typhimurium infection traced to contaminated chocolate and caused by a strain lacking the 60-megadalton virulence plasmid. Journal of Clinical Microbiology, 28, 2597–2601. Kim, J., Marshall, M. R., & Wei, C. (1995). Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry, 43, 2839–2845. Nikaido, H. (1996). Outer membrane. In: F. C. Neidhardt, (Ed.), Escherichia coli and Salmonella: Cellular and molecular biology (Vol. 1, pp. 29–47). Washington, DC: ASM Press. Pearson, L. J., & Marth, E. H. (1990). Behavior of Listeria monocytogenes in the presence of cocoa, carrageenan, and sugar in a milk medium
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incubated with and without agitation. Journal of Food Protection, 53, 30–37. Rios, J. L., Recio, M. C., & Villar, A. (1988). Screening methods for natural products with antibacterial activity: A review of the literature. Journal of Ethnopharmacology, 23, 127–149. Shelef, L. A., Jyothi, E. K., & Bulgarelli, M. A. (1984). Growth of enteropathogenic and spoilage bacteria in sage-containing broth and foods. Journal of Food Science, 49, 737–740 809. Sikkema, J., de Bont, J. A. M., & Poolman, B. (1995). Mechanism of membrane toxicity of hydrocarbons. Microbiological Reviews, 59, 201–222. Sivropoulou, A., Kokkini, S., Lanaras, T., & Arsenakis, M. (1995). Antimicrobial activity of mint essential oils. Journal of Agricultural and Food Chemistry, 43, 2384–2388. Smith-Palmer, A., Stewart, J., & Fyfe, L. (2001). The potential application of plant essential oils as natural food preservatives in soft cheese. Food Microbiology, 18, 463–470. Tuley de Silva, K. (1996). A manual on the essential oil industry. Vienna: United Nations Industrial Development Organization. Ultee, A., & Smid, E. J. (2001). Influence of carvacrol on growth and toxin production by Bacillus cereus. International Journal of Food Microbiology, 64, 373–378. Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Reviews, 56, 395–411.
LWT 41 (2008) 119–127 www.elsevier.com/locate/lwt
Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate P. Kotzekidou, P. Giannakidis, A. Boulamatsis Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Box 250, 54124 Thessaloniki, Greece Received 6 July 2006; received in revised form 24 January 2007; accepted 24 January 2007
Abstract The efficacy of commercially available plant extracts and essential oils used extensively as flavour ingredients in confectionery products were used as antimicrobials in laboratory media against the following microorganisms: Escherichia coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Using the disc diffusion method, inhibition zones in diameter 420 mm were observed by adding 10 ml of each antimicrobial substance on the following microorganisms: lemon flavour applied on E. coli O157:H7, lemongrass essences against S. aureus, plum using a B. cereus strain and strawberry flavour using a L. monocytogenes strain. E. coli O157:H7 strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Lemon flavour, lemongrass essences, pineapple and strawberry flavour inhibited the foodborne pathogens at the lowest concentration (5 ml/100 ml). Plant extracts and essential oils with potent antimicrobial activities were tested in chocolate held at different temperatures (7 and 20 1C) in dry or humidified environment, which resulted in different aw values of the product (i.e. 0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated pathogens. The most inhibitory action was observed by lemon flavour applied on chocolate inoculated with E. coli cocktail culture after storage at 20 1C for 9 days. Plant extracts tested on chocolate show an enhanced inhibitory effect during storage at 20 1C indicating that their application may provide protection in case of storage at the above temperature or even higher. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Antimicrobials; Chocolate; Essential oils; Foodborne pathogens; Plant extracts
1. Introduction Many naturally occurring compounds found in plants, herbs, and spices have been shown to possess antimicrobial functions and serve as a source of antimicrobial agents against foodborne pathogens (Deans & Ritchie, 1987). As essential oils and their constituents are extensively used as flavour ingredients in a wide variety of foods, beverages, and confectionery products, their application in controlling pathogens could reduce the risk of foodborne outbreaks and assure consumers safe food products (Burt, 2004). Consumer demand for less use of synthetic preservatives Corresponding author. Fax: +30 2310 991632.
