Antimicrobial activity of essential oils on the native microflora of organic Swiss chard

Transcription

1 ARTICLE IN PRESS Lebensm.-Wiss. u.-technol. 36 (2003) Antimicrobial activity of essential oils on the native microflora of organic Swiss chard A.G. Ponce a, *, R. Fritz b, C. del Valle b, S.I. Roura c a Comisi!on de Investigaciones Cient!ıficas de la Provincia de Buenos Aires, Argentina b Universidad Nacional de Mar del Plata, Argentina c Consejo Nacional de Investigaciones Cient!ıficas y T!ecnicas, Argentina Received 20 November 2002; accepted 18 March 2003 Abstract The antimicrobial activity of several essential oils on the native microflora of Swiss chard was evaluated. The agar diffusion technique (paper disc) was used to establish, in a rapid way, the susceptibility of the native microflora to the different essential oils and to choose those with the greatest antimicrobial activity. An optical method (turbidimetry) and a microdilution method (plate count) were used to determine the minimum bactericidal concentration (MBC) and the minimum inhibitory concentration (MIC). Both methods produced similar results. The essential oils of Eucalyptus globules, Melaleuca alternifolia, Pimpinella anisum and Syzygium aromaticum presented the largest antimicrobial activity. For these oils, the MBC values were in the range of ml/ 100 ml. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Essential oils; Leafy vegetables; Microflora; Antimicrobial activity 1. Introduction Fruits and vegetables are ecological niches for a diverse and changing microflora. Fresh produce typically contains a complex mix of bacteria, fungi and yeasts that are characteristic of that fruit or vegetable, and whose population and kinds are highly variable (Zagory, 1999). On vegetables, the microflora is dominated by soil organisms. Pathogens can contaminate raw vegetables through agricultural practices and survive during processing and distribution (Beuchat, 1996; Brackett, 1999). Which organisms will come to dominate the population on produce traded fresh will be a function of the make-up of the original population, distribution time, distribution temperature, package atmosphere (Zagory, 1999) and to post-harvest conditioning operations. To prolong the shelf-life of fresh fruits and vegetables, the growth of microbial populations must be controlled *Corresponding author. Grupo de Investigaci!on en Ingenier!ıa en Alimentos, Facultad de Ingenier!ıa, Universidad Nacional de Mar del Plata, Juan B Justo 4302, Mar del Plata 7600, Argentina. address: agponce@mdp.edu.ar (A.G. Ponce). and several post-harvest processes, such as washing and removal of damaged tissues, are employed to reduce initial high counts. It is well known that clean sanitation is essential in keeping the microbial population to a minimum, because storage life is shorter with high initial microbial loads (Bolin, Stafford, King, & Huxsoll, 1977). On the other hand, most of the evidence indicates that the native microorganisms should not be destroyed completely, because they control the growth of any contaminating pathogens (Watada, 1997). L. monocytogenes grew better on disinfected produce than on nondisinfected or water-rinsed produce (Bennik et al., 1996). Babic, Watada, and Buta (1997) noted that growth of L. monocytogenes was not restricted when spinach was autoclaved or irradiated, but was restricted if native microorganisms were present. Washing with water solutions of different sanitizing agents has been tested and some are being used. Chlorine is the primary sanitizing agent used in fruits and vegetable washing. Other sanitizing agents that have been evaluated are: chlorine dioxide (Reina, Fleming, & Humpheries, 1995; Zhang & Farber, 1996), hydrogen peroxide (Sapers & Simmons, 1996), organic acids (Karapinar, & Gonul, 1992; Nguyen-The, Halna-du-Fretay, & Silva, 1996), /03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. doi: /s (03)

