Antifungal Activity of Oregano and Thyme Essential Oils Applied as Fumigants Against Fungi Attacking Stored Grain

Transcription

1 Journal of Food Protection, Vol. 58. No.1, Pages International Association of Milk, Food and Environmental Sanitarians Antifungal Activity of Oregano and Thyme Essential Oils Applied as Fumigants Against Fungi Attacking Stored Grain NAHMAN PASTER,* MAZAL MENASHEROV, UZI RAVIOl and ENJAMIN JUVEN 1 Department of Stored Products, Agricultural Research Organization (ARO), The Volcani enter, P.O. ox 6, et Dagan 50250, Israel; 'Department of Medicinal and Spice rops, ARO, Newe Ya'ar Experimental Station, P.O. Haifa 30. Israel; and 'Department of Food Science, ARO, The Volcani enter, P.O. ox 6, et Dagan Israel (Received November 2, 1993/Accepted April II, 1994) ASTRAT Essential oils from oregano and thyme were applied for 24 h as fumigants against the mycelia and spores of Aspergillus flavus, Aspergillus niger and Aspergillus ochraceus, as well as against natural microflora of wheat grains. The minimal inhibitory concentration (MI) of oregano oil needed to inhibit the mycelial growth of the fungi was 2.0 Ill/L, while spores were eradicated following exposure to 2.0 to 2.5 Ill/L. The thyme essential oil was less efficient in controlling mycelia and growth was observed even following exposure to 4.0 Ill/L. However, the thyme essential oil was fungitoxic to spores (MI= 3.0 Ill/L). In another set of trials the efficacy of the oils and two of their constituents (carvacrol and thymol) in controlling natural microflora of surface-sterilized wheat grain was studied. Of the four materials investigated, only oregano essential oil exhibited fungicidal activity and, following 24 h exposure to J.1l/L,a significant reduction in the percent of infested grain was observed even after 5 days of incubation on potato dextrose agar. A reduction in the germinability of the grains was evident following exposure to the materials tested. When the fungicidal activity of oregano essential oil was evaluated using grains with different moisture contents (M), data revealed that the better inhibitory effect was achieved in grain with a high M. The findings emphasize the toxicity of oregano and thyme essential oils as fumigants against fungi attacking stored grain and strengthen the possibility of using them as an alternative to chemicals for preserving stored grains. Key Words: Essential oils, oregano, thyme, fungicidal activity, fungi, grain Fungi grown on stored grain can cause heavy damage, resulting in reductions in quantity and in quality. In addition, many species are capable of producing mycotoxins, secondary metabolites highly toxic to animals and humans. Fungi-inhibiting chemicals (mainly low-molecular-weight organic acids) have been used for the preservation of stored grain. However, many disadvantages are associated with the use of acids (10) and there is a worldwide trend towards limiting their use in grain and foodstuffs. Natural plant extracts may provide an alternative to these preservatives. Over the years, much effort has been devoted to the search for new antifungal materials from natural sources for food and grain preservation, and many antimicrobials occurring naturally in plants have been identified (6,). Some of them have been found also to inhibit mycotoxin formation (9,19). However, in most of the studies where the antimicrobial activity of essential oils has been studied, the assay techniques used involved application of the materials to the growth medium. While dealing with grain protection, fumigation is the preferred method for applying substances into the bulks in order to control the biotic factors which damage the grain. The objective of the present study was to evaluate the inhibitory effect of two essential oils, applied as fumigants, on the growth of three storage fungi in vitro. The ability of the materials to prevent the growth of fungi naturally infesting wheat grain was also studied. MATERIALS AND METHODS Fungal strains and growth conditions The fungi used in these studies were A. flavus Link:Fr., A. ochraceus Wilhelm and A. niger v. Tieghem. They were grown on potato dextrose agar (PDA) at 26 and agar discs (0.3 cm in diameter) covered with mycelium served as a source of inoculum. The discs were taken from the margin of 4-day-old colonies and spores were washed off from each disc using sterilized water. For spore germination assays, spore suspensions of each fungus (10 3 spores/ml counted with a hemocytometer) were prepared in sterile water. Spices and the extraction procedure The spices tested were oregano (Origanum vulgare) and thyme (oridothymus capitatus) grown for commercial use in the northern part of Israel. The essential oils were extracted as described previously (18). Fresh plant material was steam-distilled for 2.5 h in a 130-L direct steam pilot plant apparatus. The recovered oils were dried over anhydrous sodium sulfate and stored in dark at 4 to 6 (to avoid rapid oxidation). Oil constituents, carvacrol and thymol were purchased from Aldrich hemical ompany, Inc., in the United States, and arl Roth, Karlsruhe, Germany, respectively. Assay technique Glass flasks (3.4 L each) with a round bottom, closed with a glass stopper fitted with a hook, served as fumigation chambers. The test materials were applied on a small square of filter paper,