E-mail address: [email protected] (P. Kotzekidou).
has led to research and use of ‘‘naturally derived’’ antimicrobials. Certain plants and their extracts used as flavouring agents are known to possess antimicrobial activity offering a potential alternative to synthetic preservatives (Gould, 1996). In modern food industries mild processes are applied in order to obtain safe products which have a natural or ‘‘green’’ image (Burt, 2004). Under these conditions the antimicrobial effects of plant extracts and essential oils intend to reduce the proliferation of foodborne pathogens. The antimicrobial activity of plant extracts used as flavouring agents in foods is due to their essential oil fraction (Conner, 1993). Plant essential oils and their components have broad-spectrum activity against both Gram-negative and Gram-positive foodborne pathogens
0023-6438/$30.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.01.016
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(Jay & Rivers, 1984). However, most studies on antimicrobial action of plant extracts have been conducted in vitro and little information exists regarding the antimicrobial activity of commercially available plant extracts and essential oils used as flavouring agents in confectionery products. The most interesting area of application for plant extracts and essential oils is the inhibition of growth and reduction in numbers of the more serious foodborne pathogens such as Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes (Burt, 2004). Salmonellosis is a growing concern to the chocolate industry as Salmonella Typhimurium infections, caused by contaminated chocolate products, have been reported (Kapperud et al., 1990). L. monocytogenes, a pathogen of concern, causing foodborne disease grows rapidly in chocolate milk at 13 1C (Pearson & Marth, 1990). To date chocolate has not been implicated in cases of infection caused by verocytotoxin-producing E. coli (Baylis et al., 2004). The detection of the above mentioned foodborne pathogens in chocolate and cocoa products as well as the increasing consumer demand for effective, safe, and natural products underlines the importance of quantitative data on plant oils and extracts which can be applied in chocolate confectionery. As the composition of plant oils and extracts varies according to local climatic and environmental conditions (Sivropoulou, Kokkini, Lanaras, & Arsenakis, 1995) the application of commercially available plant extracts and essential oils is of importance. The aim of this study was to determine the efficacy of some commercially available plant extracts and essential oils as antimicrobials in laboratory media against E. coli O157:H7, Salmonella Enteritidis, S. Typhimurium, Staphylococcus aureus, L. monocytogenes, and Bacillus cereus strains. Plant extracts and essential oils showing potent antimicrobial activities were then tested in chocolate with different aw values and held at different temperatures (7 and 20 1C) to determine their efficacy on the fate of the inoculated pathogens. The purpose of this was to create comparable, antimicrobial data between in vitro studies and a real food system. 2. Materials and methods 2.1. Test microorganisms Microorganisms used for the experiment are from the collection of our laboratory: E. coli O157:H7 EDL-932, E. coli O157:H7 EDL-933, E. coli O157:H7 St 1, E. coli O157:H7 St 2, S. aureus, S. aureus S-6, L. monocytogenes Scott A, L. monocytogenes 1, L. monocytogenes 2, B. cereus (the above strains were kindly provided by Professor K. Genigeorgis, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki). The strains of S. Enteritidis and S. Typhimurium were obtained from the Institute of Food Hygiene, Thessaloniki, Greece.
Cocktail bacterial cultures obtained by mixing the same population of different strains of each microorganism (i.e. four strains of E. coli O157:H7, three strains of L. monocytogenes, and two strains of S. aureus) were used in the experiments concerning the fate of the inoculated pathogens on chocolate. 2.2. Media All survivor experiments were carried out on Trypticase Soy Agar supplemented with yeast extract (TSAYE: 17 g tryptone, 3 g soya peptone, 2.5 g glucose, 5 g NaCl, 2.5 g K2HPO4, 5 g yeast extract, 15 g agar in 1L distilled water, pH 7.3). All ingredients were purchased by Merck (Darmstadt, Germany). The number of viable cells was determined with plate count method after incubation at 37 1C for 24–48 h. 2.3. Plant extracts and essential oils The plant extracts and essential oils used in this study are listed in Table 1. 