2 680 ARTICLE IN PRESS A.G. Ponce et al. / Lebensm.-Wiss. u.-technol. 36 (2003) trisodium phosphate (Zhang & Farber, 1996; Zhuang & Beuchat, 1996) and ozone (Burrows, Boisrobert, & Hampson, 1999). Many of these chemicals have not been very successful at reducing the indigenous microflora (Adams, Hartley, & Cox, 1989; Brackett, 1992b; Nguyen-The & Carlin, 1994). Moreover, some of them have a negative impact on the environment and finally, some may cause the product to have an objectionable aftertaste. The exploration of naturally occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food products and a growing concern of microbial resistance towards conventional preservatives (Gould, 1995). Also, there is a growing interest in organically produced vegetables that the general public associates with healthier foods. The nature of these products precludes the use of chemical additives during post-harvest handling. Because of this, organically produced vegetables to be fresh marketed probably require preservation technologies different from those employed with their counterparts that have been produced with the aid of agrochemicals. Up to date organic farming organizations and the authorities have concentrated their efforts mainly on developing criteria for primary production. However, the subsequent steps, processing and distribution, have not enjoyed the same attention. Many spices and herbs exert antimicrobial activity due to their essential oil fractions. Nychas (1995) reported antimicrobial activity of essential oils from oregano, thyme, sage, rosemary, clove, coriander, garlic and onion against both bacteria and molds. Phenolic components present in essential oils have been known to possess antimicrobial activity and some are classified as Generally Recognized as Safe (GRAS) substances and therefore could be used to prevent postharvest growth of native and contaminant bacteria (Kabara, 1991; Singh, Singh, Bhunia, & Simmon, 2001). Skandamis, Koutsoumanis, Fasseas, and Nychas (2001) reported that oregano essential oil and EDTA inhibited the growth of Escherichia coli O157:H7 by inducing morphological changes at close to minimum bactericidal concentration (MBC). The use of essential oils, however, may be limited due to flavor, since effective antimicrobial doses may exceed acceptable levels from an organoleptical point of view. The possibility of using essential oils on fresh vegetables, as an alternative to the use of synthetic chemicals to preserve organically grown produce is investigated in this work. The antimicrobial activity of the essential oils of eucalyptus (Eucalyptus globules), tea tree (Melaleuca alternifolia), melissa (Melissa officinalis), rosemary (Rosmarinus officinalis), mint (Mentha piperita), rosa moschata (Rosa moschata), clove (Syzygium aromaticum), sweet basil (Ocimum basilicum), anise (Pimpinella anisum), lemon (Citrus limonum), oregano (Origanum vulgare) and lavender (Lavandula officinalis) on the native microflora of Swiss chard grown by organic methods is evaluated. The microbial log 10 counts in fresh organically grown Swiss chard, reported in a previous work, were for mesophyllic bacteria, for psycrotrophic bacteria, for lactic acid bacteria, for molds and yeasts, for total coliforms and for Staphylococcus spp. (Ponce, Roura, Del Valle, & Fritz, 2002). 2. Materials and methods 2.1. Raw material and inoculum preparation The raw material (fresh Swiss chard) was provided by Sierra Bachicha that produces by organic methods and is certified by the Association of Organic Producers of the Province of Buenos Aires (APROBA). This producer is located in the vicinity of Mar del Plata, in the Southeast of the Province of Buenos Aires, Argentina. Organic is a method of production that prohibits the use of synthetic fertilizers and pesticides as well as genetic manipulation of plants. These products must not be used on certified organic farms for at least 3 years prior to harvest in order for the crops to be certified organic. The raw material was received at our laboratory within 2 h of harvesting. Leaves with evident physiological damage were discarded. Ten grams of chard leaves were macerated in 90 ml phosphate buffer solution (0.1 mol/l) with a Stomacher 400 Circulator Homogenizer (ph 7.2) according to Butterfield (1960). An aliquot of leaf homogenate was transferred to 250 ml of Brain Heart Infusion (BHI) and was incubated overnight at 37 C to produce the inoculum. The final concentration of microorganisms in the media was approximately 10 8 CFU ml 1. The natural mixed cultures that growth in BHI broth were mesophilic aerobic bacteria (87/100 CFU), Enterobacteriaceae (5/100 CFU), psychrotrophic bacteria (5/100 CFU), lactic acid bacteria (2/100 CFU) and molds and yeast (1/100 CFU) Essential oils The essential oils used in this work were eucalyptus (Eucalyptus globules), tea tree (Melaleuca alternifolia), melissa (Melissa officinalis), rosemary (Rosmarinus officinalis), mint (Mentha piperita), rosa moschata (Rosa moschata), clove (Syzygium aromaticum), sweet basil (Ocimum basilicum), anise (Pimpinella anisum), lemon (Citrus limonum), oregano (Origanum vulgare) and lavender (Lavandula officinalis) (Nelson & Russell, London SW 19 9 UH, England).