2 which was suspended by the hook, together with small wire-mesh cages in which mycelial discs were placed. In studies where the effect on spore survival was assayed, spore suspensions (0 III of each fungus) were exposed in small test tubes (1.0 cm diam x 2.0 cm height) placed in plastic holders, which were suspended by the hook. In order to ensure even distribution of the oil, a magnetic stirrer was placed on the bottom of each flask, and activated gently throughout the exposure period (24 h). Studies were carried out in a room with a constant temperature of 26 ± 1. Following the treatment, mycelium discs were transferred to petri dishes (each disc was placed on a separate plate) containing PDA and the linear growth was measured daily during incubation at 26 for 14 days. For spore germination assays, three portions (2 x 50 III + I x Ill) were taken from the exposed suspensions and spread over the surface of PDA in petri dishes. Germinating spores were counted daily during incubation at 26 for 14 days. Trials with wheat grain Studies with grain included trials in which surface-sterilized grain was used and trials with non-sterilized grain at different levels of M. Surface sterilization. Lots of grain (loa g each) was dipped for 2 min in a solution of 2% commercial sodium hypochlorite and subsequently rinsed (2 x 2 min) in sterile water. The grain was then placed to dry for I h over a sterile filter paper in a sterile cabinet before being used in the studies. The M of the sterilized grain was 18%, as determined using the air-oven method (3). Adjusting grains to different levels of Me. Different levels of M were achieved with the addition of sterile water according to the procedure described by Pixton (16). The grain was then mixed thoroughly and allowed to equilibrate in a closed bin at 5 for 14 days (17). Percentage of wheat germination was determined using the ISTA method (3). Exposing grains to the test materials. Lots of grains (50 g each) were introduced into wire-mesh baskets which were suspended from the flask stopper by the hook. The grains were fumigated (for 24 h, or 24 to 72 h in the case of oregano [OEO]) in the manner described above and following each treatment, grains were placed on PDA in petri dishes (10 grains/plate). Fungal infestation, defined as the percent of fungi-yielding grains, was estimated daily during 7 days of incubation at 26. Statistical analysis Each experiment was repeated at least three times. Significance of difference was analyzed by Duncan's multiple range test at the 5% level (13). RESULTS Inhibitory effect of essential oils against mycelial growth and spore germination The inhibitory effect of OED and thyme essential oils (TEO) towards the mycelia of the test fungi is shown in Fig. 1. Oregano inhibited the growth of mycelia of A. ochraceus and A. niger at a concentration of 1.0 JlIIL and the diameters of colonies developed from mycelial discs treated with that dosage were significantly smaller than those of the control. omplete inhibition of mycelial growth of the fungi tested was achieved after exposing mycelia discs to OED at a concentration of 2.0 JlllL. Following exposure of mycelia discs to TEO, a significant reduction in colony diameter (after 8 days growth, as compared with the control) was recorded at 2.0 JlIIL (for A. jlavus) or 3.0 JlllL (for A. ochraceus and A. niger). However, complete inhibition of growth of the test fungi e e..., r- QJ Q:i e ~ :; 0.5 OREGANO ~ (Ul/I) Figure I. Radial growth of mycelia following exposure to OEO and TEO. Mycelia discs were fumigated for 24 h, placed on PDA and incubated for 14 days at 26 e. Within each horizontal group of columns, values assigned the same letter do not differ significantly, P<O.05. was not recorded even following exposure to 4.0 JlllL. When the dosages of OED or TEO applied against spores were increased up to 2.5 and 3.0 JlIIL, respectively (Fig. 2), a subsequent decrease in germination of the three test fungi was recorded. Spores failed completely to germinate after exposure to TEO at 3.0 JlllL or to OED at 2.0 JlllL (for A. ochraceus) and 2.5 JlllL (for A. jlavus and A. niger). Effect of OEO and TEO and of the constituents carvacrol and thymol on natural microjlora of wheat grain Wheat grains treated with OED at 10, 15 or JlllL (24-h exposure) did not show any fungal infestation following 2 days of incubation on PDA (Fig. 3). At that time, the rate of infestation in grains treated with TEO, carvacrol or thymol (at the three dosages indicated) was significantly lower than that of the control (untreated grain). Following an additional 3 days of incubation, the level of infestation in grains treated with OED (at JlllL) was significantly lower than that of the control, while in grains treated with TEO, thymol or carvacrol the level of infested grain did not differ significantly from that of the control. In another set of experiments, the exposure time of grain to OED (applied at different concentrations) was increased up to 48 or 72 h. The results presented in Fig. 4 indicate that the levels of infested grain were almost the same for the three exposure periods following 2 days of incubation on PDA. 4