2.4. Antibacterial assay using the disc diffusion method Screening of the plant extracts and essential oils was studied by three-point inoculations of each antimicrobial substance on TSAYE in a standard Petri dish from a 16–18 h culture grown in Trypticase Soy Broth supplemented with yeast extract incubated at 37 1C. The cells of the inoculum were in the exponential phase. The concentration of bacteria inoculated in TSAYE was 2.4–5 107 CFU/ml. All experiments were performed in duplicate. Sterilized filter paper discs (Whatman type 1, 0.6 cm in diameter) were placed on the surface of TSAYE. Each one of the antimicrobial substances was tested undiluted as well as diluted in ethanol at concentrations of 5, 15, 25, and 50 ml/100 ml, and 10 ml of each dilution was added to each filter paper disc. In the control, 10 ml ethanol was added to the filter paper. The lowest concentration of plant extracts or essential oils tested for which an inhibitory zone was observed at the points of inoculation after incubation at 37 1C for 24–48 h was recorded. The inhibition zone diameter was measured (including the filter paper disc, 6 mm in diameter) using Vernier calipers and expressed in millimetres. 2.5. Application of plant extracts and essential oils in chocolate inoculated with E. coli O157:H7, L. monocytogenes, and S. aureus Freshly produced milk chocolate bars of 1 kg (ALKH, Loutraki, Greece) were used in this experiment. The product was analysed for occurrence of E. coli O157:H7, L. monocytogenes, and S. aureus. Each chocolate bar was melted in a waterbath at 80 1C, allowed to cool to 30 1C, and inoculated with 1 ml of an exponential culture which
ARTICLE IN PRESS P. Kotzekidou et al. / LWT 41 (2008) 119–127 Table 1 Plant extracts and essential oils used in the experiment Plant extracts and essential oils
Manufacturer
Almond (bitter) Almond (bitter) Apricot Beluda flavouring Apricot Banana Bergamot flavouring oil Bergamot Cointreu Gum mastic oil
Frutarom, Israel ((FI) Greek Green Herbs (GGH) Bush Boake Allen, UK (BBA) Greek Green Herbs Frutarom, Israel Bush Boake Allen, UK Greek Green Herbs Bush Boake Allen, UK Chios Gum Mastic Growers Association, Greece Bush Boake Allen, UK TAKASAGO Europe GmbH (TE) Greek Green Herbs Greek Green Herbs Greek Green Herbs Primavera Life, Germany (PL) Bush Boake Allen, UK Greek Green Herbs Greek Green Herbs Neroli Flavouring (NF) TAKASAGO Europe GmbH Greek Green Herbs Renee` Laurent, France (RL)
Hazelnut flavouring Lemon flavour Lemon Lemongrass essences Mandarin essences Orange Pineapple Plum Rosewater Rosewater Strawberry flavour Strawberry Vanilla flavouring
was a cocktail of bacterial cultures obtained by mixing the same population of the different strains of each microorganism (i.e. E. coli O157:H7, L. monocytogenes, and S. aureus), followed by the addition of 1 ml of a plant extract or essential oil and mixed manually. In control samples 1 ml ethanol was added instead of essential oil. The mixture was dispensed in Petri dishes (each containing 20 g of the mixture) and maintained at 7 or 20 1C in dry as well as in humidified environment (obtained in a desiccator chamber containing distilled water instead of desiccant) at the above temperatures until they were analysed. Each sample was prepared in duplicate, and two independent trials were carried out for each experiment. At 0, 3, 6, 9, 12, 24, 48, 72, 96, 120, and in some experiments in addition at 168, and 216 h the content of the plate was mixed with sterile peptone water (0.1 g/100 ml) in a ratio 1:9 and homogenized in a stomacher for 2 min. The samples were plated on CT-SMAC medium, PALCAM agar, and BairdParker agar (Merck, Darmstadt, Germany) for enumeration of inoculated E. coli O157:H7, L. monocytogenes, and S. aureus counts, respectively. At the end of the experiments the pH and water activity (aw) values of the control samples were measured. The pH of the melted chocolate was measured by a pH-meter (WTW, Weilheim, Germany). The mean pH value of the control samples was 5.3. The aw was measured with a Rotronic-Hygroscop DT (Rotronic, Bassersdorf, Switzerland). The equilibrium aw values of chocolate samples held at 7 and 20 1C were 0.450 and 0.340, respectively, whereas those held at the above temperatures in humidified environment were 0.822.
