Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms

International Journal of Food Microbiology 37 (1997) 155–162

Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms a, b c a Blaise Ouattara *, Ronald E. Simard , Richard A. Holley , Gabriel J.-P. Piette , a ´ Andre´ Begin a

´ , Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Blvd. West, St. Hyacinthe, Quebec Canada, J2 S 8 E3 b ´ Departement de Science et Technologie des Aliments, Faculte´ des Sciences de l’ Agriculture et de l’ Alimentation, Universite´ Laval, ´ , Canada, G1 K 7 P4 Quebec c Department of Food Science, Faculty of Agricultural and Food Sciences, University of Mannitoba, Winnipeg, Mannitoba, Canada, R3 T 2 N2 Received 2 December 1996; received in revised form 16 May 1997; accepted 10 June 1997

Abstract The antibacterial activity of selected fatty acids and essential oils was examined against two gram-negative (Pseudomonas fluorescens and Serratia liquefaciens), and four gram-positive (Brochothrix thermosphacta, Carnobacterium piscicola, Lactobacillus curvatus, and Lactobacillus sake) bacteria involved in meat spoilage. Various amounts of each preservative were added to brain heart infusion or MRS (deMan, Rogosa and Sharpe) agars, and the minimum inhibitory concentration was determined for each organism. Essential oils were analysed by gas–liquid chromatography to determine the concentration of selected components commonly found in spices. B. thermosphacta, P. fluorescens and S. liquefaciens were not affected by fatty acids, and generally overcame the inhibitory effect of essential oils after 24 h of exposure. Among the fatty acids, lauric and palmitoleic acids exhibited the greatest inhibitory effect with minimum inhibitory concentrations of 250 to 500 mg / ml, while myristic, palmitic, stearic and oleic acids were completely ineffective. For essential oils, clove, cinnamon, pimento, and rosemary were found to be the most active. The 1 / 100 dilution of those oils inhibited at least five of the six tested organisms. A relationship was found between the inhibitory effect of essential oils and the presence of eugenol and cinnamaldehyde.  1997 Elsevier Science B.V. Keywords: Fatty acids; Essential oils; Meat; Spoilage; Bacteria

1. Introduction The problem of safe preservation in the meat industry has grown to be more complex as today’s *Corresponding author. Tel: 514 773 1105; Fax: 514 773 8461.

products require longer shelf-life and greater assurance of protection from microbial spoilage. Many attempts have been made to control microbial growth at the surface of meat and meat products with antimicrobial chemicals. For example, significant reductions of microbial growth were obtained by

0168-1605 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00070-6

156

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

dipping or spraying meat with organic acid solutions (Abugroun et al., 1993; Anderson and Marshall, 1989). However, preservatives could not be stabilized at the surface of food due to evaporation, neutralization (Siragusa and Dickson, 1992), and diffusion into the matrix (Torres et al., 1985). Fatty acids and essential oils have also been shown to possess antibacterial and antifungal activities against many plant and food microorganisms (Kabara, 1981; Shelef et al., 1980; Russel, 1991). Gram-negative bacteria were shown to be generally more resistant than gram-positive ones to the antagonistic effects of fatty acids and essential oils because of their cell wall lipopolysaccharide (Kabara, 1979; Branen et al., 1980; Russel, 1991) but this was not always true (Karapinar and Aktug, 1987). In addition, most studies to date have been done with pathogens such as Salmonella typhimurium and Staphylococcus aureus (Karapinar and Aktug, 1987; Paster et al., 1990; Juven et al., 1994), Listeria monocytogenes (Aureli et al., 1992; Wang and Johnson, 1992), Vibrio parahaemoliticus (Karapinar and Aktug, 1987; Shelef et al., 1980), and Clostridium botulinum (Ababouch et al., 1992), and little is known about the effect of these compounds on meat spoilage bacteria such as Carnobacterium piscicola, Lactobacillus curvatus and Lactobacillus sake. An investigation is currently under way in our laboratory to develop active packaging materials for the preservation of meat products. As a first step, it was necessary to know how the regular meat flora was affected by antibacterial agents currently approved for food use, in particular fatty acids and essential oils. The purpose of the present study was therefore to evaluate the efficacy of various fatty acids and essential oils to control the growth of meat spoilage organisms.