3 THYME (I) --- Oregono --0- Thyme --0- Thymol an-ero. A. niger o c: o 0 ;: III c: E a- D Ol OREGANO D ~... &. '-;ffl'~'>1~,.". --.~-.-'- III i'!,---~.-, '.. 1"..-'1 I~' > i! i i! I i 40 Ii, II ; I i I '.. o,...j.~ I ;''''p'.~ concentration!: ochraceus \! I f! 1/s'. ' 0.5 1" :T,:'" ' 1.5. '--V ' o ':~, /) <~~'!": (Ill/I) Figure 2. Spore germination (% of control) following a 24-h exposure to OED and TEO. Spore suspensions were fumigated and then spread on PDA. Data represent counts made following 7 days of incubation at 26. Within each horizontal group of columns, values assigned the same letter do not differ significantly, P<O.05. Following an additional 3 days of incubation, differences in infestation levels were recorded only between grain exposed to 10 III for 24 h and those exposed to the same OEO dosage but for 48 h. None of the other treatments, at each of the dosages applied, differed significantly. Effect of OEO, TEO, carvacrol and thymol on wheat germination Wheat germination was significantly reduced following exposure to OEO applied at concentrations of 5 Ill/L and above (exposure time 24 h) or to carvacrol and thymol applied at a concentration of Ill/L. Extending the exposure time of wheat to OEO (up to 48 or 72 h) (Fig. 5) also resulted in a significant reduction in germination, with the values recorded for the different dosages applied at those exposure periods being similar to those achieved following the 24 h exposure. ~ 40., c: '; "a ] :ro 0 10 IS concentration (111/1) Figure 3. Percent of infested wheat grain following a 24-h exposure to OED and TEO and to two of their constituents, carvacrol and thymol. The grains were surface sterilized before use. Infestation was recorded following 2 (I) and 5 (II) days of incubation. on PDA at 26. Values assigned the same letter do not differ significantly, P<O.05. Fungicidal activity of OEO in relation to grain moisture content Figure 6 shows the levels of infested wheat grains recorded after exposing grains with different Ms to OEO. The data indicate that a significant reduction in the rate of infested grain was recorded in grains with 15% M and above, after 2 days of incubation on PDA. However, following an additional 3 days of incubation, only grains with % M exhibited a significantly lower rate of infestation. DISUSSION The ability of oregano and thyme essential oils and of two of their major constituents to inhibit grain fungi was evaluated. The materials were applied as fumigants, a form of application, which is commonly utilized with grain protectants (e.g., methyl bromide and phosphine) and is the preferred form to be implemented in silos. Therefore, acquiring knowledge on the fumigant toxicity of natural materials toward grain fungi could pave the way for their implementation. Most studies concerning the antimicrobial activity of natural products, including oregano and thyme, have been conducted by testing the plant extracts as contact materials mostly by supplementing growth media or by inoculation IS

4 H4 PASTER, MENASHEROV, RA VID AND JUVEN --- Orepno (I) - A --0- Thyme -b- ~ Thymol 90 -b an1iroi --'6-- 72br --40 so A- ~.. A- lii ~ A- t., ajo Q 11 0 A- ; III J '5.!.. ~IOO (II) 70 -b- 24IIr -b- --fr- 7ZIlr 1IO concentration (111/1) 15 o concentration (111/1) Figure 4. Percent of infested wheat grain following exposure for 24, 48 or 72 h to OEO. Infestation was recorded following 2 (I) and 5 (II) days of incubation on PDA at 26. Values assigned the same letter do not differ significantly, P<0.05. Figure 5. (A) The effect of OEO and TEO and of carvacrol and thymol, on wheat grain germination. Grains were exposed for 24 h. Values assigned the same letter do not differ significantly, P<0.05. () The effect of OEO, applied for 24, 48 or 72 h, on wheat grain germination. Values assigned the same letter do not differ significantly, P<0.05. been conducted to compare the activity of plant extracts and their constituents, with different results presented in the literature according to the materials tested. It should be noted, however, that several factors e.g., the extraction technique, oxidative deterioration and the assay method, could partially explain the conflicting results related to antimicrobial activity of natural products and their constituof the plant itself (12). However, these methods do not reflect the actual activity of the volatile fraction alone (14). In this respect, therefore, the present work, aimed at studying the fumigant toxicity of the materials against grain fungi, seems to be relatively novel. The results of the in vitro experiments showed clearly that both OEO and TEO have fungicidal activity, with OEO being superior in controlling mycelia and spores of the three test organisms. This was reflected by the lower dosages of OEO needed to control these fungal structures as compared with those of TEO. These data corroborate earlier reports on OEO's fungicidal effect when applied in the growth medium (1,4,15). The results of the in vitro studies encouraged us to conduct experiments with grain. In such studies the penetration ability of the tested materials into the grain to control internal microflora is of utmost importance and, therefore, studies were conducted with surface-sterilized grain. The findings showed that OEO was highly effective in controlling the internal wheat fungi, while TEO and the constituents carvacrol and thymol were less effective. This might indicate that the inhibitory effect of both OEO and TEO is largely accounted for by these two main components, as was indicated earlier for carvacrol (21) and thymol (7,11). Moreover, the in vivo results of OEO being superior to TEO confirm the in vitro results. In reference to the lesser activity of the constituents, several studies have 110 ~~ 'n c.~.... ~ 40 ~.: II grain molslure conlenl (%) 2 day. IIiI 5 day. Figure 6. Percent of infestation of wheat grain at different moisture contents, following a 24-h exposure to OEO ( IlVL). Infestation was recorded following 2 or 5 days of incubation on PDA at 26. For each period of infestation recording, values assigned the same letter do not differ significantly, P<0.05.

