Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products

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1 Journal of Applied Microbiology 2003, 94, Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products M. E. Guynot, A. J. Ramos, L. Setó, P. Purroy, V. Sanchis and S. Marín Food Technology Department, Lleida University, Rovira Roure, Lleida, Spain 2002/273: received 3 July 2002, revised 17 January 2003 and accepted 31 January 2003 ABSTRACT M. E. GUYNOT, A. J. RAMOS, L. SETÓ, P. PURROY, V. SANCHIS AND S. MARÍN Aims: To investigate the volatile fractions of 16 essential oils for activity against the more common fungi causing spoilage of bakery products, Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. Methods and Results: The study applied 50 ll of pure essential oils in a sterilized filter paper, were carried out at ph 6 and at different water activity levels (0Æ80 0Æ90). First, a wheat flour based agar medium was used, where cinnamon leaf, clove, bay, lemongrass and thyme essential oils where found to totally inhibit all microorganisms tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. Conclusion: Five essential oils showed potential antifungal capacity against all species tested, over a wide range of water availability. Their activity, however, seems to be substrate-dependent. More research is needed to make them work in real bakery products, as in the preliminary study limited effectiveness was found. Significance and Impact of the Study: The potential of the cinnamon leaf, clove, bay, lemongrass and thyme essential oils against species belonging to Eurotium, Aspergillus and Penicillium genus has been demonstrated. Keywords: bakery products, essential oils, fungi, volatile compounds, water activity. INTRODUCTION In bakery processing, the most common type of microbial spoilage is mould growth and in many cases it is the major factor governing shelf-life (Earle and Putt 1984). As products of intermediate moisture (0Æ75 0Æ90a w, a w being the water activity) (Marín et al., 2002a), cakes are particularly sensitive to xerophilic moulds spoilage (Seiler 1988; Beuchat and Hocking 1990; Pitt and Hocking 1997; Fustier et al. 1998). Historically, many plant oils and extracts have been used as topical antiseptics, or have been reported to have antimicrobial properties (Hoffman 1987; Lawless 1995). Correspondence to: A. J. Ramos, Food Technology Department, Lleida University, UTPV-CeRTA, Rovira Roure 191, Lleida, Spain ( ajramos@tecal.udl.es). Restrictions imposed by the food industry and regulatory agencies on the use of some synthetic food additives have led to renewed interest in searching for alternatives, as natural antimicrobial compounds, particularly those derived from plants (Delaquis and Mazza 1995; Hammer et al. 1999). Essential oils and their constituents have been used extensively as flavour ingredients in a wide variety of foods, beverages, and confectionery products. Many of such compounds are classified as Generally Recognised As Safe (Kim et al. 1995). The antimicrobial activity of spices and essential oils is well recognized. Several studies have reported results on their preservative action (Mishra and Dubey 1994; Paster et al. 1995; Montes-Belmont and Carvajal 1998; Vázquez et al. 2001). For this reason, these substances can be used as safe and effective alternatives to synthetic preservatives (Wilson et al. 1997). Essential oils ª 2003 The Society for Applied Microbiology