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Each experiment was repeated on two separate occasions, in duplicate. 3. Results The 22 plant extracts and essential oils tested showed various degrees of inhibition against the 12 bacterial strains using the disc diffusion method as presented in Table 2. Plant extracts with an enhanced inhibitory effect in decreasing order were: strawberry (GGH) which inhibited all strains; strawberry flavour and almond (GGH) (inhibition of 11 strains); apricot Beluda flavouring, apricot, bergamot, plum, rosewater (GGH), and pineapple (inhibition of nine strains); lemon flavour; almond bitter (FI) and banana (inhibition of seven strains); hazelnut flavouring and rosewater (NF) (inhibition of six strains); cointreu (inhibition of five strains); lemon (GGH) and lemongrass essences (inhibition of four strains); bergamot flavouring oil, and gum mastic oil (inhibition of three strains); mandarin essences (inhibition of two strains); orange and vanilla flavouring which inhibited one strain. Inhibition zones in diameter 420 mm were observed by adding 10 ml of each antimicrobial substance using inoculum of 2.4–5 107 CFU/ml of the following microorganisms: lemon flavour against a strain of E. coli O157:H7 (zone diameter 21.8 mm), lemongrass essences against S. aureus (zone diameter 25.1 mm), plum using a B. cereus strain (zone diameter 20.2 mm) and strawberry flavour using a L. monocytogenes strain (zone diameter 28.2 mm). E. coli strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Plant extracts and essential oils which were the most inhibitory against the bacterial strains tested as well as those producing a clear zone of inhibition 420 mm (in diameter) were selected for determining their inhibitory action diluted in ethanol obtaining a concentration of 5, 15, 25, 50 ml/100 ml, respectively. Table 3 shows the lowest concentration of 11 plant extracts and oils against the foodborne pathogens tested by the disc diffusion assay. Lemon flavour, pineapple, and rosewater (GGH) with the minimum inhibitory concentration of 5 ml/100 ml had an inhibitory action against E. coli O157:H7 EDL-933. S. Typhimurium was inhibited by 5 ml/100 ml of lemon flavour and lemongrass essences. Lemon flavour and lemongrass essences at concentrations of 15 ml/100 ml inhibited strains of S. aureus. Strains of L. monocytogenes and S. Enteritidis were inhibited by 15 ml/100 ml of lemon flavour, whereas B. cereus was inhibited by 15 ml/100 ml of plum. The results show that the zone of inhibition is a practical approach for screening different concentrations of potential antimicrobial substances. Based on the disc diffusion studies apricot, lemon, lemongrass, plum, and strawberry were selected for further studies in chocolate. Since in disc diffusion experiment the different bacterial strains of E. coli O157:H7, S. aureus, and L. monocytogenes responded differently to the action of the
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Table 2 Zone of inhibition of growth of foodborne pathogensa by plant extracts and essential oils Plant extracts and essential oils
E. coli EDL-933
E. coli O157:H7 St 1
E. coli O157:H7 St 2
S. Enteritidis
S. Typhimurium
S. auureus
S. aureus S-6
L. monocytogenes Scott A
L. monocytogenes 1
L. monocytogenes 2
B. cereus
7.070.40c
n.i.d
9.270.3
7.570.9
n.i.
6.170.6
7.570.6
n.i.
n.i.
10.171.1
7.270.6
n.i.
8.370.3
8.470.4
9.170.7
9.871.0
9.270.5
8.270.7
9.870.9
10.271.1
7.670.8
10.270.9
n.i.
7.270.7
11.270.2
7.370.4
10.471.2
11.371.2
9.570.8
6.370.5
10.171.0
11.371.4
n.i.
11.371.3
n.i.
n.i.
10.570.5 13.270.2 5.170.1
9.570.3 8.470.6 n.i.
11.671.1 n.i. n.i.
9.670.8 10.271.1 n.i.
8.470.6 7.770.8 n.i.
11.470.8 9.571.1 6.670.7
7.370.6 n.i. 14.271.3
n.i. n.i. n.i.
n.i. n.i. n.i.
12.470.9 9.270.8 n.i.
n.i. n.i. n.i.
8.170.9 7.270.8 n.i.
9.670.6 7.770.7 n.i. 5.170.1
12.370.5 13.770.6 n.i. n.i.
13.570.9 14.171.3 n.i. 6.770.9
10.270.6 10.771.2 n.i. 7.370.8
10.271.2 12.371.2 n.i. 6.270.6
12.271.3 n.i. 6.770.8 n.i.
n.i. n.i. 13.571.4 n.i.
15.271.5 n.i. 12.471.3 n.i.
n.i. n.i. n.i. n.i.
11.270.9 n.i. n.i. 7.270.8
n.i. n.i. n.i. 14.171.2
8.170.9 n.i. n.i. n.i.
n.i. n.i. n.i.
13.470.6 21.871.2 n.i.
n.i. 10.371.2 n.i.
n.i. 11.470.7 n.i.
n.i. 11.170.9 n.i.
6.470.6 9.671.2 8.570.7
13.271.1 7.470.8 15.271.3
n.i. n.i. 25.171.8
n.i. n.i. 15.271.3
n.i. 9.370.7 n.i.
15.371.4 n.i. n.i.
n.i. n.i. n.i.
n.i.
n.i.
n.i.
n.i.
6.570.5
n.i.
n.i.
