Food Research International 39 (2006) 639644 www.elsevier.

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Antimicrobial activity of whey protein based edible lms incorporated with oregano, rosemary and garlic essential oils
A.C. Seydim *, G. Sarikus
Department of Food Engineering, Faculty of Agriculture, Suleyman Demirel University, 32260 Isparta, Turkey Received 22 November 2005; accepted 22 January 2006

Abstract The use of edible lms to release antimicrobial constituents in food packaging is a form of active packaging. Antimicrobial properties of spice extracts are well known, however their application to edible lms is limited. In this study, antimicrobial properties of whey protein isolate (WPI) lms containing 1.04.0% (wt/vol) ratios of oregano, rosemary and garlic essential oils were tested against Escherichia coli O157:H7 (ATCC 35218), Staphylococcus aureus (ATCC 43300), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (NCTC 2167) and Lactobacillus plantarum (DSM 20174). Ten millilitres of molten hard agar was inoculated by 200 ll of bacterial cultures (colony count of 1 108 CFU/ml) grown overnight in appropriate medium. Circular discs of WPI lms containing spice extracts, prepared by casting method, were placed on a bacterial lawn. Zones of inhibition were measured after an incubation period. The lm containing oregano essential oil was the most eective against these bacteria at 2% level than those containing garlic and rosemary extracts (P < 0.05). The use of rosemary essential oil incorporated into WPI lms did not exhibit any antimicrobial activity whereas inhibitory eect of WPI lm containing garlic essential oil was observed only at 3% and 4% level (P < 0.05). The results of this study suggested that the antimicrobial activity of some spice extracts were expressed in a WPI based edible lm. 2006 Elsevier Ltd. All rights reserved.
Keywords: Whey protein isolate; Edible lm; Oregano; Garlic; Rosemary; Essential oils; Antimicrobial packaging

1. Introduction In recent years, packaging research has focused more on biodegradable lms, including lms made from plant and animal edible protein sources such as corn zein, wheat gluten, soy and peanut protein, cottonseed, albumin, gelatin, collagen, casein and whey proteins (Tharanathan, 2003). Investigations for their ability to produce edible lms and especially properties of milk protein based edible lms have been extensively reviewed by McHugh and Krochta (1994a), Chen (1995) and Gennadios et al. (1994). Milk protein based edible lms have good mechanical strength and are excellent oxygen, lipid, and aroma barriers; however, due to their hydrophilic nature, they have poor moisture barrier properties (Chen, 1995; Chick & Ustunol,
*

Corresponding author. Tel.: +90 246 211 1669; fax: +90 246 237 0437. E-mail address: aseydim@sdu.edu.tr (A.C. Seydim).

1998; Gennadios, McHugh, Weller, & Krochta, 1994; Krochta & DeMulder-Johnston, 1997; Miller & Krochta, 1997). This property was improved by the incorporation of hydrophobic materials such as lipids (McHugh & Krochta, 1994b; Perez-Gago & Krochta, 1999; Shellhammer & Krochta, 1997). Antimicrobial packaging is a form of active packaging that could extend the shelf-life of product and provides microbial safety for consumers (Rooney, 1995). It acts to reduce, inhibit, or retard the growth of pathogen microorganisms in packed foods and packaging material (Vermeiren, Devlieghere, van Beest, de Kruiif, & Debevere, 1999). In order to control undesirable microorganisms on food surfaces: (1) volatile and non-volatile antimicrobial agents can be incorporated into polymers or (2) coating or adsorbing antimicrobial onto polymer surfaces can be applied (Appendini & Hotchkiss, 2002). The coating can serve as a carrier for antimicrobial compounds and/or antioxidants