2. Materials and methods

2.1. Organisms and cultures The following organisms were obtained from the American Type Culture Collection (Rockville, MD, USA); Carnobacterium piscicola (ATCC 43224), Lactobacillus curvatus (ATCC 25601), and Lactobacillus sake (ATCC 15521). Pseudomonas fluorescens and Brochothrix thermosphacta were

isolated from beef stored at 48C (Farber and Idziak, 1984). Serratia liquefaciens was isolated from vacuum packaged bologna (Food Research and De´ velopment Centre, St. Hyacinthe, Quebec). P. fluorescens, B. thermosphacta, and S. liquefaciens were first inoculated and grown aerobically on brain heart infusion agar (BHI, Difco Laboratories, Detroit, MI, USA). C. piscicola, L. curvatus, and L. sake were similarly inoculated and grown on lactobacilli MRS agar (Difco), in an atmosphere enriched in hydrogen and carbon dioxide (Gaspak Anaerobic System; Becton Dickinson, Cockeysville, MD, USA). All incubations were done at 208C. Bacterial cells were subsequently harvested and resuspended in reconstituted skim milk (skim milk powder in deionized water, 20% w / v final concentration), containing 5% sucrose (w / v), and lyophilized to obtain stock cultures. To prepare working cultures, stock cultures were standardized through two successive 24 h growth cycles in the appropriate broth (BHI or MRS) without agitation. Cells from the standardized cultures were then inoculated in fresh medium and incubated (208C without agitation) for 6 h (C. piscicola for 9 h) to obtain working cultures containing approximately 10 7 colony forming units (CFU) / ml.

2.2. Preparation of the antibacterial media Analytical grade free fatty acids [lauric (C 12:0 ), myristic (C 14:0 ), palmitic (C 16:0 ), palmitoleic (C 16:1 ), stearic (C 18:0 ), oleic (C 18:1 ), linoleic (C 18:2 ), and linolenic (C 18:3 )], with a purity $ 98% were obtained from Sigma (St. Louis, MO, USA). The acids were first dissolved in 95% ethanol (Ababouch et al., 1992), and the solutions were added to 250 ml bottles of sterile BHI or MRS molten agar in concentrations ranging from 100 to 2500 mg / ml, in increments of 50 mg / ml from 100 to 500 mg / ml, and of 100 mg / ml from 500 to 1000 mg / ml. The contents of each bottle were then dispensed into sterile Petri plates and left to solidify. The maximum concentration of ethanol in the agar was 2.5% (v / v), which was shown in preliminary trials to have no inhibitory effect upon the microorganisms used in this study. Eight essential oils (cinnamon, ELB 40404; clove, ELB 41312; cumin, ELB 41402; garlic, EB 40892;

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

oregano, ELB 41401; black pepper, EB 33423; pimento, ELB 41441; thyme, ELB 41403) were provided by Food Ingredients (Mississauga, Ontario, Canada). Rosemary oil, 8136-L was obtained from Kalsec (Kalamazoo, MI, USA). Oils were manually mixed with sterile molten BHI or MRS agar maintained at 458C, to dilutions of 1 / 10, 1 / 100, and 1 / 1000. The molten agars containing essential oils were poured into sterile Petri plates and left to solidify.

2.3. Growth inhibition experiments Petri plates of BHI or MRS agar containing various concentrations of fatty acids or essential oils were inoculated with the selected organisms. The working cultures were diluted (1 / 100) in peptone water, and 0.1 ml of the diluted cultures was spread on the surface of the solidified agar plates. The positive controls for growth consisted of BHI and MRS agar without preservative, inoculated with the diluted working cultures. Uninoculated plates containing either fatty acids or essential oils, served as negative controls. Test and control plates were then incubated at 208C under aerobic conditions for B. thermosphacta, P. fluorescens, and S. liquefaciens, or in H 2 - and CO 2 -enriched atmosphere for C. piscicola, L. curvatus, and L. sake. Three Petri plates were used to test the inhibitory effect for each organism and each level of each preservative, and the experiment was performed twice. Plates were checked for presence or absence of colonies after incubation for 24 and 48 h. The absence of colonies on all the three plates of a treatment was considered as an inhibitory effect. The lowest concentration of fatty acids or essential oils required to inhibit the growth of the test microorganisms was designated as the minimum inhibitory concentration.