12.271.1
n.i.
n.i.
n.i.
n.i.
n.i. 6.27 0.1 5.270.2 11.870.8 11.670.6 10.47 0.4
n.i. 9.470.6 10.771.1 10.970.6 8.370.4 11.271.5
n.i. 11.270.9 11.370.7 11.571.1 10.770.8 9.671.0
n.i. 8.570.8 11.771.2 10.370.9 8.470.7 9.370.8
n.i. 9.370.7 8.670.7 11.271.3 8.770.8 9.570.6
n.i. 11.271.1 6.270.4 8.670.9 7.570.6 6.470.5
n.i. 5.370.4 n.i. n.i. n.i. 10.271.2
12.371.2 n.i. 9.470.9 11.370.8 n.i. 10.570.7
n.i. n.i. n.i. n.i. n.i. 15.171.6
n.i. n.i. 12.371.2 12.271.3 n.i. 9.570.9
n.i. 6.470.7 n.i. n.i. n.i. 9.570.8
n.i. 6.370.5 20.271.9 7.270.8 n.i. 10.271.3
12.770.7
13.771.1
13.471.3
10.671.3
n.i.
9.770.8
7.370.7
12.470.8
28.271.9
12.271.4
18.271.5
8.670.9
5.370.3
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
Obtained by adding 10 ml of each undiluted antimicrobial substance. The diameter of the filter paper discs (6 mm) is included. Inoculum level: 2.4–5 107 CFU/ml. c Values are mean7standard deviation. d No zone of inhibition observed. b
ARTICLE IN PRESS
a
E. coli EDL-932
P. Kotzekidou et al. / LWT 41 (2008) 119–127
Almond (bitter) FI Almond (bitter) GGH Apricot Beluda flavouring Apricot GGH Banana Bergamot flavouring oil Bergamot GGH Cointreu Gum mastic oil Hazelnut flavouring Lemon GGH Lemon flavour Lemongrass essences Mandarin essences Orange Pineapple Plum Rosewater GGH Rosewater (NF) Strawberry GGH Strawberry flavour Vanilla flavouring
Inhibition zone diameter (mm)b
Table 3 Zones of inhibition of growth of foodborne pathogens obtained by the lowest concentration (i.e. 5, 15, 25, or 50 ml/100 ml) of plant extract and essential oil Plant extracts and essential oils
Inhibition zone diameter (mm)a Con. ml/100 ml
Control Almond (bitter) GGH
Apricot Beluda flavouring
Apricot GGH
Lemongrass essences
Pineapple
Plum
Rosewater GGH
Strawberry GGH
Strawberry flavour
a
E. coli O157:H7 St 1
E. coli O157:H7 St 2
S. Enteritidis
S. Typhimurium
S. aureus
S. aureus S6
L. monocytogenes Scott A
L. monocytogenes 1
L. monocytogenes 2
B. cereus
n.i.b n.i. n.i. 12.371.2 14.171.4 n.i. n.i. n.i. 17.271.5 n.i. n.i. n.i. n.i. n.i. n.i. 11.271.3 15.470.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.371.0 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.571.9
n.i. n.i. n.i. n.i. 13.271.0 n.i. n.i. n.i. 12.470.9 n.i. n.i. 10.671.1 12.371.3 n.i. n.i. n.i. 12.571.4 18.471.9 22.371.8 23.271.7 25.471.6 n.i. n.i. n.i. n.i. 11.271.3 11.171.1 12.271.0 13.471.5 n.i. 14.371.6 14.270.9 15.171.2 11.270.8 13.670.7 12.670.8 14.271.2 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.371.3
n.i. n.i. n.i. 10.270.7 13.271.4 n.i. n.i. 9.370.8 10.171.2 n.i. 8.470.9 11.270.8 15.171.2 n.i. n.i. n.i. 10.670.9 n.i. n.i. n.i. 10.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.571.4 n.i. n.i. n.i. 13.271.1 n.i. n.i. n.i. 15.371.2 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 16.471.6
n.i. n.i. n.i. n.i. 12.270.9 n.i. n.i. 11.370.7 14.270.6 n.i. n.i. n.i. 12.170.5 n.i. n.i. n.i. 10.370.9 n.i. n.i. n.i. 16.571.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.470.8 n.i. n.i. n.i. 16.570.9 n.i. n.i. n.i. n.i. n.i. n.i. 12.270.9 12.371.1 12.171.2 13.671.0 14.771.3 15.470.9
n.i. n.i. n.i. n.i. 12.270.8 n.i. n.i. n.i. 13.470.8 n.i. n.i. 11.370.9 12.171.1 n.i. n.i. 10.270.9 13.471.1 n.i. 12.371.2 13.471.1 12.570.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.270.9 n.i. 10.270.8 11.470.9 12.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.471.0 n.i. n.i. n.i. n.i.
n.i. n.i. n.i. 8.370.6 10.170.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.170.9 9.270.7 10.370.8 11.271.0 10.371.2 11.271.1 12.471.5 13.670.9 n.i. n.i. n.i. 11.270.8 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.370.9
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.070.5 9.270.6 10.370.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 17.270.8 n.i. n.i. n.i. n.i. n.i. 19.370.9 21.271.8 23.471.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 15.170.9 16.271.0 18.371.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.270.9 9.470.8 11.271.0 15.47 1.5 22.571.8
n.i. n.i. n.i. n.i. 10.370.5 n.i. n.i. 11.570.7 13.270.9 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.370.8 n.i. 11.570.9 9.370.6 8.470.4 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.270.8 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.370.7 n.i. n.i. n.i. n.i.