5 ents. att et al. (5) tested the effect of a volatile fraction of carrot seed oil and its components on the growth of Aspergillus parasiticus and found that the growth inhibition caused by limonene or terpinene (two major constituents) was more pronounced than that caused by the whole volatile fraction. On the other hand, Alderman and Marth (2) found that orange and lemon oils were more inhibitory to mold growth and aflatoxin production than was d-limonene, the main constituent of the two peel oils. ullerman et al. (8), working with cinnamon and clove oils and their two major constituents, cinnamic aldehyde and eugenol, found these materials to be inhibitors of mold growth. The authors state that cinnamic aldehyde and eugenol apparently are the active compounds responsible for the inhibitory activity of the whole extract but raise the possibility that interactive effects of the other compounds present in smaller quantities with the major constituents may also be a possibility. In our studies, carvacrol and thymol were less effective in inhibiting fungal growth than were the whole-plant extracts, although thymol is a major constituent of the plants tested (OED contained 54% thymol and 5% carvacrol, while thyme contained 41 % thymol and 4% carvacrol). It, therefore, seems likely that the activity of the extracts results mainly from a synergistic or cumulative effect existing between the plant components, as was noted for other spices (8), but further studies are needed to elucidate the mode of action of the essential oils in order to identify such a possible synergism. Since the M of the grains used in the early studies was relatively high (18%), as a consequence of their being moistened during the surface sterilization, it was of interest to study whether the activity of OED, which was chosen as the test material, could be correlated with the grain M. A series of trials in which grains at different Ms were used, showed that the most active inhibition occurred at the high M levels. It might be assumed that penetration of the oils into the internal parts of the grain is improved in the presence of water (thymol volatilizes in water vapors), and therefore pathogens could be more easily controlled in the inner parts of the moist grains. This argument is strongly supported by the better fungitoxic activity achieved when using surface-sterilized (Fig. 3) versus non-sterilized grains (Fig. 6). All the materials tested caused, to different extents, a reduction in wheat germination. The mode of action in preventing germination is not known, but it is clear that the materials cannot be used for seed preservation but only to preserve grains destined for human or animal consumption. The efficacy of OED and to a lesser degree of TEO, applied as fumigants against grain microflora, was evident in our study. These materials as well as other natural products should therefore be considered as alternatives for grain and food preservation. REFERENES I. Akgul, A. and M. Kivanc Inhibitory effect of selected Turkish spices and oregano components on some foodborne fungi. Int. J. Food Microbiol. 6: Alderman, G. G. and E. H. Marth Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils. Z. Lebensm. Unters. Forsch. 1: Anonymous International rules for seed testing. Proceedings of the International Seed Testing Association, Wageningen, the Netherlands. 31, No. I. 4. Azzouz, M. A. and L.. ullerman omparative antimycotic effects of selected herbs, spices, plant components and commercial antifungal agents. J. Food Prot. 45:1298-\ att,., M. Solberg and M. eponis Effect of volatile components of carrot seed oil on growth and aflatoxin production by Aspergillus parasiticus. J. Food Sci. 48: euchat, L. R. and D. A. Golden Antimicrobials occurring naturally in foods. Food Technol. 43: \ uchanan, R. L. and A. J. Shepherd Inhibition of Aspergillus parasiticus by thymol. J. Food Sci. 46: ullerman, L.., F. Y. Lieu and S. A. Seier Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde and eugenol. J. Food Sci. 42: ullerman, L.., L. L. Schroeder and K. Park Formation and control of mycotoxins in food. J. Food Prot. 47: hristensen,. M. and D.. Sauer Microflora. pp In. M. hristensen (ed.). Storage of cereal grains and their products. American Association of ereal hemists, Inc., St. Paul, MN. ] I. Hitokoto, H., S. Morozumi, T. Wauke, S. Sakai and H. Kurata. ]9. Inhibitory effects of spices on growth and toxin production of toxigenic fungi. Appl. Environ. Microbiol. 39:8] ]2. Llewellyn, G.., M. L. urkett and T. Eadie. ]981. Potential mold growth, aflatoxin production and antimycotic activity of selected natura] spices and herbs. J. Assoc. Off. Anal. hern. 64: ]3. Miller, R. G. ]966. Simultaneous statistical inference. McGraw - Hill, New York, NY. ]4. Mo]eyer, V. and P. Narashimam. ]986. Antifunga] activity of some essential oil components. Food Microbiol. 3:33] ]5. Paster, N.,. J. Juven, E. Shaaya, M. Menasherov, R. Nitzan, H. Weisse]owicz and U. Ravid. ]990. Inhibitory effect of oregano and thyme essential oils on molds and foodborne bacteria. Lett. Appl. Microbiol. ]] : ]6. Pixton, S. W. ]967. Moisture content - its significance and measurement in stored products. J. Stored Prod. Res. 3: Pixton, S. W. and S. Warburton Moisture content relative humidity equilibrium, at different temperatures of some oil seeds of economic importance. J. Stored Prod. Res. 7: Ravid, U. and E. Putievsky Essential oils of Israeli wild species of Labiatae. pp In. Svendsen and J. J.. Scheffer (eds.). Essential oils and aromatic plants. Martinus Nijhoff, Dordrecht, the Netherlands. 19. Rusal, G. and E. H. Marth Food additives and plant components control growth and aflatoxin production by toxigenic Aspergilli: A review. Mycopathologia 101: Shelef, L. A Antimicrobial effects of spices. J. Food Safety 6: Thompson, D. P Fungitoxic activity of essential oil components on food storage fungi. Mycologia 81:

6 81 Journal of Food Protection, Yolo 58. No. I, Pages International Association of Milk, Food and Environmental Sanitarians Antifungal Activity of Oregano and Thyme Essential Oils Applied as Fumigants Against Fungi Attacking Stored Grain NAHMAN PASTER, MAZAL MENASHEROV, UZI RAVID 1 and ENJAMIN JUVE~ Depanmenl of Stored Products, Agricultural Research Organization (ARO), The Yolcani enter, P.O. ox 6, et Dagan 50250, Israel; 'Depanment of Medicinal and Spice rops, ARO, Newe Ya'ar Experimental Station, P.O. Haifa 30, Israel; and 2Department of Food Science, ARO, The Yolcani enter, P.O. ox 6, et Dagan 50250, Israel (Received November 2, 1993/Accepted April II, 1994) ASTRAT Essential oils from oregano and thyme were applied for 24 h as fumigants against the mycelia and spores of Aspergillus jlavus, Aspergillus niger and Aspergillus ochraceus, as well as against natural microflora of wheat grains. The minimal inhibitory concentration (MI) of oregano oil needed to inhibit the mycelial growth of the fungi was 2.0 IlIIL, while spores were eradicated following exposure to 2.0 to 2.5 IlIIL. The thyme essential oil was less efficient in controlling mycelia and growth was observed even following exposure to 4.0 ~l/l. However, the thyme essential oil was fungitoxic to spores (MI= 3.0 IlIIL). In another set of trials the efficacy of the oils and two of their constituents (carvacrol and thymol) in controlling natural microflora of surface-sterilized wheat grain was studied. Of the four materials investigated, only oregano essential oil exhibited fungicidal activity and, following 24 h exposure to jliil, a significant reduction in the percent of infested grain was observed even after 5 days of incubation on potato dextrose agar. A reduction in the germinability of the grains was evident following exposure to the materials tested. When the fungicidal activity of oregano essential oil was evaluated using grains with different moisture contents (M), data revealed that the better inhibitory effect was achieved in grain with a high M. The findings emphasize the toxicity of oregano and thyme essential oils as fumigants against fungi attacking stored grain and strengthen the possibility of using them as an alternative to chemicals for preserving stored grains. Key Words: Essential oils, oregano, thyme, fungicidal activity, fungi, grain Fungi grown on stored grain can cause heavy damage, resulting in reductions in quantity and in quality. In addition, many species are capable of producing mycotoxins, secondary metabolites highly toxic to animals and humans. Fungi-inhibiting chemicals (mainly low-molecular-weight organic acids) have been used for the preservation of stored grain. However, many disadvantages are associated with the use of acids (10) and there is a worldwide trend towards limiting their use in grain and foodstuffs, Natural plant extracts may provide an alternative to these preservatives. Over the years, much effort has been devoted to the search for new antifungal materials from natural sources for food and grain preservation, and many antimicrobials occurring naturally in plants have been identified (6,). Some of them have been found also to inhibit mycotoxin formation (9,19). However, in most of the studies where the antimicrobial activity of essential oils has been studied, the assay techniques used involved application of the materials to the growth medium. While dealing with grain protection, fumigation is the preferred method for applying substances into the bulks in order to control the biotic factors which damage the grain. The objective of the present study was to evaluate the inhibitory effect of two essential oils, applied as fumigants, on the growth of three storage fungi in vitro. The ability of the materials to prevent the growth of fungi naturally infesting wheat grain was also studied. MATERIALS AND METHODS Fungal strains and growth conditions The fungi used in these studies were A. jlavus Link:Fr., A. ochraceus Wilhelm and A. niger v. Tieghem. They were grown on potato dextrose agar (PDA) at 26 and agar discs (0.3 cm in diameter) covered with mycelium served as a source of inoculum. The discs were taken from the margin of 4-day-old colonies and spores were washed off from each disc using sterilized water. For spore germination assays, spore suspensions of each fungus (10 3 spores/ml counted with a hemocytometer) were prepared in sterile water. Spices and the extraction procedure The spices tested were oregano (Origanum vulgare) and thyme (oridothymus capitatus) grown for commercial use in the northern part of Israel. The essential oils were extracted as described previously (18). Fresh plant material was steam-distilled for 2.5 h in a l30-l direct steam pilot plant apparatus, The recovered oils were dried over anhydrous sodium sulfate and stored in dark at 4 to 6 (to avoid rapid oxidation). Oil constituents, carvacrol and thymol were purchased from Aldrich hemical ompany, Inc., in the United States, and arl Roth, Karlsruhe, Germany, respectively, Assay technique Glass flasks (3.4 L each) with a round bottom, closed with a glass stopper fitted with a hook, served as fumigation chambers. The test materials were applied on a small square of filter paper,