2.4. Analysis of essential oils Seventeen substances commonly found in spices were used for this experiment: allylsulfide, carvacrol, camphor, cineole, eugenol, 4-iso-propylbenzaldehyde, trans-cinnamaldehyde, myrcene, a-terpineol, and thymol were purchased from Aldrich Chemical (Milwaukee, WI, USA); geraniol, linalool, and a-terpinene from Sigma; camphene, carvon,

157

limonene, and g-terpinene were obtained from Fluka Chemika–Biochemika (Buchs, Switzerland). Camphene, linalool, a-terpinene, and g-terpinene were of technical quality (90–95% purity) while all the others compounds were at least 97% pure. A Hewlett-Packard model 5890 gas chromatograph equipped with a 1-mm DB-1 fused-silica column 30 m 3 0.316 mm (J and W Scientific, Folsom, CA, USA) was used to determine the concentration of the seventeen substances in the selected essential oils. The split injector was set at ratio of 18:1, and the carrier gas (He) flow at 1.0 ml / min. The oven temperature was programmed to rise 2 C8 / min from 908C to 1158C, 5 C8 / min from 1158C to 2008C, and remained isothermal at the final temperature (2008C) for 4 min. Samples of essential oils injected in the gas chromatograph consisted of 1 ml of 250 mg / ml (rosemary), and 50 mg / ml (other essential oils) solutions in ethyl acetate.

3. Results

3.1. Fatty acids All the fatty acids failed to inhibit B. thermosphacta, P. fluorescens, and S. liquefaciens at concentrations up to 2500 mg / ml (results not shown). The inhibitory effects against the three other bacteria (C. piscicola, L. curvatus, and L. sake) are presented in Table 1. All were unaffected by myristic, palmitic, stearic, and oleic acids at the concentrations tested. Lauric, palmitoleic, linoleic, and linolenic acids exhibited various inhibitory activity with lauric and palmitoleic acids having the greatest effect. Among

Table 1 Minimum inhibitory concentration (mg / ml) of fatty acids against meat spoilage bacteria Fatty acids

C. piscicola

L. curvatus

L. sake

Lauric C 12:0 Myristic C 14:0 Palmitic C 16:0 Palmitoleic C 16:1 Stearic C 18:0 Oleic C 18:1 Linoleic C 18:2 Linolenic C 18:3

250 NI a NI 350 NI NI 600 500

500 NI NI 450 NI NI 650 500

500 NI NI 450 NI NI 650 650

a

NI: No inhibition at concentrations up to 2500 mg / ml.

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

158

the organisms which were affected by fatty acids, C. piscicola was the most susceptible.

3.2. Essential oils All the essential oils tested for antibacterial activity were ineffective at the 1 / 1000 dilution (results not shown). The inhibitory properties observed with the 1 / 100 and 1 / 10 dilutions are shown in Table 2. The strongest effects were obtained with clove, cinnamon, pimento, and rosemary oils, for which the 1 / 100 dilution inhibited at least five of the six tested organisms. However, pimento oil was not able to maintain the inhibitory effect over 24 h. All the other oils were weakly active. Gram-positive and gram-negative bacteria were generally affected in the same manner within 24 h of exposure, but extension of the inhibitory effects up to 48 h was less often observed with the gramnegative bacteria (Table 2). For example, P. fluorescens and S. liquefaciens, which were affected by the 1 / 100 dilution of cinnamon, clove, and rosemary were no longer inhibited after 48 h, except for S. liquefaciens in the presence of clove oil. In contrast, three of the four gram-positive organisms (C. piscicola, L. curvatus, and L. sake) continued to be inhibited by the same oils at that dilution. Of the gram-positive bacteria, B. thermosphacta exhibited resistance similar to those of the two gram-negative bacteria tested. The contents of the essential oils in the seventeen selected substances is shown in Table 3. In general, the sum of selected substances which were identified and quantified constituted a small proportion of the