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.170.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 9.470.7 11.270.6 13.570.9 20.271.4
n.i. n.i. n.i. n.i. 8.570.6 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 8.470.7 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 7.270.3 n.i. 10.170.4 12.470.6 17.370.6 n.i. n.i. n.i. n.i. n.i. n.i. n.i. 7.570.5 n.i. n.i. n.i. n.i.
No zone of inhibition observed. Values are mean7standard deviation.
b
ARTICLE IN PRESS
Lemon flavour
E. coli EDL-933
P. Kotzekidou et al. / LWT 41 (2008) 119–127
Bergamot GGH
5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50 5 15 25 50
E. coli EDL-932
123
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124
above plant extracts, a cocktail of bacterial cultures obtained by mixing the same population of the different strains of each microorganism was used in the experiments concerning the fate of the inoculated pathogens in chocolate. For practical application of plant extracts or essential oils in chocolate, it is important to know if very low concentrations (i.e. 0.1 ml/100 g) have any antimicrobial effects. Plant extracts differed significantly in their ability to inhibit the growth of E. coli pooled inoculum in chocolate with aw 0.450 held at 7 1C. Among the four extracts (i.e. apricot, lemon, plum, and strawberry) added at concentration of 0.1 ml/100 g, lemon was the most inhibitory to E. coli O157:H7. At low temperature storage (7 1C), a nearly 1.7-log reduction in E. coli O157:H7 was observed in samples inoculated with ca. 105 CFU/g and treated with lemon. A quick repression of the inoculum was observed within 2 days (Fig. 1). The results showed that strawberry is able to suppress E. coli O157:H7 by 1-log during 7 days of storage at 7 1C. In contrast, plum completely inhibited the growth of the inoculated E. coli O157:H7 showing bacteriostatic results and a reduction of 0.4-log after 9 days of storage. Apricot resulted in no clear decline of the population of E. coli O157:H7. In control samples of chocolate a slight increase of the inoculated population was observed during storage at 7 1C. In chocolate samples held at 20 1C with aw 0.340, lemon added at concentration of 0.1 ml/100 g suppressed quickly the inoculated population of E. coli O157:H7 (ca. 105 CFU/g) within 24 h by about 1.8-log cycles. The most inhibitory/bactericidal action was observed after 9 days of storage causing a 3.3-log reduction of the inoculum (Fig. 1). Strawberry and plum possessed the ability to suppress E. coli O157:H7. However, the reduction in populations was not as much as with lemon treatment. Chocolate samples treated with apricot extract suppressed growth of E. coli O157:H7 and reduced the inoculum (ca. 105 CFU/g) after 9 days of storage by about 1-log
cycle. During the first 24 h after inoculation of the control chocolate samples a slight increase of the inoculated bacterial population was observed (by about 0.6-log), followed by reduction of E. coli O157:H7 population after prolonged storage of chocolate at 20 1C, due to the low aw (0.340) of the product. As shown in Fig. 1 increased storage temperature enhanced the inhibitory effect of the plant extracts tested on chocolate. Especially, application of lemon flavour on chocolate resulted in 3.3-log decrease of the inoculated E. coli O157:H7 after prolonged storage at 20 1C. According to this observation, plant extracts may be more effective in case of abused storage temperatures. The high initial population of L. monocytogenes (inoculum ca. 105 CFU/g) reduced by about 0.6 to 1.2-log cycles in control samples as well as in chocolate containing strawberry flavour (0.1 ml/100 g) during the first 9 h of storage (Fig. 2). The most drastic effect (i.e. a reduction of 1.5-log) was observed in chocolate containing strawberry flavour held at 20 1C after 5 days of storage. The effect of strawberry flavour was bactericidal and the reduced L. monocytogenes population remained at low levels during prolonged storage in dry or humidified environment at 7 or 20 1C. In control samples the inoculated L. monocytogenes was reduced in the first 24 h, but later during storage at 7 1C the population of L. monocytogenes was slightly increased in dry as well as in humidified environment, whereas this effect was less pronounced at 20 1C. In samples where 0.1 ml/100 g strawberry extract was added a reduction of 1.5- and 1.2-log cycles occurred in dry and humidified environment, respectively. Lemongrass essences added in chocolate held in humidified or dry environment at 7 as well as at 20 1C (Fig. 3) had a slight bactericidal effect on inoculated S. aureus (inoculum level ca. 105 CFU/g) but there was no great difference between samples where lemongrass essences were added and control samples. The reduced population of inoculated pathogen fluctuated during prolonged storage at the different environmental conditions. The