2 894 M. E. GUYNOT ET AL. of thyme, cinnamon, bay and clove had been proved to possess antimicrobial activity (Farag et al. 1989). The antifungal activity of essential oils upon solution contact as measured by the broth dilution and agar dilution methods has been studied by many researchers. However, the activity by vapour contact has been reported more rarely (Matsuoka et al. 1990; Inouye et al. 2000). Several spices, herbs and fruits contain volatile antimicrobial compounds (Zaika 1988). Some volatile components of citrus fruit essential oils show a high degree of fungal inhibition (Caccioni et al. 1998). Wilson et al. (1987) found that a number of the volatiles that emanate from peaches, as they ripen, are fungicidal. Volatile antimicrobial substances (essential oils and oleoresins) from mustard, cinnamon and garlic have proven to be efficient in the control of spoilage by common bread spoiling fungi (Nielsen and Rios 2000). Earlier, some investigators reported the antifungal activity of black mustard powder (Goi et al. 1985) or mustard extract (Sekiyama et al. 1996) by gas contact. Arras and Usai (2001) found that thyme essential oil vapours had some antifungal activity. The authors also recorded alteration in Penicillium digitatum morphology. Although different results have been observed depending on testing conditions, target microorganisms and antimicrobial substance origin, some essential oil compounds such as eugenol, geraniol, thymol and cinnamaldehide have consistently been reported to have antifungal activity (Bullerman et al. 1977; Farag et al. 1989; Salmeron and Pozo 1991; Mahmound 1994; Chao and Young 2000; Chang et al. 2001). The objective of this work was to evaluate the antifungal activity of 16 essential oils by vapour contact against the most common fungal species causing bakery products spoilage, under different water activity conditions. MATERIALS AND METHODS Fungal isolates A total of seven isolates from different bakery products were used. Five of them, Eurotium amstelodami (3Æ205), Eurotium herbariorum (3Æ209), Eurotium rubrum (3Æ228), Aspergillus flavus (3Æ226) and Aspergillus niger (3Æ227) were isolated by Abellana et al. (1997) from Spanish bakery products. These isolates belong to the Food Technology Department microorganisms collection of the Lleida University. The other two isolates, Eurotium repens (IBT18000) and Penicillium corylophilum (IBT6978) were kindly provided by the Department of Biotechnology of the Technical University of Denmark and had been isolated from Danish bakery products. Study in wheat flour agar Essential oils. The essential oils used in this study were of lemon, aniseed, mandarin, grapefruit, cinnamon leaf, lemongrass, rosemary, thyme, basil, sweet fennel, peppermint, ginger, bay, clove, sage and orange. They were all supplied by F. D. Copelans & Sons, Ltd (London, UK). Experimental design. The factors assayed were different essential oils as a qualitative factor and a w (0Æ80, 0Æ85 and 0Æ90) as a quantitative factor. The a w values include the range of a w variation of bakery products. The response recorded was colony diameter. A full factorial design was used and all treatments were repeated twice. Media preparation. The basic