7 82 PASTER, MENASHEROV, RA VID AND JUVEN which was suspended by the hook, together with small wire-mesh cages in which mycelial discs were placed. In studies where the effect on spore survival was assayed, spore suspensions (0 IIIof each fungus) were exposed in small test tubes (1.0 cm diam X 2.0 cm height) placed in plastic holders, which were suspended by the hook. In order to ensure even distribution of the oil, a magnetic stirrer was placed on the bottom of each flask, and activated gently throughout the exposure period (24 h). Studies were carried out in a room with a constant temperature of 26 ± 1. Following the treatment, mycelium discs were transferred to petri dishes (each disc was placed on a separate plate) containing PDA and the linear growth was measured daily during incubation at 26 for 14 days. For spore germination assays, three portions (2 X 50 III + 1 X Ill) were taken from the exposed suspensions and spread over the surface of PDA in petri dishes. Germinating spores were counted daily during incubation at 26 for 14 days. o i e '-' OREGANO Trials with wheat grain Studies with grain included trials in which surface-sterilized grain was used and trials with non-sterilized grain at different levels of M. Surface sterilization. Lots of grain ( g each) was dipped for 2 min in a solution of 2% commercial sodium hypochlorite and subsequently rinsed (2 X 2 min) in sterile water. The grain was then placed to dry for 1 h over a sterile filter paper in a sterile cabinet before being used in the studies. The M of the sterilized grain was 18%, as determined using the air-oven method (3). Adjusting grains to different levels of Me. Different levels of M were achieved with the addition of sterile water according to the procedure described by Pixton (16). The grain was then mixed thoroughly and allowed to equilibrate in a closed bin at 5 for 14 days (17). Percentage of wheat germination was determined using the ISTA method (3). Exposing grains to the test materials. Lots of grains (50 g each) were introduced into wire-mesh baskets which were suspended from the flask stopper by the hook. The grains were fumigated (for 24 h, or 24 to 72 h in the case of oregano [OEO]) in the manner described above and following each treatment, grains were placed on PDA in petri dishes (10 grains/plate). Fungal infestation, defined as the percent of fungi-yielding grains, was estimated daily during 7 days of incubation at 26. Statistical analysis Each experiment was repeated at least three times. Significance of difference was analyzed by Duncan's multiple range test at the 5% level (13). RESULTS Inhibitory effect of essential oils against mycelial growth and spore germination The inhibitory effect of OEO and thyme essential oils (TEO) towards the mycelia of the test fungi is shown in Fig. 1. Oregano inhibited the growth of mycelia of A. ochraceus and A. niger at a concentration of 1.0 f.1l/l and the diameters of colonies developed from mycelial discs treated with that dosage were significantly smaller than those of the control. omplete inhibition of mycelial growth of the fungi tested was achieved after exposing mycelia discs to OEO at a concentration of 2.0 f.1l/l. Following exposure of mycelia discs to TEO, a significant reduction in colony diameter (after 8 days growth, as compared with the control) was recorded at 2.0 f.1l/l (for A. flavus) or 3.0 f.1l/l (for A. ochraceus and A. niger). However, complete inhibition of growth of the test fungi (Ul/l) Figure 1. Radial growth of mycelia following exposure to OEO and TEO. Mycelia discs were fumigated for 24 h, placed on PDA and incubated for 14 days at 26. Within each horizontal group of columns, values assigned the same letter do not differ significantly, P<O.05. was not recorded even following exposure to 4.0 f.1ljl. When the dosages of OEO or TEO applied against spores were increased up to 2.5 and 3.0 f.1l/l,respectively (Fig. 2), a subsequent decrease in germination of the three test fungi was recorded. Spores failed completely to germinate after exposure to TEO at 3.0 f.1l/l or to OEO at 2.0 f.1l/l (for A. ochraceus) and 2.5 f.1l/l (for A. flavus and A. niger). Effect of OEO and TEO and of the constituents carvacrol and thymol on natural micro flora of wheat grain Wheat grains treated with OEO at 10, 15 or f.1l/l (24-h exposure) did not show any fungal infestation follow~ ing 2 days of incubation on PDA (Fig. 3). At that time, the rate of infestation in grains treated with TEO, carvacrol or thymol (at the three dosages indicated) was significantly lower than that of the control (untreated grain). Following an additional 3 days of incubation, the level of infestation in grains treated with OEO (at f.1l/l) was significantly lower than that of the control, while in grains treated with TEO, thymol or carvacrol the level of infested grain did not differ significantly from that of the control. In another set of experiments, the exposure time of grain to OEO (applied at different concentrations) was increased up to 48 or 72 h. The results presented in Fig. 4 indicate that the levels of infested grain were almost the same for the three exposure periods following 2 days of incubation on PDA. 4