7° C
0
0 -1
Log[N(t)/No]
Log[N(t)/No]
20° C
1
1
-2 -3
-1 -2 -3 -4
-4
-5
-5 0
24
48
72
96 120 144 Time (h)
168
192
216
0
24
48
72
96
120 144 168 192 216
Time (h)
Fig. 1. Inhibition of E. coli O157:H7 pooled inoculum by plant extracts added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C and aw 0.450 and (b) at 20 1C and aw 0.340. Symbols: (x) control experiment without plant extract added; (n) lemon flavour; (&) plum; (J) strawberry flavour; (B) apricot Beluda flavouring. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
ARTICLE IN PRESS P. Kotzekidou et al. / LWT 41 (2008) 119–127
7° C
1
20° C
1 0 Log[N(t)/No]
0 Log[N(t)/No]
125
-1 -2
-1 -2 -3
-3 0
12
24
36
48
60
72
84
96 108 120
0
12
24
36
48
Time (h)
60
72
84
96 108 120
Time (h)
Fig. 2. Inhibition of L. monocytogenes pooled inoculum by strawberry flavour added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C at aw 0.450 (), and aw 0.822 (’); (b) at 20 1C at aw 0.340 (m), and aw 0.822 (’). Open symbols: control experiment without essential oil added. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
7° C
0 Log[N(t)/No]
Log[N(t)/No]
0
-1
-1
-2
-2
-3
20° C
1
1
0
12
24
36
48
60
72
84
96 108 120
Time (h)
-3
0
12
24
36
48
60
72
84
96 108 120
Time (h)
Fig. 3. Inhibition of S. aureus pooled inoculum by lemongrass essences added in concentration of 0.1 ml/100 g in chocolate: (a) at 7 1C at aw 0.450 (), and aw 0.822 (’); (b) at 20 1C at aw 0.340 (m), and aw 0.822 (’). Open symbols: control experiment without essential oil added. Inoculum concentration: 2–5 105 CFU/g. Error bars indicate the mean7standard deviation.
strongest inhibitory effect (reduction of ca. 1.4-log) was observed in chocolate samples containing lemongrass essences and held at 20 1C in humidified environment for 5 days. In some cases the inhibitory effect observed within the first 12 h could be overcome by inoculated microorganisms during prolonged storage time. Therefore, lemongrass alone cannot provide complete protection against S. aureus in chocolate. 4. Discussion Extracts from plants are widely used in the food industry and are considered GRAS. They usually contain more than a single compound with antimicrobial activity. Hence, using extracts has, as a consequence, to take advantage of all active compounds present in extracts as reported by Hao, Brackett, and Doyle (1998). Plant-derived essential oils due to their content of antimicrobial compounds
possess potential as natural agents for food preservation. Their antimicrobial activity is assigned to a number of small terpenoid and phenolic compounds which, due to their lipophilic character, accumulate in bacterial membranes causing energy depletion (Conner, 1993). Our results show that Gram-positive and Gram-negative organisms were affected by the plant extracts and essential oils tested. S. Typhimurium (Gram-negative) was the most sensitive strain as it was inhibited by 17 of the compounds tested, while L. monocytogenes Scott A (Gram-positive) was the most resistant strain, as it was inhibited by four of the compounds tested. Our results agree with the observation of Dorman and Deans (2000) that the susceptibility of bacteria to plant volatile oils and the Gram reaction appears to have little influence on growth inhibition. Similar observations were reported by Hao et al. (1998) that the Gram-negative Aeromonas hydrophila is more sensitive to plant extracts and essential oils than the
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Gram-positive L. monocytogenes. Other researchers reported that essential oils are slightly more active against Gram-positive than Gram-negative bacteria as Gramnegative organisms are less susceptible to the action of antibacterials (Shelef, Jyothi, & Bulgarelli, 1984; Vaara, 1992). Gram-negative bacteria possess a hydrophilic outer membrane, due to the presence of lipopolysaccharide molecules (Nikaido, 1996). Small hydrophilic solutes are able to pass the outer membrane through abundant porin proteins providing hydrophilic trans-membrane channels, whereas the outer membrane serves as a penetration barrier towards macromolecules and to hydrophobic compounds (Nikaido, 1996). But the outer membrane is not totally impermeable to hydrophobic molecules, some of which can slowly traverse through porins. Bypassing the outer membrane is a prerequisite for any solute to exert bactericidal activity. Thus, it is worthwhile to evaluate antimicrobial compounds in a real food system, because low molecular lipophilic compounds, despite their limited solubility in water, are able to penetrate Gram-negative bacteria and influence their proliferation in food-related environments (Helander et al., 1998). Plant extracts and essential oils tested may