medium used was a 2% wheat flour agar. It was adjusted to achieve the desired a w level and to ph 6 (as a mean ph for bakery products) as previously described by Marín et al. (2002a). Sterile media were poured into 9-cm diameter sterile Petri plates. The final a w and ph levels of the media were checked with an AquaLab (Decagon, Pullman, WA, USA), and a Crison microph2000 ph-meter (Crison, Barcelona, Spain), respectively. Inoculation, incubation and measurement. Plates were kept in an inverted position. A sterilized filter paper (square of 1 1 cm) was placed in the centre of the lid and 50 ll of pure essential oil were added to the paper. Blank were made by adding 50 ll of water to it. For each fungal isolate, a conidial spore suspension of 10 6 spores ml )1 was prepared and Petri plates were needle-inoculated in the centre. Plates were tightly sealed with parafilm and incubated for 42 days at 25 C. Diameters of the growing colonies were measured weekly with the aid of a binocular magnifier. Chemical analysis of the essential oils. Analysis of essential oils having antifungal activity in the wheat flour agar study was performed by gas chromatography. The Consejo Superior de Investigaciones Científicas (CSIC, Institute of General Organic Chemistry, Madrid, Spain) kindly performed the analysis, using a Hewlett-Packard chromatograph (Hewlett Packard Co. Geneva, Switzerland), model 6890 coupled with mass spectrometer selective detector 5973N. Studies in cake analogues Pure essential oils found to be effective in the agar study were checked (at the same levels of a w and ph) in a Spanish cake dough representative, prepared in the laboratory as previously reported by Abellana et al. (1999). The dough after baking had a ph of about 7Æ5, and its initial a w was ca. 0Æ75. To reach the required ph (6Æ0), citric acid was added to the mixed solid ingredients before baking, whereas a w was

3 ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS 895 adjusted to 0Æ80, 0Æ85 and 0Æ90 by placing the cake analogues in Petri plates containing water glycerol agar (1Æ5% of agar and different amounts of glycerol depending on the desired a w, as indicated in Table 1). Previously, two calibration curves were plotted, one to determine the amount of citric acid necessary to reach the desired ph (Marín et al. 2002b) and the other to determine the concentration of glycerol in the agar needed to increase and maintain the a w of the dough during the incubation period (data not shown). After baking, the cakes were aseptically cut into 5 5-cm squared pieces, placed in 9-cm sterile Petri plates containing water glycerol agar and after being sealed with parafilm, incubated at 25 C for a 48-h equilibration period. The procedures for adding the essential oils (50 ll) and for making the blanks were the same as described above, but in this case, paper was placed on the water the glycerol agar beside the cake analogue, as Petri plates had to be incubated in an upright position. The inoculation and incubation procedures, and the measurements of fungal growth were the same as described above. The experiment was performed in triplicate. Table 1 Composition of the water glycerol agar used to adjust a w level of the cake analogues a w of cake analogues 0Æ80 0Æ85 0Æ90 Glycerol (g) per 100 ml of water 105Æ8 69Æ0 39Æ1 Statistical treatment of