0963-9969/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2006.01.013

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A.C. Seydim, G. Sarikus / Food Research International 39 (2006) 639644

compounds in order to maintain high concentrations of preservatives on the food surfaces (Oussallah, Caillet, Salmieri, Saucier, & Lacroix, 2004; Siragusa, Cutter, & Willett, 1999). Several compounds have been proposed for antimicrobial activity in food packaging, including organic acids, enzymes such as lysozyme, and fungicides such as benomyl, imazalil and natural antimicrobial compounds such as spices (Tharanathan, 2003; Weng & Hotchkiss, 1992). These compounds carry mostly antimicrobial and some antioxidant properties. Natural compounds, such as nisin and lysozyme, have been studied as potential food preservatives added to the edible lms that are safe for human consumption (Cagri, Ustunol, & Ryser, 2004; Dawson, Carl, Acton, & Han, 2002; Homan, Han, & Dawson, 2001; Min, Harris, Han, & Krochta, 2005; Padgett, Han, & Dawson, 1998; Padget, Han, & Dawson, 2000). Spices are rich in phenolic compounds, such as avonoids and phenolic acids (Dadalioglu & Evrendilek, 2004). Generally, the essential oils possessing the strongest antibacterial properties against foodborne pathogens contain higher concentrations of phenolic compounds such as carvacrol, eugenol (2-methoxy-4-(2-propenyl) phenol) and thymol (Burt, 2004). These compounds exhibit a wide range of biological eects including antioxidant and antimicrobial properties. The mode of action is generally considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron ow, active transport, and/or coagulation of cell contents (Burt, 2004). Some spice essential oils incorporated into packaging materials can control microbial contamination in beef muscle by reducing the growth of Escherichia coli O157:H7 and Pseudomonas spp. (Oussallah et al., 2004). Essential oil fractions of oregano and pimento are ecient against various food borne bacteria such as Salmonella (Helander et al., 1998; Paster et al., 1990), E. coli O157:H7 (Burt & Reinders, 2003). Spice extracts from oregano, sage, rosemary, garlic, thyme and pimento are also reported to possess antioxidant properties (Dorman & Deans, 2000; Hammer, Carson, & Riley, 1999). Experiments on the antimicrobial properties of spices and herbs and their compounds have been well documented (Zaika, 1988) and interest continues to the present; however, limited information is available for their biological activity in the edible lms. Therefore, the purpose of the study was to incorporate of oregano, garlic and rosemary essential oils into whey protein isolate lms (WPI) and to determine the inhibitory eects of lms incorporated with these essential oils against selected food pathogens. 2. Materials and methods 2.1. Materials Whey protein isolate was obtained from Davisco Foods International Inc. (BiPRO, Le Sueur, MN, USA). Candelilla wax was provided from Strahl and Pitsch Inc. (West

Babylon, NY, USA). Glycerol, NaOH, De Man, Rogosa, Sharpe (MRS) agar, Brain Heart Infusion (BHI) (broth and agar) were purchased from Merck Co. (Darmstadt, Germany). 2.2. Culture preparation Lactobacillus plantarum (DSM 20174), Salmonella enteritidis (ATCC 13076), E. coli O157:H7 (ATCC 35218), Listeria monocytogenes (NCTC 2167) and Staphylococcus aureus (ATCC 43300) cultures were obtained from the culture collections of the Ministry of Health, Rek Saydam Hygiene Centre Contagious Diseases Research Centre (Ankara, Turkey) on tryptone soy agar (TSA) slants. Overnight cultures of L. plantarum were grown in MRS broth at 32 C at CO2 chamber (New Brunswick Scientic Co, Edison, NJ, USA) and E. coli O157:H7, L. monocytogenes, S. aureus, and S. enteritidis were grown aerobically in BHI broth at 37 C. 2.3. Essential oil production Leaves and aerial parts of oregano (Origanum minutiorum) and rosemary (Rosmarinus ocianalis L.) were collected from the district of Sutculer, Isparta-Turkey. Garlic (Allium sativum L.) bulbs were purchased from a local market (herbalist) in Isparta. Oregano and rosemary owers and leaves were separated from their stem parts and air-dried until use. Garlic bulbs were broken into cloves at the time of extraction. Oregano, rosemary and garlic essential oils were obtained by steam-distillation for 3 h using a Clevenger-type apparatus according to the method of Dadalioglu and Evrendilek (2004). The obtained essential oils were closed under nitrogen gas and stored in airtight glass vials covered with aluminium foil at 4 C. 2.4. Whey protein isolate-based lm preparation The WPI lms were formed with the modication of the method described by Kim and Ustunol (2001). Whey protein isolate (5% wt/vol) was dissolved in distilled water, and glycerol (5% wt/vol) was added. The pH was adjusted to 8.0 with 2 N NaOH. Then, solutions were heated to 90 2 C while being stirred continuously. Candelilla wax (0.8% wt/vol) was added during heating. The lm solutions were ltered through a layer of cheesecloth. Oregano, rosemary and garlic oils in 1%, 2%, 3%, and 4% ratios were added to the lm solutions as essential oil concentrations per lm. After homogenizing for 2 min using a Heidolph DIAX 900 homogenizer (Kelheim, Germany) solutions were cooled to room temperature for 1.5 h. Vacuum was applied for 30 min to remove dissolved air in the solutions. The lms forming solutions (27.5 mL) were casted on 18.5 cm circular Teon surfaces, and then dried overnight at 35 C and 45 5% RH. Dried weight of the lm was 6.83 g. Dried lms were peeled and stored at room temperature and 40 5% RH. The nal concentrations in the