total mass of each essential oil, with values ranging from 0.15% for rosemary oil to 19.94% for clove oil. In addition, three of the four most active essential oils (which inhibited more than five organisms at the 1 / 1000 dilution) contained a significant amount of eugenol: clove (19.81%); pimento (9.33%); and cinnamon (5.38%). Cinnamon oil also contained large amounts of cinnamaldehyde (5.37%). The least most active oil (rosemary oil) contained camphor as its major component, but in a low amount (0.10%). Among the less active essential oils (those which inhibited only two or less than two organisms at the 1 / 1000 dilution), only thyme oil contained eugenol and cinnamaldehyde, but these were present in small amounts (0.01% for each of the two components). On the other hand, greater concentrations of other components were found in those oils: carvacrol in oregano oil (5.19%); a-terpinene in cumin and thyme oils (1.15% and 1.64%, respectively); and thymol in thyme oil (2.40%).

4. Discussion The two gram-negative bacteria (P. fluorescens, and S. liquefaciens) were unaffected by fatty acids at concentrations up to 2500 mg / ml. This was to be expected since several other studies (Kabara, 1979, 1981; McKellar et al., 1992) reported that gramnegative bacteria were resistant to the inhibitory effects of medium and long chain fatty acids and their derivatives. This resistance has been attributed to the presence of cell wall lipopolysaccharides, which can screen out the fatty acids; the lipids are

Table 2 Inhibitory properties of diluted essential oils toward meat spoilage bacteria a Essential oils Cinnamon Clove Cumin Garlic Oregano Black pepper Pimento Rosemary a

B. thermosphacta b

11 (1 1) 1c 11 1 1 1 11

P. fluorescens

S. liquefaciens

C. piscicola

L. curvatus

L. sake

11 11 1 1 1 1 11 11

11 (1 1) 1 11 1 1 11 11

(1 1) (1 1) 1 1 1 1 11 (1 1)

(1 1) (1 1) 1 1 1 11 11 (1 1)

(1 1) (1 1) 1 1 1 11 11 (1 1)

Bacteria were tested at 10 5 CFU / ml, at both 24 and 48 h. 1 1 : Inhibition by 1 / 100 dilution of essential oils. c 1 : Inhibition by 1 / 10 dilution of essential oils. () Inhibition extended to 48 h by 1 / 100 dilution of essential oils. b

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

159

Table 3 Quantitative determination of selected authentic antibacterial components in essential oils a Most efficient oils

Least efficient oils

Compound

Clove

Cinnamon

Pimento

Rosemary

Garlic

Black pepper

Oregano

Cumin

Thyme

Total identified Allylsulfide Camphene Camphor Carvacrol Carvon Cinnamaldehyde Eugenol Fenchon Geraniol 4-Isopropylbenzaldehyde Limonene–cineole Linalool Myrcene a-Terpinene g-Terpinene a-Terpineol Thymol

19.94 –b – – – – – 19.81 – – 0.05 – 0.02 – – 0.06 0.03 –

11.20 – 0.02 – – – 5.37 5.38 – – 0.01 0.05 0.15 0.03 0.06 – 0.04 0.09

9.83 – – – 0.03 – – 9.33 0.02 – 0.02 0.11 0.11 0.03 0.06 0.05 0.04 0.03

0.15 – 0.10 0.01 – – – – – – – – 0.01 – – 0.02 0.01

0.26 0.22 0.02 – – – – – – – – – – – – – 0.02 –

2.94 – 0.05 – – 0.07 – – 0.04 – – 1.12 0.12 0.93 0.04 0.49 0.08 –

6.49 – – 0.09 5.19 0.08 – – 0.02 – – 0.08 0.14 0.05 0.29 0.11 0.07 0.37

5.50 – – – 0.04 – – – 0.03 – 1.95 1.24 0.07 0.55 1.15 0.04 0.43 –

6.78 – 0.09 0.10 0.31 0.01 0.01 0.01 0.02 – – 1.31 0.26 0.49 1.64 0.08 0.05 2.40

a b

The results are given in % of the total mass of oils. Component not present in the oil.