the results Analyses of variance of the effect of a w and essential oil volatile fraction with the colony radius measures at the different incubation days as co-variable, were performed for each isolate separately by using SAS (Statistical Analysis System) program version 8Æ02 (SAS Institute Inc., Cary, NC, USA). RESULTS Studies in wheat flour agar The combination of natural preservatives in the surrounding atmosphere and reduced a w, had a significant impact on the growth of all isolates tested (Tables 2 and 3). The behaviour of all species was quite similar and, as expected, the growth was favoured by increasing levels of water activity. Aspergillus spp. and P. corylophilum were unable to grow at 0Æ80a w.at0æ85a w A. niger was markedly affected by low water availability, and then fungal growth was most easily prevented (Table 2). Among the essential oils, the ones that totally inhibited growth of all species, were cinnamon leaf, lemongrass, thyme, bay and clove, regardless of a w level. Peppermint and ginger were also able to inhibit fungal growth but their activity was restricted to the lower a w levels (0Æ80 0Æ85). The remaining essential oils were, in general, ineffective or only Table 2 Mean colony diameter (mm) of Eurotium spp. growing on wheat flour agar medium exposed to different essential oil atmospheres and a w levels, after 42 days of incubation at 25 C E. amstelodami E. herbariorum E. repens E. rubrum Essential oil 0Æ80a w 0Æ85a w 0Æ90a w 0Æ80a w 0Æ85a w 0Æ90a w 0Æ80a w 0Æ85a w 0Æ90a w 0Æ80a w 0Æ85a w 0Æ90a w Blank 29Æ5 a 43Æ1 a 69Æ6 a 0Æ2 b 30Æ3 ab 44Æ3 a 14Æ8 a 28Æ5 ab 61Æ2 ab 5Æ4 bc 27Æ2 ab 60Æ6 a Lemon 18Æ8 abc 44Æ3 a 65Æ5 a 0Æ0 b 29Æ8 ab 46Æ1 a 0Æ0 b 27Æ7 ab 59Æ7 ab 19Æ7 ab 38Æ7 ab 48Æ7 a Mandarin 21Æ9 ab 44Æ2 a 56Æ3 a 3Æ4 a 37Æ8 a 58Æ0 a 11Æ1 a 26Æ1 ab 64Æ8 a 26Æ1 a 52Æ3 a 66Æ4 a Aniseed 16Æ5 abcd 44Æ0 a 61Æ6 a 0Æ0 b 19Æ4 abc 50Æ0 a 7Æ8 ab 33Æ2 ab 61Æ1 ab 6Æ8 bc 37Æ2 ab 63Æ7 a Sweet fennel 13Æ9 bcde 35Æ7 a 59Æ3 a 0Æ2 b 18Æ1 abc 55Æ0 a 7Æ4 ab 19Æ6 abc 48Æ0 abc 12Æ8 abc 39Æ1 ab 62Æ1 a Orange 13Æ0 bcde 47Æ1 a 70Æ9 a 1Æ4 ab 35Æ1 a 54Æ8 a 13Æ1 a 26Æ8 ab 63Æ9 ab 12Æ7 abc 47Æ1 ab 67Æ1 a Grapefruit 7Æ7 cde 43Æ8 a 68Æ0 a 0Æ0 b 23Æ9 ab 50Æ3 a 1Æ6 b 36Æ6 a 58Æ4 ab 9Æ9 abc 34Æ8 ab 53Æ7 b Rosemary 3Æ0 de 24Æ2 ab 59Æ4 a 0Æ0 b 49Æ0 ab 56Æ5 a 0Æ0 b 26Æ4 ab 55Æ6 ab 9Æ6 abc 24Æ6 abc 62Æ3 a Basil 2Æ5 e 36Æ6 a 59Æ5 a 1Æ1 ab 42Æ8 ab 54Æ4 a 0Æ0 b 20Æ3 abc 44Æ2 abc 16Æ6 abc 29Æ8 ab 61Æ8 a Sage 2Æ3 e 20Æ7 ab 64Æ3 a 1Æ3 ab 0Æ0 abc 47Æ1 a 1Æ3 b 21Æ2 abc 33Æ3 bc 9Æ1 abc 20Æ8 bc 69Æ7 a Ginger 0Æ0 e 23Æ5 ab 63Æ4 a 0Æ0 b 21Æ4 abc 51Æ6 a 0Æ0 b 16Æ2 bcd 49Æ7 abc 0Æ0 c 40Æ5 ab 65Æ0 a Peppermint 0Æ0 e 0Æ6 b 20Æ2 b 0Æ0 b 12Æ9 bc 52Æ7 a 0Æ0 b 3Æ7 cd 20Æ5 cd 0Æ0 c 0Æ0 c 49Æ7 a Lemongrass 0Æ0 e 0Æ0 b 0Æ0 b 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b 0Æ0 d 0Æ0 d 0Æ0 c 0Æ0 c 0Æ0 b Clove 0Æ0 e 0Æ0 b 0Æ0 b 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b 0Æ0 d 0Æ0 d 0Æ0 c 0Æ0 c 0Æ0 b Cinnamon leaf 0Æ0 e 0Æ0 b 0Æ0 b 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b 0Æ0 d 0Æ0 d 0Æ0 c 0Æ0 c 0Æ0 b Bay 0Æ0 e 0Æ0 b 0Æ0 b 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b 0Æ0 d 0Æ0 d 0Æ0 c 0Æ0 c 0Æ0 b Thyme 0Æ0 e 0Æ0 b 0Æ0 b 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b 0Æ0 d 0Æ0 d 0Æ0 c 0Æ0 c 0Æ0 b Data in the column followed by different letters are significantly different in LSMEANS test.