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dried lms were 68.3, 136.6, 204.9 and 273.2 mg/g lm, respectively. The concentration of essential oil per disc was calculated using essential oil concentration per gram multiplied by the average weight of the discs. 2.5. Film thickness and disc weight Film thicknesses were measured with a digital micrometer (Digimatic Micrometer, Mitutoyo, Japan) to the nearest 0.0001 mm. Measurements were taken at twelve random locations of the lms sheets. Average lm thickness was 0.2458 mm. Circular discs were cut from the WPI edible lms using a cutting well (diameter = 8.64 mm). Disc weights were measured at twelve random location of lm on each lm making. Average disc weight was 0.0149 g. 2.6. Determination of antimicrobial eects of WPI lms The zone of inhibition assay on solid media was used for determination of the antimicrobial eects of lms against L. plantarum (DSM 20174), S. enteritidis (ATCC 13076), E. coli O157:H7 (ATCC 35218), L. monocytogenes (NCTC 2167) and S. aureus (ATCC 43300). Ten millilitres of molten BHI agar was inoculated by 200 lL of bacterial cultures (colony count of 1 108 CFU/mL). Test discs were placed on the bacterial lawns. The plates were incubated at 37 C for 24 h in the appropriate incubation chamber. The plates were examined for zone of inhibition of the lm discs, and the diameter of the zone was measured with a calliper. The area of the whole zone was calculated then subtracted from the lm disc area and this dierence in area was reported as the zone of inhibition. 2.7. Statistical analysis Three observations were performed at each level (1%, 2%, 3%, and 4%) of essential oil treatment to WPI lms, and each experiment was replicated 3 times. The signicance of added essential oils based on the area of inhibition was determined using analysis of variance (ANOVA) at the 5% level. Means were compared with Duncans multiple range test of SAS statistical analysis system (SAS, 1999). 3. Results and discussion Increasing levels of oregano essential oils were incorporated to WPI lms and were tested against microorganisms for the zone of inhibition area (Table 1). Film containing 1% of oregano essential oil was not eective against any test microorganisms. The minimum amount of oregano essential oil level that showed inhibition was 2% for all test microorganisms. The greatest zone of inhibition was observed at 4% level against S. aureus, S. enteritidis, and L. monocytogenes (P < 0.05). As the concentration increased, the zone of inhibition also increased signicantly for S. enteritidis, E. coli O157:H7, L. monocytogenes and S. aureus, whereas an increase in concentration was not

Table 1 Antimicrobial activity of WPI lms incorporated with essential oils against test microorganisms Bacteria Percentage concentration (wt/vol) in lm solution 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Inhibitory zone (mm2) Oregano Garlic Rose mary 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Escherichia coli O157:H7