8 OREGANO AND THYME ESSENTIAL OILS AGAINST GRAIN FUNGI 83 THYME -- Oregano --D-- TbYIIIf Thymol -.-- arvacrol A, niger -~oc o :;:: III E.. 4» 01 o OREGANO i!: ochraceus 3 ~ 40 I: '; i ] 5.5 (11) ~ ~... ~ "~ffl:.... i '. ---::?-~~.- i,i 1.' \~,! 'i i 11:!! I 1 40 Ai I!! i, --..._--- J I i I j.'; i" : I I I.,< _ o...,.j.,~ 'II il-v~--'~'._~ ~) /~;>~~. o "" I.-8.. o 5 ',. :, -'r :: 8'. 1.. :. I.,;'., :~ concentration (J.IIII) Figure 2. Spore germination (% of control) following a 24-h exposure to OED and TEO. Spore suspensions were fumigated and then spread on PDA. Data represent counts made following 7 days of incubation at 26. Within each horizontal group of columns, values assigned the same letter do not differ significantly, P<O.05. Following an additional 3 days of incubation, differences in infestation levels were recorded only between grain exposed to to ~l for 24 h and those exposed to the same OEO dosage but for 48 h. None of the other treatments, at each of the dosages applied, differed significantly. Effect of OEO, TEO, carvacrol and thymol on wheat germination Wheat germination was significantly reduced following exposure to OEO applied at concentrations of 5 ~l/l and above (exposure time 24 h) or to carvacrol and thymol applied at a concentration of ~. Extending the exposure time of wheat to OEO (up to 48 or 72 h) (Fig. 5) also resulted in a significant reduction in germination, with the values recorded for the different dosages applied at those exposure periods being similar to those achieved following the 24 h exposure concentration (III/I) Figure 3. Percent of infested wheat grain following a 24-h exposure to OED and TEO and to two of their constituents, carvacrol and thymol. The grains were surface sterilized before use. Infestation was recorded following 2 (1) and 5 (II) days of incubation. on PDA at 26. Values assigned the same letter do not differ significantly, P<O.05. Fungicidal activity of OEO in relation to grain moisture content Figure 6 shows the levels of infested wheat grains recorded after exposing grains with different Ms to OEO. The data indicate that a significant reduction in the rate of infested grain was recorded in grains with 15% M and above, after 2 days of incubation on PDA. However, following an additional 3 days of incubation, only grains with % M exhibited a significantly lower rate of infestation. DISUSSION The ability of oregano and thyme essential oils and of two of their major constituents to inhibit grain fungi was evaluated. The materials were applied as fumigants, a form of application, which is commonly utilized with grain protectants (e.g., methyl bromide and phosphine) and is the preferred form to be implemented in silos. Therefore, acquiring knowledge on the fumigant toxicity of natural materials toward grain fungi could pave the way for their implementation. Most studies concerning the antimicrobial activity of natural products, including oregano and thyme, have been conducted by testing the plant extracts as contact materials mostly by supplementing growth media or by inoculation

9 84 PASTER, MENASHEROY, RA YID AND JUVEN...- Onpno (I) - A --0- TIl, TIIymoI!lO =ni 10 --'6-72br.., 70 ~40 50 A I. A...40! A Iii 1:130 0 ;::z 0 A III. 1:1 J ZO ZO.~.E -6- & (II) /It--,.. 10 A 40 o 5 10 concentration (11111) 15 zo A o ZO concentration (11111) Figure 4. Percent of infested wheat grain following exposure for 24, 48 or 72 h to OEO. Infestation was recorded following 2 (I) and 5 (ll) days of incubation on PDA at 26. Values assigned the same letter do not differ significantly, P<0.05. Figure 5. (A) The effect of OEO and TEO and of carvacrol and thymol, on wheat grain germination. Grains were exposed for 24 h. Values assigned the same letter do not differ significantly, P<0.05. () The effect of OEO, applied for 24, 48 or 72 h, on wheat grain germination. Values assigned the same letter do not differ significantly, P<0.05. of the plant itself (12). However, these methods do not reflect the actual activity of the volatile fraction alone (14). In this respect, therefore, the present work, aimed at studying the fumigant toxicity of the materials against grain fungi, seems to be relatively novel. The results of the in vitro experiments showed clearly that both OEO and TEO have fungicidal activity, with OEO being superior in controlling mycelia and spores of the three test organisms. This was reflected by the lower dosages of OEOneeded to control these fungal structures as compared with those of TEO. These data corroborate earlier reports on OEO's fungicidal effect when applied in the growth medium (1,4,15). The results of the in vitro studies encouraged us to conduct experiments with grain. In such studies the penetration ability of the tested materials into the grain to control internal microflora is of utmost importance and, therefore, studies were conducted with surface-sterilized grain. The findings showed that