4 896 M. E. GUYNOT ET AL. A. flavus A. niger P. corylophilum Essential oil 0Æ85a w 0Æ90a w 0Æ85a w 0Æ90a w 0Æ85a w 0Æ90a w Blank 31Æ1 a 74Æ9 a 14Æ0 a 55Æ2 ab 1Æ4 a 8Æ6 b Lemon 14Æ2 bc 62Æ9 a 0Æ0 b 69Æ0 ab 0Æ0 b 29Æ0 a Mandarin 6Æ3 cd 61Æ3 a 0Æ0 b 73Æ2 a 0Æ0 b 3Æ2 b Aniseed 15Æ2 bc 55Æ6 ab 0Æ0 b 66Æ4 ab 0Æ0 b 7Æ1 b Sweet fennel 19Æ4 ab 72Æ2 a 0Æ0 b 72Æ5 a 0Æ0 b 1Æ1 b Orange 21Æ4 ab 75Æ2 a 2Æ8 b 75Æ5 a 0Æ0 b 7Æ7 b Grapefruit 18Æ7 abc 63Æ8 a 0Æ0 b 72Æ3 a 0Æ0 b 5Æ8 b Rosemary 12Æ1 bcd 68Æ8 a 0Æ0 b 66Æ3 ab 0Æ0 b 22Æ7 a Basil 24Æ4 ab 72Æ2 a 0Æ0 b 72Æ9 a 0Æ0 b 5Æ6 b Sage 0Æ0 d 54Æ0 ab 0Æ5 b 66Æ9 ab 0Æ0 b 5Æ5 b Ginger 18Æ0 ab 56Æ1 ab 0Æ0 b 69Æ9 ab 0Æ0 b 3Æ5 b Peppermint 0Æ0 d 36Æ0 b 0Æ7 b 45Æ2 b 0Æ0 b 2Æ9 b Lemongrass 0Æ0 d 0Æ0 c 0Æ0 b 0Æ0 c 0Æ0 b 6Æ4 b Clove 0Æ0 d 0Æ0 c 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b Cinnamon leaf 0Æ0 d 0Æ0 c 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b Bay 0Æ0 d 0Æ0 c 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b Thyme 0Æ0 d 0Æ0 c 0Æ0 b 0Æ0 c 0Æ0 b 0Æ0 b Table 3 Mean colony diameter (mm) of Aspergillus spp. and Penicillium corylophilum growing on wheat flour agar medium exposed to different essential oil atmospheres and a w levels, after 42 days of incubation at 25 C Data in the column followed by different letters are significantly different in LSMEANS test. delayed growth of some of the species. Sage essential oil slowed E. amstelodami and E. repens growth at 0Æ80a w, although it favoured E. herbariorum and E. rubrum growth at the same a w level (Table 2); A. niger and A. flavus growth were also inhibited by sage essential oil at 0Æ85a w (Table 3). The most sensitive species to essential oils at low a w were E. repens and A. niger, as they were inhibited by almost all essential oils at 0Æ80a w and at 0Æ85a w, respectively. On the other hand, some promoting effect on fungal growth by the addition of some essential oils was observed; the ones that stood out were mandarin favouring E. herbariorum and E. rubrum growth at low a w, orange favouring E. herbariorum also at low a w, lemon favouring P. corylophilum at 0Æ90a w and to a lesser extent E. repens at 0Æ80a w. Finally P. corylophilum grew more at 0Æ90a w when rosemary essential oil was added. Composition of the essential oils with antifungal activity in agar medium The main components of cinnamon leaf, clove, bay, thyme and lemongrass essential oils were identified by GC-mass spectrometer analyses. Their percentage compositions, calculated as the ratio of the peak area to the total chromatographic area, are listed in Table 4. Eugenol was the main component of clove (83Æ9%), cinnamon leaf (78Æ5%) and bay (57%) essential oils. Geraniol and neral represented the 50Æ5 and 29Æ4%, respectively, of lemongrass essential oil. Finally thyme essential oil was mainly composed of thymol (53Æ9%) and p-cimene (25Æ2%). Studies in cake analogues Both factors, the presence of essential oil and a w, and their interaction had a significant impact on the growth of all isolates tested. Further separate analyses were performed for each water activity level and the effect of different essential oils was analysed by the LSMEANS test (Table 5, E. repens). In general, a slight trend to delay fungal growth was observed only at 0Æ80a w. Moreover, in some cases, as a w increased fungal growth was favoured. All isolates had similar responses. DISCUSSION The ability of 16 essential oils to inhibit bakery spoilage fungi was evaluated. Most studies concerning the antimicrobial activity of natural products have been conducted by testing the plant extracts as contact materials, mostly by supplementing growth media or by incorporation of the plant material itself (Paster et al. 1995). However, these methods do not reflect the activity of the volatile fraction alone (Moleyer and Narashimam 1986; Delespaul et al. 2000). To this end, therefore, the present work aims to study the activity of the volatile portion of the essential oils against fungal growth. Previous experiments were carried out with the same set of essential oils used in this study, but they were added diluted directly to 2% wheat flour agar ( ppm). Results showed clearly that cinnamon leaf, bay, clove, rosemary and thyme had some antifungal activity; they