0d 18.99c 28.62b 37.09a 0d 33.65c 37.09b 43.07a 0d 30.28c 34.33b 40.59a 0d 29.61c 34.33b 41.65a 0d 9.30c 11.96b 13.45a

0c 0c 9.30b 11.36a 0c 0c 11.36b 13.45a 0c 0c 8.43b 10.48a 0c 0c 9.89b 11.96a 0c 0c 6.13b 9.21a

Staphylococcus aureus

Salmonella enteritidis

Listeria monocytogenes

Lactobacillus plantarum

Values (n = 9) with dierent superscript letters in each row are signicantly dierent (P < 0.05).

eective for L. plantarum (P > 0.05) above the 2% level. Sample pictures of the inhibitory eect of WPI lms incorporated with 2% oregano essential oil against all test microorganisms in comparison with the control were shown in Fig. 1. Dadalioglu and Evrendilek (2004) reported a strong inhibitory eect by the direct application of oregano essential oil to E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus using the same source of oregano as the current study. These researchers found the inhibitory eect of oregano was due to the relatively high concentration of carvacrol and p-cymene. In the current study, this inhibitory eect was incorporated and expressed in a exible bio-based lm. Mode of action of carvacrol was explained by Burt (2004) that it disintegrates the outer membrane of gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane to ATP. In another study, 1% of oregano essential oil incorporated into a calcium caseinate WPI-carboxymethyl cellulose lm was found to be eective against E. coli O157:H7 and Pseudomonas spp. on the surface of beef muscle pieces (Oussallah et al., 2004). The current study suggests that the use of 2% oregano essential oil in WPI lm was the minimum inhibitory level against S. aureus, S. enteritidis, L. monocytogenes, L. plantarum, and E. coli O157:H7. It is concluded that source and concentration of active compounds of the plant extracts, and the

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Fig. 1. Representative picture of inhibitory zones of WPI lm incorporated with 2% (wt/vol) oregano essential oil compared to control against test microorganisms. (a) Control, (b) S. aureus, (c) E. coli O157:H7, (d) L. monocytogenes, (e) L. plantarum and (f) S. enteritidis.

compositions of lm material have crucial eect on their biological activity in edible lms. WPI lms containing 1% and 2% of garlic essential oil were not inhibitory against any of the test microorganisms (Table 1). When the concentration of garlic essential oil in WPI lms was increased to 3%, signicant inhibition was observed against all test strains (P < 0.05). At 4% a greater inhibitory eect was observed on S. aureus followed by L. monocytogenes with zone area of 13.45 mm2 and 11.96 mm2, respectively. These results veried the study of Pranoto, Salokhe, and Rakshit (2005) who reported that S. aureus and Bacillus cereus were more sensitive to garlic oil-incorporated alginate-based lm as compared to E. coli and S. typhimurium. Previous studies on antimicrobial activity of garlic essential oils in culture media showed

some inhibitory eect on certain pathogen bacteria (Ross, OGara, Hill, Sleightholme, & Maslin, 2001; Warade & Shinde, 1998). Garlic oil, extracted from garlic bulbs using steam distillation, is composed primarily of diallyl disulde (60%), diallyl trisulde (20%), allyl propyl disulde (16%), a small quantity of disulde and probably diallyl polysulde (Warade & Shinde, 1998) named allicin. Han, Lawson, Han, and Han (1995) reported that the antibiotic activity of 1 mg allicin has been equated to that of 15 IU of penicillin. Rosemary essential oils incorporated WPI lms did not show any inhibitory eect against the microorganisms tested. Antimicrobial activity of rosemary essential oil against some food-borne pathogens in liquid media was reported by Smith-Palmer, Stewart, and Fyfe (1998),