exhibit different modes of action against the bacterial strains, such as interference with the phospholipid bilayer of the cell membrane which has as a consequence a permeability increase and loss of cellular constituents, damage of the enzymes involved in the production of cellular energy and synthesis of structural components, and destruction or inactivation of genetic material (Kim, Marshall, & Wei, 1995). In general, the mechanism of action is considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents (Davidson, 1997; Sikkema, de Bont, & Poolman, 1995). Comparison of published data is complicated, as the outcome of a test is affected by factors such as the volume of inoculum, growth phase, culture medium used, pH of the media, and incubation time and temperature (Friedman, Henika, & Mandrell, 2002; Rios, Recio, & Villar, 1988). Especially, during their application in foods the lower water content of food compared to laboratory media may hamper the progress of antibacterial agents to the target site in the bacterial cell (Smith-Palmer, Stewart, & Fyfe, 2001). The plant extracts and essential oils added to chocolate not only affected the progress of bacterial growth but also resulted in a greater than 1-log reduction of the initial viable bacterial population. Carbohydrates in foods do not appear to protect bacteria from the action of essential oils as much as fat do (Shelef et al., 1984). In artificially contaminated chocolate verocytotoxin-producing strains E. coli O157:H7, O111:H-, and O26:H11 survived in products stored at 22 1C for up to 90 days and in products stored at 10 1C they could be detected after 366 days of storage (Baylis et al., 2004). Our results revealed that the
plant extracts and essential oils applied to chocolate were bactericidal to E. coli O157:H7, L. monocytogenes, and S. aureus. Application of the above antimicrobial substances resulted in an immediate reduction in population of pathogens. The results indicate that lemon flavour was the most effective extract against E. coli O157:H7, and that strawberry flavour had a more inhibitory effect than plum and apricot extract. Especially in case of S. aureus and B. cereus, it is of importance that essential oils cause inhibition of toxin production, due to lower specific growth rate which has as a consequence that the cells use all the available energy to sustain viability, leaving little left for toxin production, whereas if toxin excretion is an active process, there may be insufficient ATP or proton motive force to export it from the cell (Ultee & Smid, 2001). In conclusion, our results show that plant extracts and essential oils alone cannot provide complete protection against pathogens in chocolate. Their antimicrobial activity diminishes in food systems as they appeared less efficacious when added to chocolate. But, they might prove to be more effective in food products that have a low level of bacterial contamination. Increased storage temperature enhanced the inhibitory effect of all plant extracts tested on chocolate, indicating that the application of the tested plant extracts may provide protection in case of storage at 20 1C or higher temperature. The use of plant extracts and essential oils in consumer goods is expected to increase in the future due to the rise of ‘‘green consumerism’’, which stimulates the use and development of products derived from plants (Tuley de Silva, 1996), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials. References Baylis, C. L., MacPhee, S., Robinson, A. J., Griffiths, R., Lilley, K., & Betts, R. P. (2004). Survival of Escherichia coli O157:H7, O111:H- and O26:H11 in artificially contaminated chocolate and confectionery products. International Journal of Food Microbiology, 96, 35–48. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology, 94, 223–253. Conner, D. E. (1993). Naturally occurring compounds. In P. M. Davidson, & A. L. Branen (Eds.), Antimicrobials in foods (pp. 441–468). New York: Marcel Dekker. Davidson, P. M. (1997). Chemical preservatives and natural antimicrobial compounds. In M. P. Doyle, L. R. Beuchat, & T. J. Montville (Eds.), Food microbiology. Fundamentals and frontiers (pp. 520–556). Washington, DC: ASM Publications. Deans, S. G., & Ritchie, G. A. (1987). Antimicrobial properties of plant essential oils. International Journal of Food Microbiology, 5, 165–180. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308–316. Friedman, M., Henika, P. R., & Mandrell, R. E. (2002). Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella enterica. Journal of Food Protection, 65, 1545–1560. Gould, G.W., 1996. Industry perspectives on the use of natural antimicrobials and inhibitors for food applications. Journal of Food Protection (suppl.), 82–86.
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