3 ARTICLE IN PRESS A.G. Ponce et al. / Lebensm.-Wiss. u.-technol. 36 (2003) Determination of sensitivity The sensitivity of microorganisms to the essential oils was determined by the agar diffusion method. Sterile paper discs (Whatman No. 40; 6.0 mm in diameter) were soaked with pure essential oils and placed on the surface of the inoculated BHI agar plates. The dishes were incubated at 37 C for 24 h and the zones of inhibition were measured. The sensitivity to the different oils was classified by the diameter of the inhibition halos as: not sensitive ( ) for diameters less than 8 mm; sensitive (+) for diameters 9 14 mm; very sensitive (++) for diameters mm and extremely sensitive (+++) for diameters larger than 20 mm. 3. Results and discussion The susceptibility of the native microflora of Swiss chard to 12 different essential oils, as determined by the agar diffusion method, showed that clove produced an inhibition halo of mm in diameter (Fig. 1A), making it the oil with the highest inhibitory effect. The 2.4. Optical density method Test tubes with 10 ml of BHI broth were inoculated with 0.1 ml of inoculum. Essential oils were added to the tubes to give the following final concentrations: 1.5, 0.75, 0.37, 0.18, 0.09, 0.04, 0.02, 0.01 and ml/ 100 ml. After 24 h of incubation at 37 C, the optical density of the broths at 615 nm was measured with a UV visible spectrophotometer (Shimadzu Corporation UV 1601 PC UV Visible, Kyoto, Japan). A blank curve of absorbance at 615 nm vs. CFU/mL was prepared Microdilution agar plate method Aliquots of 90 ml tempered BHI agar were agitated vigorously with essential oils to achieve the following final oil concentrations: 2.5, 2.0, 1.5, 1.0, 0.6, 0.25, 0.15, 0.10, 0.05 and ml/100 ml. Approximately 15 ml of each of these mixtures were transferred with 1 ml of inoculum to agar plates. The plates were incubated at 37 C for 24 h and numbers of colonies were determined. Each assay was performed by duplicate on two separate experimental runs Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The minimum inhibitory concentration (MIC) is defined as the minimum level of essential oil concentration that produces a 90% reduction in the growth (populations) of microbial colonies and was determined by the optical density method and by the microdilution agar plate method. The MBC is defined as the minimum level of essential oil concentration that produces at least a 99.9% reduction in the growth (population) of microbial colonies (Skandamis et al., 2001). The MIC and MBC were determined by the optical density and the microdilution agar plate methods. Fig. 1. Inhibition halos obtained by the agar diffusion method for different essential oils on native microflora of Swiss chard Rosa moschata and clove (A), eucalyptus and tea tree (B). The central paper disc corresponds to the control sample and was soaked with sterile distillated water.

4 682 ARTICLE IN PRESS A.G. Ponce et al. / Lebensm.-Wiss. u.-technol. 36 (2003) susceptibility of the microflora to eucalyptus, tea tree, rosemary, lemon and mint was also appreciable, with inhibition zones with diameters of 1671, 1772, 15+1, 1771, and 1771 mm, respectively. Fig. 1B shows typical inhibition halos obtained for eucalyptus and tea tree. On the other hand, the native microflora of Swiss chard was less susceptibility to melissa, anis, sweet basil, oregano and lavender. The diameters of the inhibition halos for these oils were 971, 971, 1071, 1171 and 1171 mm, respectively. Finally, no inhibition zone was detected for rosa moschata (Fig. 1A), indicating that this oil does not have any apparent effect on the microflora of Swiss chard. Essential oils derived from many plants are known to possess antifungal (Thomson, 1989) and antibacterial activity (Janssen, Scheffer, & Svendsen, 1987). Essential oils extracted from spices and herbs are generally recognized as containing active antimicrobial compounds. Allicin is a component in garlic oil that inhibits the growth of both gram-negative and gram-positive bacteria. Sulfur-containing compounds found in onions, leeks and chives are also antimicrobial components. Eugenol, carvacrol and thymol are phenolic compounds in cinnamon, cloves, sage and oregano that present antimicrobial activity. The exact cause effect relation for the mode of action of phenolic compounds has not been determined, but Davidson (1993) indicates that they may inactivate essential enzymes, reacting with the cell membrane or disturbing material functionality. Zaika (1988) reported differences in the antimicrobial activity of clove and oregano with clove exerting a strong inhibitory effect and oregano, a mild one. The essential