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Hammer et al. (1999) and Pintore et al. (2002). The minimum inhibitory concentration was greater than the levels in this research. The dierent inhibitory eects of essential oils may be attributed to the dierences in the biological properties of the main compounds in the essential oils. Notably, rosemary essential oil did not maintain its known antimicrobial activity in WPI based edible lms. 4. Conclusions Direct addition of essential oils to food will result in immediate reduction of bacterial population, and may alter the sensory characteristics of added food. Thus, incorporation of essential oils to edible lms may have supplementary applications in food packaging. The use of essential oils in the WPI lm production is very promising. Biologically active substances of some essential oils were eective in WPI lms based on the zone of inhibition assay. Oregano and garlic essential oil added lms exhibited larger inhibitory zones on S. aureus, S. enteritidis, L. monocytogenes, E. coli and L. plantarum as compared to rosemary essential oil incorporated lms. Further investigation is needed for other spice and plant extracts for their ecacy in bio-based lms. Such application of edible lms containing oregano and garlic essential oils in the sausage casings and individually wrapped process sliced cheese packaging may have great benets. Acknowledgements This research was supported by The Scientic and Technical Research Council of Turkey (TUBITAK-TOGTAG Project # 3343). The authors like to thank Drs Paul L Dawson, Gu n Akdemir Evrendilek, Yesim Ekinci and lsu Zeynep Guzel-Seydim for their valuable support. References
Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science and Emerging Technologies, 3, 113126. Burt, S. A., & Reinders, R. D. (2003). Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Letters in Applied Microbiology, 36, 162167. Burt, S. A. (2004). Essential oils: their antibacterial properties and potential applications in foods: a review. International Journal of Food Microbiology, 94, 223253. Cagri, A., Ustunol, Z., & Ryser, E. T. (2004). Antimicrobial edible lms and coatings. Journal of Food Protection, 67(4), 833848. Chen, H. (1995). Functional properties and applications of edible lms made form milk proteins. Journal of Dairy Science, 78, 25632583. Chick, J., & Ustunol, Z. (1998). Mechanical and barrier properties of lactic acid and rennet-precipitated casein-based edible lms. Journal of Food Science, 63(6), 10241027. Dadalioglu, I., & Evrendilek, G. (2004). Chemical compositions and antibacterial eects of essential oils of Turkish oregano (Origanum minutiorum), bay laurel (Laurus nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum vulgare) on common foodborne pathogens. Journal of Agriculture and Food Chemistry, 52, 82558260. Dawson, P. L., Carl, G. D., Acton, J. C., & Han, I. Y. (2002). Eect of lauric acid and nisin-impregnated soy-based lms on the growth of