thus prevented from accumulating on the transporting cell membrane, and from entering into the cells (Kabara, 1979; Branen et al., 1980; Russel, 1991). B. thermosphacta, a gram-positive bacterium, also exhibited resistance to fatty acids, but little information is available about its sensitivity to challenge by fatty acids. Macaskie (1982) reported that growth rates and numbers of B. thermosphacta were both reduced in the presence of 0.5 mmol / l of palmitic acid. However, both the determination of palmitic acid uptake and the determination of the inhibition of substrate uptake by palmitic acid failed to explain the mechanism by which B. thermosphacta was inhibited. Similar resistance of gram-positive organisms was reported by Tsuchido et al. (1993) working with Bacillus subtilis. They found mutants which were tolerant to the lytic action of sucrose esters of long-chain fatty acids. Among the saturated fatty acids under study, lauric acid exhibited the greatest inhibitory effect against C. piscicola, L. curvatus, and L. sake while all the other saturated fatty acids with chain length between C 14 and C 18 were completely ineffective. These results are consistent with previous reports about the antibacterial activity of saturated fatty acids with lauric acid being the most effective

(Kabara, 1979; Branen et al., 1980; Babic et al., 1994). For saturated fatty acids, hydrophobic groups have been shown to have the greatest influence on antibacterial activity (Branen et al., 1980), but increasing hydrophobicity with longer chain length may reduce their solubility in aqueous systems. Thus hydrophobic groups may be prevented from reaching sufficient concentration to interact with hydrophobic proteins or lipids on the bacterial cell surface (Wang and Johnson, 1992). Lauric acid has been reported to have the best balance between hydrophobic and hydrophilic groups (Branen et al., 1980; Kabara et al., 1977). It is known that unsaturated fatty acids with chain lengths of C 14 or longer are more active against microorganisms than the corresponding saturated fatty acids (Kabara, 1981). Also, the inhibitory effects of unsaturated fatty acids are increased as the number of double bonds in the molecule increases (Kabara, 1979). In agreement with that observation, palmitoleic acid was found to be more active than myristic and palmitic acids (this study), and the antibacterial efficacies of C 18 unsaturated fatty acids were in the following order: linolenic (C 18:3 ) . linoleic (C 18:2 ) . oleic (C 18:1 ). Similar results have been reported by Wang and Johnson (1992) who

160

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

found that linolenic acid was more effective against Listeria monocytogenes than linoleic and oleic acids. The fact that palmitoleic acid and C 18 unsaturated fatty acids are active in spite of their long carbon chain suggests that the hydrophobic / hydrophilic balance alone cannot explain the observed inhibitory effects. This activity may be related to other factors such as a peroxidative process involving hydrogen peroxide and bacterial iron as reported by Wang and Johnson (1992). In the study on the antibacterial activity of essential oils, no obvious difference in susceptibility was found between gram-negative and gram-positive bacteria after 24 h of exposure to essential oils. Data, however, showed that the extent of the inhibitory effect up to 48 h was mostly observed with grampositive organisms. This is supported by many other reports on the greater susceptibility of gram-positive bacteria to the inhibitory effect of essential oils and their components (Shelef et al., 1980; Farag et al., 1989; Chanegriha et al., 1994). As reported for fatty acids, the cell wall lipopolysaccharides of gramnegative bacteria may prevent active components from reaching the cytoplasmic membrane. However, the greater resistance of gram-negative bacteria may not be an overall trend since B. thermosphacta (gram-positive) was as resistant as S. liquefaciens (gram-negative). Similar results have been reported by Kim et al. (1995b) who found that L. monocytogenes (gram-positive) was more resistant to the inhibitory effects of eleven essential oil constituents than the gram-negative bacteria tested under the same conditions, including Escherichia coli, E. coli O157:H7, Salmonella typhimurium and Vibrio vulnificus. It seems that the variability of the resistance of gram-positive bacteria to the inhibitory effect of essential oils may be due to differences between strains of the same bacterial species. This hypothesis was recently confirmed by Sivropoulou et al. (1996) with two strains of Staphylococcus aureus in the presence of carvacrol and thymol. ¨ (1988) reviewed the literature reporting the Zaıka antimicrobial activity of many spices and classified their activities as strong, medium, or weak. According to this ranking, several studies (Conner, 1993; Aureli et al., 1992; Shelef et al., 1980) showed that cinnamon, clove, pimento, thyme, oregano, and rosemary had strong and consistent inhibitory effects against various pathogens and spoilage bacteria. In