oil fraction is particularly high in clove and eugenol and accounts for 95% of that fraction (Shelef, 1983). Essential oils from different plants, including eucalyptus, present diverse effects on gram-positive and gram-negative microorganisms, but do not inhibit the growth of Pseudomona aeruginosa (Alzamora, Morales, Armas, & Fern!andez, 2001). Paster, Menasherov, Ravid, and Juven (1995) have found that the essential oils of oregano and thyme exhibit antifungal activity and proposed their use as an alternative to chemicals for the preservation of stored grain. Cox, Mann, Markham, Bell, and Gustafson (2000) reported that exposing gramnegative bacterium E. coli AG100, gram-positive bacterium Staphylococcus aureus NCTC 8325 and yeast Candida albicans to MIC and MBC of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasm and yeast plasma membranes. The inhibitory effects of essential oils on the native microbial populations of Swiss chard were determined by the disk diffusion method that proved to be a semiqualitative and fast tool. To evaluate the MIC and MBC, only essential oils with inhibition halos with a diameter of 9 mm or higher were tested. Lavender was discarded because of its strong aroma that would limit its use in food preservation. For essential oils with high flavor impact, their active principle could be used (Lambert, Skandamis, Cootel, & Nychas, 2001). The optical density method using a reference curve of absorbance at 615 nm against the microbial concentration, as CFU/mL, was constructed and used to test the effect of essential oils on the microbial populations. The resulting points were fitted with a logarithmic tendency line of the form Absorbance ¼ 0:2592 lnðcfu=mlþ 0:3639: The optical densities of cultures of the inoculums in BHI broth with different concentrations of essential oils were determined and the equation above was used to infer the microbial populations. In that way, profiles of microbial counts vs. essential oil concentration were obtained. As an example, Fig. 2A shows the profile for lemon essential oil. The microbial populations for oil-free tests were CFU/mL (reference value). Therefore the MIC was established as the one that resulted in a microbial concentration of CFU/ml and, likewise, the MBC as the one that resulted in microbial concentration of CFU/mL. For all tested essential oils, the antimicrobial activity was found to be concentration dependent, most showing that, at low concentrations, small changes in concentration produced large changes in the microbial growth while, at high concentration, large changes in concentrations were needed to produce only minor changes in microbial growth. This is reflected by the changing slope Fig. 2. Absorbance at 615 nm of culture media incubated for 24 h with different concentrations of lemon essential oil (A) and oregano essential oil (B).

5 ARTICLE IN PRESS A.G. Ponce et al. / Lebensm.-Wiss. u.-technol. 36 (2003) in the pattern of the profiles (Fig. 2A). Exceptions to this behavior were found for oregano and rosemary, for which a straight line best represents the microbial populations vs. oil concentration profiles within the range of oil concentrations tested. The profile obtained for oregano essential oil is shown in Fig. 2B. Prindle and Wright (1997) reported that the antimicrobial activity of phenolic compounds was concentration dependent, affecting enzymatic activity related to energy production at low concentrations and causing protein precipitation at high concentrations. As it can be observed in Fig. 2A, the essential oil of lemon yielded a MIC of about 0.05 ml/100 ml and the MBC was not reached at the highest concentration tested. The values of MIC and MBC for the different oils are presented in Table 1. The essential oil with the lowest MIC and MBC was that of clove, with an MIC of ml/100 ml and an MBC of ml/100 ml. This would be in accordance with the results of the agar diffusion method in which clove presented the largest inhibition halo. The MBC of sweet basil, lemon, rosemary and oregano would be higher than 1.5 ml/ 100 ml. For melissa the MBC was reached at that concentration. Hammer and Carson (1999) reported that a concentration of 2.0 ml/100 ml of the essential oil from oregano was needed to inhibit the growth of all microorganisms. Chaibi, Ababouch, BalasrI, Boucetta, and Busta (1997) found that the germination and vegetative growth of Bacillus cereus and Clostridium spp. spores were arrested by rosemary and eucalyptus at concentrations of and ml/100 ml, respectively. These concentrations are much lower than the MIC and MBC values obtained in our study for these essential oils. Among other factors, a