5 ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS 897 Table 4 Qualitative and quantitative composition (expressed as percentage) of essential oils having antifungal activity Component Bay Cinnamon leaf Clove Lemongrass Thyme Limonene 2Æ2 3Æ8 1Æ1 Cariophyllene 0Æ9 4Æ7 12Æ6 4Æ1 <1 Neral 29Æ4 Geraniol 50Æ5 Geranial acetate 1Æ6 Geranial 5Æ0 Cariophylene oxide <1 <1 Humelene 1Æ4 Eugenol 57Æ0 78Æ5 83Æ9 a-cubenene <1 Linalool 2Æ6 2Æ6 3Æ5 2-propenil benzodioxol 1Æ4 3-phenil propenal 1Æ1 3-phenil propenol acetate 1Æ1 Eugenil acetate 1Æ7 Mirceno 12Æ4 1Æ7 Cineol 1Æ6 1Æ0 1-octen-3-ole 1Æ0 Cadinene 0Æ5 Metil eugenol 1Æ9 c-terpinene 3Æ4 p-cimene 25Æ2 Bornil-acetate 1Æ8 a-terpineol <1 Borneol <1 Thymol 53Æ9 Carvacrol 2Æ9 Non-identified compounds 15Æ7 Table 5 Mean colony diameter (mm) of Eurotium repens growing on sponge cake analogues, exposed to different essential oils volatile fraction, after 42 days of incubation at 25 C Essential oil 0Æ80a w 0Æ85a w 0Æ90a w Control 14Æ6 a 25Æ2 b 39Æ9 a Thyme 10Æ9 b 26Æ9 ab 39Æ7 a Lemongrass 8Æ8 bc 29Æ5 ab 39Æ3 a Cinnamon leaf 8Æ1 bc 29Æ2 ab 39Æ3 a Bay 10Æ6 bc 25Æ6 b 39Æ8 a Clove 7Æ3 c 31Æ9 a 41Æ0 a Data in the column followed by different letters are significantly different at P <0Æ01 in LSMEANS test. prevented or delayed mycelium growth of the seven isolates tested (M. E. Guynot, S. Marín, L. Setó, V. Sanchis, A. J. Ramos, unpublished data). The present study confirmed the antifungal activity of cinnamon leaf, bay, clove and thyme essential oils. Although no growth inhibition was observed by applying rosemary essential oil, lemongrass was found to delay fungal growth by vapour contact. Nielsen and Rios (2000) had proven that the volatile substances from mustard, cinnamon, garlic and clove essential oils were efficient in the control of common bread spoilage fungi. Inouye et al. (2000) also found fungicidal activity in lemongrass essential oil followed by cinnamon bark and thyme oils, by vapour contact. However, comparison of the data obtained by different studies is difficult, because of differences in plants extract compositions, in methodologies followed to assess antimicrobial activity and in microorganisms chosen to be tested (Hammer et al., 1999). Several investigators support the best antifungal activity of volatile compounds by gaseous contact than by aqueous solution or by agar contact (Goi et al. 1985; Sekiyama et al. 1996; Inouye et al. 2000). It is difficult to differentiate the direct effect of vapour on fungi from the indirect effect of vapour acting after absorption by the medium during a long incubation period (Inouye et al. 2000). Due to the hydrophobic nature of compounds that constitute the volatile fraction of essential oil, it could be expected that their dilution in the agar medium during incubation period was not so important. Sekiyama et al. (1996) measured the amount of allyl isothiocyanate (active principle of some species) in a potato dextrose agar exposed to vapour released

6 898 M. E. GUYNOT ET AL. from a mustard essential oil, and they detected that only the 15 20% of the extract used was absorbed by the culture medium. Due to the high lipophilic nature of mycelia coupled with a large surface area relative to the volume of a fungus, vapours of essential oils may act mainly by accumulation on mycelia than in the agar (Inouye et al. 2000). In general, the inhibitory effect has been attributed to the most abundant components and not to the other associated substances of the oil (Farag et al. 1989). However, some workers found more antifungal activity when adding the whole extract to the medium than when adding only the principal