agreement with this finding, four of the essential oils tested in this study (cinnamon, clove, pimento, and rosemary) exhibited a strong inhibitory effect toward selected meat spoilage bacteria. The antibacterial activities have been attributed to the presence of some volatile constituents in the oils. Bullerman et al. (1977) found that cinnamon and clove contained cinnamaldehyde and eugenol as major constituents which represented 65–75% and 93–95% of the total volatile oils, respectively, and which were responsible for the antibacterial effect. In oregano and thyme, the major antibacterial constituents have been identified as carvacrol (62–79%), and thymol (42%) respectively (Farag et al., 1989; Sivropoulou et al., 1996). The means by which microorganisms are inhibited by essential oils seems to involve different modes of action. The most frequent inhibitions involve phenolic components of oils which sensitize the phospholipid bilayer of the cell membrane, causing an increase of permeability and leakage of vital intracellular constituents (Kim et al., 1995b; Juven et al., 1994), or impairment of bacterial enzyme systems (Wendakoon and Sakaguchi, 1995; Farag et al., 1989). A number of reports indicated that essential oils containing carvacrol, eugenol, or thymol had the highest antibacterial performances (Kim et al., 1995b; Lattaoui and Tantaoui-Elaraki, 1994; Suresh et al., 1992). For example, Suresh et al. (1992) found that eugenol was more bactericidal against Escherichia coli, Enterobacter sakazakii, and Klebsiella pneumoniae than several antibiotics including ampicillin, erythromycin, and sulphamethizole. Among non-phenolic compounds of essential oils, cinnamaldehyde has been shown to possess antibacterial properties by inhibiting amino acid decarboxylase activity (Didry et al., 1993; Wendakoon and Sakaguchi, 1995). Baranowski and Nagel (1982) reported that allylhydroxycinnamates, which are quite similar to cinnamaldehyde inhibited P. fluorescens by a specific mode of action related to cellular energy depletion. The antibacterial activity of eugenol and cinnamaldehyde was supported by the results obtained by the gas–liquid chromatographic analysis of the essential oils, although components quantified constituted only a small proportion of the oils. Cinnamon and clove oils which were among the most active oils contained the largest amounts of eugenol and cinnamal-

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

dehyde. Also eugenol and cinnamaldehyde were slightly or not present in the oils which produced small inhibitory effects (inhibition of two or less than two organism at 1 / 1000 dilution). Therefore, the presence of eugenol or cinnamaldehyde was directly related to the antibacterial properties of tested essential oils. Our results, however, failed to confirm the inhibitory effect of oregano and thyme although those oils contained high concentrations of phenolic compounds (carvacrol and thymol respectively). Juven et al. (1994) reported that in presence of a high oxygen tension, thyme and oregano oils may be inactivated by oxidation of their phenolic components. In the present study, their effectiveness was not enhanced when the test was done under anaerobic conditions (inhibition test against C. piscicola, L. curvatus, and L. sake). Therefore, the low antibacterial activity reported here for thyme and oregano oils could not be explained in terms of the oxygen tension hypothesis. It is most likely that the weak efficacy of carvacrol and thymol-containing oils (oregano and thyme) found in the present study may be due to some other factors such as insolubility in aqueous media (Juven et al., 1994), pH of the medium (Thompson, 1990), or seasonal and intraspecific variation of essential oil composition (McGimpsey et al., 1994; Kokkini and Vokou, 1989; Sivropoulou et al., 1996). For example, an essential oil from Origanum vulgare has been reported by Sivropoulou et al. (1996) to eliminate S. aureus at dilutions up to 1 / 10000, but the oil sample used contained carvacrol at a concentration of 79.58% compared to 5.19% in the commercial oregano oil tested in the present study. In the present study, rosemary oil was as inhibitory as cinnamon and clove oils. Yet, rosemary did not contain cinnamaldehyde nor eugenol, and all the other background components screened were present in small amounts. Camphor (0.10%) was the only component which was present in rosemary oil in concentrations higher than in the other oils under study. Therefore, the antibacterial efficacy of rosemary oil could be at least partly related to the presence of camphor. This is supported by the report of Lattaoui and Tantaoui-Elaraki (1994) who found that in some essences, minor compounds could have a huge antibacterial impact. Also, the small amount of the total identified components (0.15%) in rosemary