possible explanation for this would reside on the fact that the oils were tested on the heterogeneous pool of microorganisms that constitute the native microflora of Swiss chard. Differences in the composition of essential oils, in the interactions of their constituents and in their extraction Table 1 Values of MIC and MBC for different oils obtained by the optical density method Essential oils MBC a (ml/100 ml) MIC b (ml/100 ml) Eucalyptus globulus Melaleuca alternifolia Melissa officinalis Ocimum basilicum > Pimpinella anisum Citrus limonum > Rosmarinus officinalis > Syzygium aromaticum Origanum vulgare > a Minimum bactericidal concentration. b Minimum inhibitory concentration. Table 2 Values of MIC and MBC for different oils obtained by the agar dilution method Essential oils MBC (ml/100 ml) MIC (ml/100 ml) Eucalyptus globulus Melaleuca alternifolia Syzygium aromaticum and purification operations affect their antimicrobial activity (Manou, Bouillard, Devleeschouwer, & Barel, 1998). The MIC and MBC were determined by the agar dilution method in those oils for which a MBC could be found by the optical density method, that is, eucalyptus, tea tree and clove. Anise was not tested because of its strong aroma at the concentrations required to achieve the MBC. The MIC and MBC values, as determined by this method, are presented in Table 2. Both methods produced similar results for both the MIC and MBC values except for the MBC of tea tree, that resulted 3 4 times higher by the optical density method. Several causes can be cited to explain this difference. The size of bacterial cells, the presence of damaged cells and the oxidation of essential oils may affect the absorbance of the growth media (Skandamis et al., 2001). The agar medium may contribute to achieve a good contact between the microorganisms and the essential oils (Mann, 1998). 4. Conclusions The effect of essential oils on the native microflora of organic Swiss chard was investigated using different methods. The agar diffusion method provided a rapid semi-quantitative tool to evaluate the sensitivity of the microflora to the different essential oils. The optical density method was a rapid and easy-to-achieve procedure to determine the MIC and MBC of essential oils that produced similar results to the more laborious and time-demanding microdilution agar plate method. Results would indicate that Rosa moschata and pinnus do not present any significant antimicrobial activity and that high concentrations of melissa, rosemary, mint, oregano and lemon would be needed to achieve bactericidal effects. On the other hand, eucalyptus, tea tree and clove present antimicrobial activity that indicates clearly that these oils have the potential to become technologically useful products as sanitizing agents. However, the in vitro study presented in this work suggested that some oils showed promising activity as preservatives in organically produced chard; further research will be needed to establish the technical feasibility of their use as natural sanitizing agents in the post-harvest processing of vegetables.

6 684 ARTICLE IN PRESS A.G. Ponce et al. / Lebensm.-Wiss. u.-technol. 36 (2003) References Adams, M. R., Hartley, A. D., & Cox, L. J. (1989). Factors affecting the efficacy of washing procedures used in the production of prepared salads. Food Microbiology, 6, Alzamora, L., Morales, L., Armas, L., & Fern!andez, G. (2001). Actividad antimicrobiana in vitro de los aceites esenciales extra!ıdos de algunas plantas arom!aticas. Anales de la Facultad de Medicina Universidad Nacional Mayor de San Marcos, ISSN Babic, I., Watada, A. E., & Buta, J. G. (1997). Growth of Listeria monocytogenes restricted by native microorganisms and other properties of fresh-cut spinach. Journal of Food Protection, 60, 912. Bennik, M. H. J., Peppelenbos, H. W., Nguyen-THE, C., Carlin, F., Smid, E. J., & Gorris, L. G. M. (1996). Microbiology of minimally processed, modified-atmosphere packaged chicory endive. Postharvest Biology and Technology, 9, 209. Beuchat, L. R. (1996). Pathogenic microorganisms associated with fresh produce. Journal of Food Protection, 59, Bolin, H. R., Stafford, A. E., King Jr., A. D., & Huxsoll, C. C. (1977). Factors affecting the storage stability of shredded lettuce. Journal of Food Science, 42, Brackett, R. E. (1992b). Shelf stability and safety of fresh produce as influenced by sanitation and disinfection. Journal of Food Protection, 55(10), Brackett, R. (1999). Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biology and Technology, 15, Burrows, J., Yuan, J., Novak, J., Boisrobert, C., & Hampson, B. (1999). Ozone applications in food processing. Fresh Cut, Butterfield, C. T. (1960). A selection of dilution water for bacteriological examinations. Journal of Bacteriology, 23, Chaibi, A., Ababouch, L. H., BalasrI, K., Boucetta, S., & Busta, F. F. (1997). Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62 A spores by essential oils. Food Microbiology, 14, Cox, S. D., Mann, J. L., Markham, H. C., Bell, J. E., & Gustafson, J. R. (2000). The mode of antimicrobial action of the essential oil of Melaleuca alternifolia. Journal of Applied Microbiology, 88, Davidson, P. M. (1993). Parabens and phenolic compunds. In: P. M. Davidson, & A. L. Branen (Eds.), Antimicrobials in foods (pp ). New York, Marcel Dekker, Inc. Gould, G. W. (1995). Homeostatic mechanisms during food preservation by combined methods. In: G. Barbosa-C!anovas, & J. Welti- Chanes (Eds.), Food preservation by moisture control (pp ). Lancaster, Techromic Publishing Co., Inc. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Influence of organic matter, cations and surfactants on the antimicrobial activity of Melaleuca alternifonlia oil in vitro. Journal of Applied Microbiology, 86, Janssen, A. M., Scheffer, J. J. C., & Baerheim Svendsen, A. (1987). Antimicrobial activity of essentials oils. Plant Medicine, 53, Kabara, J. J. (1991). Phenols and chelators. In: N. J. Rusell, & G. W. Gould (Eds.), Food preservatives (200pp.). London, Blackie. Karapinar, M., & Gonul, S. A. (1992). Removal of Yersinia enterocolitica from parsley by washing with acetic acid vinegar. International Journal of Food Microbiology, 16, Lambert, R. J. W., Skandamis, P. N., Cootel, P. J., & Nychas, J. E. (2001). A study of minimum inhibitory concentration and mode of oregano essential oil, thymol and cavacrol. Journal of Applied Microbiology, 91, Mann, K. (1998). A new method for determining the minimum inhibitory concentration of essential oils. Journal of Applied Microbiology, 84, Manou, I., Bouillard, L., Devleeschouwer, M. J., & Barel, A. O. (1998). Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test. Journal of Applied Microbiology, 84, Nguyen-The, C., & Carlin, F. (1994). The microbiology of minimally processed fresh fruits and vegetables. Critical Review of Food Science and Nutrition, 34, Nguyen-The, C., Halna-du-Fretay, B., & Silva, A. A. D. (1996). The microbiology of mixed salad containing ingredients without dressing. International Journal of Food Science and Technology, 31, Nychas, G. J. E. (1995). Natural antimicrobials from plants. In: G.W. Gould (Ed.), New methods of food preservation (58pp.). London, Blackie, Academic Professional. Paster, N., Menasherov, M., Ravid, U., & Juven, B. (1995). Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking stored grain. Journal of Food Protection, 58, Ponce, A. G., Roura, S. I., Del Valle, C. E., & Fritz, R. (2002). Characterization of native microbial population of Swiss Chard (Beta vulgaris, type cicla). Food Science and Technology, 35, Prindle, R. F., & Wright, E. S. (1997). Phenolic compounds. In S. S. Block (Ed.), Disinfection, sterilization and preservation. Philadelfia: Lea & Febiger. Reina, L. D., Fleming, H. P., & Humpheries, E. G. (1995). Microbiological control of cucumber hydrocooling water with chlorine dioxide. Journal of Food Protection, 58, Sapers, G. M., & Simmons, G. F. (1996). Hydrogen peroxide as an alternative to chloride for disinfection of minimally processed fruits and vegetables. Agricultural Research Service. Shelef, L. A. (1983). Antimicrobial effects of spices. Journal of Food Safety, 6, Singh, A., Singh, R. K., Bhunia, A. K., & Simmon, J. E. (2001). Use of plant essential oils as antimicrobial agents against Listeria monocytogenes in hotdogs. Program listing. The 2001 IFT Annual Meeting, New Orleans, LO. Skandamis, P., Koutsoumanis, K., Fasseas, K., & Nychas, G. J. E. (2001). Inhibition of oregano essential oil and EDTA on E. coli O157: H7. Italian Journal of Food Science, 13, Thomson, D. P. (1989). Fungitoxic activity of essential oil components on food storage fungi. Mycolog!ıa, 81(1), Watada, A. E. (1997). Quality maintenance of fresh-cut fruits and vegetables. Foods and Biotechnology, 6(4), Zagory, D. (1999). Effects of post-processing handling and packaging on microbial populations. Postharvest Biology and Technology, 15, Zaika, L. L. (1988). Spices and herbs: Their antimicrobial activity and its determination. Journal of Food Safety, 9, Zhang, S., & Farber, J. M. (1996). The effects of various disinfectants against Listeria monocytogenes on fresh vegetables. Food Microbiology, 13, Zhuang, R. Y., & Beuchat, L. R. (1996). Effectiveness of trisodium phosphate for killing Salmonella montevideo on tomatoes. Journal of Applied Microbiology, 22,