Listeria monocytogenes on turkey bologna. Poultry Science, 81, 721726. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308316. Gennadios, A., McHugh, T. H., Weller, C. L., & Krochta, J. M. (1994). Edible coating and lms based on properties. In J. M. Krochta, E. Baldwin, & M. Nisperos-Carriedo (Eds.), Edible coating and lms to improve food quality (pp. 201277). Lancaster, PA: Technomic Publishing Co. Han, J., Lawson, L., Han, G., & Han, P. (1995). A spectrophotometric method for quantitative determination of allicin and total garlic thiosulnates. Annals of Biochemistry, 225, 157160. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology, 86, 985990. Helander, I. M., Alokomi, 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 Agriculture and Food Chemistry, 46, 35903595. Homan, K. L., Han, I. Y., & Dawson, P. L. (2001). Antimicrobial eects of corn zein lms impregnated with nisin, lauric acid, and EDTA. Journal of Food Protection, 64(6), 885889. Kim, S.-J., & Ustunol, Z. (2001). Sensory attributes of whey protein isolate and candelilla wax emulsion edible lms. Journal of Food Science, 66(6), 909911. Krochta, J. M., & DeMulder-Johnston, C. (1997). Edible and biodegradable polymer lms; Challenges and opportunities. Food Technology, 51(2), 6174. McHugh, T. H., & Krochta, J. M. (1994a). Milk protein based edible lms and coatings. Food Technology, 48(1), 97103. McHugh, T. H., & Krochta, J. M. (1994b). Water vapor permeability properties of edible whey proteinlipid emulsion lms. Journal of AOAC International, 71, 307312. Miller, K. S., & Krochta, J. M. (1997). Oxygen and aroma barrier properties of edible lms: a review. Trends in Food Science and Technology, 8, 228237. Min, S., Harris, L. J., Han, J. H., & Krochta, J. M. (2005). Listeria monocytogenes inhibition by whey protein lms and coatings incorporating lysozyme. Journal of Food Protection, 68(11), 23172325. Oussallah, M., Caillet, S., Salmieri, S., Saucier, L., & Lacroix, M. (2004). Antimicrobial and antioxidant eects of milk protein based lm containing essential oils for the preservation of whole beef muscle. Journal of Agriculture and Food Chemistry, 52, 55985605. Padgett, T., Han, I. Y., & Dawson, P. L. (1998). Incorporation of food grade antimicrobial compounds into biodegradable packaging lms. Journal of Food Protection, 61(10), 13301335. Padget, T., Han, I. Y., & Dawson, P. L. (2000). Eect of lauric acid addition on the antimicrobial ecacy and water permeability of corn zein lms containing nisin. Journal of Food Processing and Preservation, 24(5), 423432. Paster, N., Juven, B. J., Shaaya, E., Menasherov, M., Nitzan, R., Weisslowiez, H., et al. (1990). Inhibitory eect of oregano and thyme essential oils on moulds and foodborne bacteria. Letters in Applied Microbiology, 11, 3337. Perez-Gago, M. B., & Krochta, J. M. (1999). Water permeability of whey protein emulsion lms as eected by pH. Journal of Food Science, 64(4), 695698. Pintore, G., Usai, M., Bradesi, P., Juliano, C., Boatto, G., Tomi, F., et al. (2002). Chemical composition and antimicrobial activity of Rosmarinus ocinalis L. oils from Sardinia and Corsica. Flavour and Fragrance Journal, 17, 519. Pranoto, Y., Salokhe, V. M., & Rakshit, S. K. (2005). Physical and antibacterial properties of alginate-based edible lm incorporated with garlic oil. Food Research International, 38, 267272. Rooney, M. L. (1995). Overview of active food packaging. In M. L. Rooney (Ed.), Active food packaging (pp. 137). Glasgow, Ireland: Blackie Academic and Professional.

644

A.C. Seydim, G. Sarikus / Food Research International 39 (2006) 639644 important food-borne pathogens. Letters in Food Microbiology, 26, 118122. Tharanathan, R. N. (2003). Biodegradable lms and composite coatings: past, present and future. Trends in Food Science Technology, 14, 7178. Vermeiren, L., Devlieghere, F., van Beest, M., de Kruiif, N., & Debevere, J. (1999). Developments in the active packing of foods. Trends in Food Science and Technology, 10, 7786. Warade, S. D., & Shinde, K. G. (1998). In D. K. Salunke & S. S. Kadam (Eds.), Handbook of vegetable science and technology (pp. 397413). USA: Marcel Dekker Inc. Weng, Y. M., & Hotchkiss, J. H. (1992). Inhibition of surface molds on cheese by polyethylene containing the antimycotic imazalil. Journal of Food Protection, 55, 367369. Zaika, L. L. (1988). Spices and herbs: their antimicrobial activity and its determination. Journal of Food Safety, 9, 97118.

Ross, Z. M., OGara, E. A., Hill, D. J., Sleightholme, H. V., & Maslin, D. J. (2001). Antimicrobial properties of garlic oil against human enteric bacteria: evaluation of methodologies and comparisons with garlic oil suldes and garlic powder. Applied and Environmental Microbiology, 67, 475480. SAS. (1999). SAS OnlineDoc, Version 8. Cary, NC: SAS Institute Inc. Siragusa, G. R., Cutter, C. N., & Willett, J. L. (1999). Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat. Food Microbiology, 16, 229235. Shellhammer, T. H., & Krochta, J. M. (1997). Whey protein emulsion lm performance: Eect of lipid type and amount. Journal of Food Science, 62(2), 390394. Smith-Palmer, A., Stewart, J., & Fyfe, L. (1998). Antimicrobial properties of plant essential oils and essences against ve