161

oils suggests that some other components may have contributed to its high antibacterial action. The present study on the inhibitory effects of fatty acids and essential oils on meat spoilage bacteria was done under specific, controlled conditions (BHI and MRS agars). Even though some of these compounds showed consistent antibacterial activities against meat spoilage bacteria, the extrapolation of these results to meat systems must be done with caution. Bacteria present on meat surfaces may attach firmly resulting in reduced exposure to essential oils or fatty acids. Proteins and lipid components of meat can also interact with the active components of antibacterial compounds as reported by Kim et al. (1995a). Also, for subsequent use as components of active packages, additional experiments must be done to determine the ease with which fatty acids and essential oils can be incorporated into packaging films and their diffusion rates from the surface of the product to the interior must be characterized.

Acknowledgments This research was made possible by the financial support of Programme Canadien des Bourses de la ´ Francophonie, Agence Canadienne du Developpement International, Ottawa, Ontario. The expert technical assistance of Yves Raymond is greatly appreciated

References Ababouch, L., Chaibi, A., Busta, F.F., 1992. Inhibition of bacterial spore growth by fatty acids and their salts. J. Food Protect. 55, 980–984. Abugroun, H.A., Cousin, N.A., Judge, M.D., 1993. Extended shelf life of unrefrigerated prerigor cooked meat. Meat Sc. 33, 207–229. Anderson, M.E., Marshall, R.T., 1989. Interaction of concentration and temperature of acetic acid solution on reduction of various species of microorganisms on beef surfaces. J. Food Protect. 52, 312–315. Aureli, P., Costantini, A., Zolea, S., 1992. Antibacterial activity of some plant essential oils against Listeria monocytogenes. J. Food Protect. 55, 344–348. Babic, I., Nguyen-the, C., Amiot, M.J., Aubert, S., 1994. Antibacterial activity of shredded carrot extracts on food-borne bacteria and yeast. J. Appl. Bacteriol. 76, 135–141. Baranowski, J.D., Nagel, C.W., 1982. Inhibition of Pseudomonas

162

B. Ouattara et al. / International Journal of Food Microbiology 37 (1997) 155 – 162

fluorescens by hydroxycinnamic acids and their alkyl esters. J. Food Sci. 47, 1587–1589. Branen, A.L., Davidson, P.M., Katz, B., 1980. Antibacterial properties of phenolic antioxidants and lipids. Food Technol. 34, 42; 44; 46; 51–53; 63. Bullerman, L.B., Lieu, F.Y., Seier, S.A., 1977. Inhibition of growth and aflatoxin production by cinnamon and clove oils. Cinnamic aldehyde and eugenol. J. Food Sci. 42, 1107–1109. Chanegriha, N., Sabaou, N., Baaliouamer, A., Meklati, B.Y., 1994. ´ Activite´ antibacterienne et antifongique de l’huile essentielle du cypres d’Algerie. Riv. Ital. Eppos. 12, 5–12. Conner, D.E., 1993. Naturally occuring compounds. In: Davidson, P.M., Branen, A.L. (Eds.), Antimicrobials in foods, 13. Marcel Dekker, New York, pp. 441–468. ´ Didry, N., Dubreuil, L., Pinkas, M., 1993. Activite´ antibacterienne du thymol, du carvacrol et de l’aldehyde cinnamique seuls ou ´ Pharmazie 48, 301–304. associes. Farag, R.S., Daw, Z.Y., Hewedi, F.M., El-Baroty, G.S.A., 1989. Antibacterial activity of some Egyptian spice essential oils. J. Food Protect. 52, 665–667. Farber, J.M., Idziak, E.S., 1984. Attachment of psychrotrophic meat spoilage bacteria to muscle surfaces. J. Food Protect. 47, 92–95. Juven, B.J., Kanner, J., Sched, F., Weisslowicz, H., 1994. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J. Appl. Bacteriol. 76, 626–631. Kabara, J.J., 1979. Fatty acids and derivatives as antimicrobial agents—a review. AOCS Monograph. 5, 1–14. Kabara, J.J., 1981. Food-grade chemicals for use in designing food preservative systems. J. Food Protect. 44, 633–647. Kabara, J.J., Vrable, R., Lie Ken Jie, M., 1977. Antimicrobial lipids: Natural and synthetic fatty acids and monoglycerides. Lipids 12, 753–759. Karapinar, M., Aktug, S.E., 1987. Inhibition of food borne pathogens by thymol, eugenol, menthole and anethole. Int. J. Food Microbiol. 4, 161–166. Kim, J.M., Marshall, M.R., Cornell, J.A., Preston III, J.F., Wei, C.I., 1995a. Antibacterial activity of carvacrol, citral, and geraniol against Salmonella typhimurium in culture medium and on fish cubes. J. Food Sci. 60, 1364–1368; 1374. Kim, J., Marshall, M.R., Wei, C., 1995. Antibacterial activity of some essential oils components against five foodborne pathogens. J. Agric. Food Chem. 43, 2839–2845. Kokkini, S., Vokou, D., 1989. Carvacrol rich plants in Greece. Flavour Fragr. J. 4, 1–7. Lattaoui, N., Tantaoui-Elaraki, A., 1994. Individual and combined antibacterial activity of the main components of three thyme essential oils. Riv. Ital. Eppos. 8, 13–19.