compound, suggesting a synergistic effect of some minor constituents of the oil (Bullerman et al. 1977; Paster et al. 1995; Chang et al. 2001). The composition of the extracts obtained from a plant is known to vary according to the species and variety of the plant, the agronomic conditions, the harvest time and the type of processing followed (Mishra and Dubey 1994; Guillén and Cabo 1996; Hammer et al. 1999). However, in general, the results obtained from the GC analysis of the essential oils used in this experiment were as expected. Eugenol reported by different workers to be the main component of clove, cinnamon leaf and bay essential oils and is also responsible for the antifungal effect of these oils (Ross 1976; Bullerman et al. 1977; Farag et al. 1989; Montes- Belmont and Carvajal, 1998). Bullerman et al. (1977) found eugenol to be the active compound responsible for fungal inhibition by clove essential oil, but they raised the possibility that interactive effects of other compounds present in smaller quantities may also contribute. On the other hand, Mahmound (1994) reported that 1000 ppm of different essential oils constituents, namely geraniol and thymol, completely inhibited fungal growth. Later Paster et al. (1995) found thymol, the main component of thyme essential oil, to be active against Aspergillus spp. when it was added to the medium. Vázquez et al. (2001) reported a total inhibition of Penicillium citrinum adding 2000 ppm of eugenol and thymol in a liquid medium. Several studies have been conducted to understand the mechanism of action of essential oils (Chang et al. 2001), however it is still unclear. Some studies suggest that compounds penetrate inside the cell, where they interfere with cellular metabolism (Marino et al. 2001). Other studies suggest that they disturb the cellular membrane and react with active sites of enzymes or act as a H + carrier, depleting adenosine triphosphate pool (Farag et al. 1989; Chang et al., 2001; Ultee et al. 2002). The promising results of the agar culture study encouraged us to conduct experiments in a sponge cake analogue. It was found, however, that all five essential oils had low antifungal activity, as they inhibited fungal growth only if they were combined with low water availability. Actually, bibliography exists reporting cases of low antimicrobial activity by this type of compounds when they are applied in foodstuff (Kim et al. 1995; Del Campo et al. 2000). Cake dough lipids may affect the activity of essential oils because of the hydrophobic properties of their active compounds. Arras and Usai (2001) showed that thyme essential oil was less effective on orange fruits than in vitro against P. digitatum. Earlier, Kim et al. (1995) found differences in the bactericidal effectiveness of three essential oil compounds (geraniol, citral and carvacol) when they were added in a culture medium or on inoculated fish cubes. Authors argue that this minor activity in vivo could be because of interactions of essential oils active compounds (known to be lipophilic) with food components, such as proteins and lipids, decreasing their level. As a conclusion, volatile substances from cinnamon leaf, clove, bay, lemongrass and thyme essential oils had a potential antifungal activity against common fungi causing spoilage of bakery products. Due to the poor activity observed when they were used in a sponge cake analogue, further studies are needed before their application in bakery products. Combining them with other preservatives or modified atmosphere packaging could be a valuable alternative. ACKNOWLEDGEMENTS The authors are grateful to the European Union (PL ), to the Spanish government (CICYT, ALI ) and to the Catalonian government (CIRIT) for their financial support. REFERENCES Abellana, M., Torres, L., Sanchis, V. and Ramos, A.J. (1997) Caracterización de diferentes productos de bollería industrial. II. 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