Macaskie, L.E., 1982. Inhibition of growth of Brochothrix thermosphacta by palmitic acid. J. Appl. Bacteriol. 52, 339–343. McGimpsey, J.A., Douglas, M.H., van Klink, J.W., Beauregard, D.A., Perry, N.B., 1994. Seasonal variation in essential oil yield and composition from naturalized Thymus vulgaris L. in New Zealand. Flavour Fragr. J. 9, 347–352. McKellar, R.C., Paquet, A., Ma, C.Y., 1992. Antimicrobial activity of fatty N-acylamino acids against gram-positive foodborne pathogens. Food Microbiol. 9, 67–76. Paster, N., Juven, B.J., Shaaya, E., Menasherov, M., Nitzan, R., Weisslowicz, H., Ravid, U., 1990. Inhibitory effect of oregano and thyme on moulds and foodborne bacteria. Lett. Appl. Microbiol. 11, 33–37. Russel, A.D., 1991. Mechanisms of bacterial resistance to nonantibiotics: food additives and food pharmaceutical preservatives. J. Appl. Bacteriol. 71, 191–201. Shelef, L.A., Naglik, O.A., Bogen, D.W., 1980. Sensitivity of some common food-borne bacteria to the spices sage, rosemary, and allspice. J. Food Sci. 45, 1042–1044. Siragusa, G.R., Dickson, J.S., 1992. Inhibition of Listeria monocytogenes on beef tissue by application of organic acids immobilized in a calcium alginate gel. J. Food Sci. 57, 293– 296. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T., Arsenakis, M., 1996. Antimicrobial and cytotoxic activities of Origanum essential oils. J. Agric. Food Chem. 44, 1202–1205. Suresh, P., Ingle, V.K., Vijayalakshmi, V., 1992. Antibacterial activity of eugenol in comparison with other antibiotics. J. Food Sci. Technol. 29, 254–256. Thompson, D.P., 1990. Influence of pH on the fungitoxic activity of naturally occuring compounds. J. Food Protect. 53, 428– 429. Torres, J.A., Mitoki, M., Karel, M., 1985. Microbial stabilization of intermediate moisture food surfaces. I. Control of surface preservative concentration. J. Food Proc. Pres. 9, 75–92. Tsuchido, T., Yokosuka, N., Takano, M., 1993. Isolation and characteristics of a Bacillus subtilis mutant tolerant to the lytic action of sucrose esters of long-chain fatty acids. J. Ferment. Bioeng. 75, 191–193. Wang, L., Johnson, E.A., 1992. Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58, 624–629. Wendakoon, C.N., Sakaguchi, M., 1995. Inhibition of amino acid decarboxylase activity of Enterobacter aerogenes by active components in spices. J. Food Protect. 58, 280–283. ¨ L.L., 1988. Spices and herbs: their antibacterial activity and Zaıka, its determination. J. Food Safety 23, 97–118.