OEO was highly effective in controlling the internal wheat fungi, while TEO and the constituents carvacrol and thymol were less effective. This might indicate that the inhibitory effect of both OEO and TEO is largely accounted for by these two main components, as was indicated earlier for carvacrol (21) and thymol (7,11). Moreover, the in vivo results of OEO being superior to TEO confirm the in vitro results. In reference to the lesser activity of the constituents, several studies have lill tl'." c.;..... ~ 40.!!.: grain moisture (ontent (%) 2 day. ll 5 day. Figure 6. Percent of infestation of wheat grain at different moisture contents, following a 24-h exposure to OEO ( JLVL). Infestation was recorded following 2 or 5 days of incubation on PDA at 26. For each period of infestation recording, values assigned the same letter do not differ significantly, P<0.05. been conducted to compare the activity of plant extracts and their constituents, with different results presented in the literature according to the materials tested. It should be noted, however, that several factors e.g., the extraction technique, oxidative deterioration and the assay method, could partially explain the conflicting results related to antimicrobial activity of natural products and their constitu- JOURNAL OF FOOD PROTET/ON, YOLo 58, JANUARY 1995

10 OREGANO AND THYME ESSENTIAL OILS AGAINST GRAIN FUNGI 85 ents. att et al. (5) tested the effect of a volatile fraction of carrot seed oil and its components on the growth of Aspergillus parasiticus and found that the growth inhibition caused by limonene or terpinene (two major constituents) was more pronounced than that caused by the whole volatile fraction. On the other hand, Alderman and Marth (2) found that orange and lemon oils were more inhibitory to mold growth and aflatoxin production than was d-limonene, the main constituent of the two peel oils. ullerman et al. (8), working with cinnamon and clove oils and their two major constituents, cinnamic aldehyde and eugenol, found these materials to be inhibitors of mold growth. The authors state that cinnamic aldehyde and eugenol apparently are the active compounds responsible for the inhibitory activity of the whole extract but raise the possibility that interactive effects of the other compounds present in smaller quantities with the major constituents may also be a possibility. In our studies, carvacrol and thymol were less effective in inhibiting fungal growth than were the whole-plant extracts, although thymol is a major constituent of the plants tested (OEO contained 54% thymol and 5% carvacrol, while thyme contained 41 % thymol and 4% carvacrol). It, therefore, seems likely that the activity of the extracts results mainly from a synergistic or cumulative effect existing between the plant components, as was noted for other spices (8), but further studies are needed to elucidate the mode of action of the essential oils in order to identify such a possible synergism. Since the M of the grains used in the early studies was relatively high (18%), as a consequence of their being moistened during the surface sterilization, it was of interest to study whether the activity of OEO, which was chosen as the test material, could be correlated with the grain Me. A series of trials in which grains at different Ms were used, showed that the most active inhibition occurred at the high M levels. It might be assumed that penetration of the oils into the internal parts of the grain is improved in the presence of water (thymol volatilizes in water vapors), and therefore pathogens could be more easily controlled in the inner parts of the moist grains. This argument is strongly supported by the better fungitoxic activity achieved when using surface-sterilized (Fig. 3) versus non-sterilized grains (Fig. 6). All the materials tested caused, to different extents, a reduction in wheat germination. The mode of action in preventing germination is not known, but it is clear that the materials cannot be used for seed preservation but only to preserve grains destined for human or animal consumption. The efficacy of OEO and to a lesser degree of TEO, applied as fumigants against grain microflora, was evident in our study. These materials as well as other natural products should therefore be considered as alternatives for grain and food preservation. REFERENES I. Akgul, A. and M. Kivanc Inhibitory effect of selected Turkish spices and oregano components on some foodbome fungi. Int. J. Food Microbiol. 6: Alderman, G. G. and E. H. Marth Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils. Z. Lebensm. Unters. Forsch. 1: Anonymous International rules for seed testing. Proceedings of the International Seed Testing Association, Wageningen, the Netherlands. 31, No. I. 4. Azzouz, M. A. and L.. ulierman omparative antimycotic effects of selected herbs, spices, plant components and commercial antifungal agents. J. Food Prot. 45: I. 5. att,., M. Solberg and M. eponis Effect of volatile components of carrot seed oil on growth and aflatoxin production by Aspergillus parasiticus. J. Food Sci. 48: euchat, L. R. and D. A. Golden Antimicrobials occurring naturally in foods. Food Technol. 43: uchanan, R. L. and A. J. Shepherd. 198I. Inhibition of Aspergillus parasiticus by thymol. J. Food Sci. 46: ulierman, L.., F. Y. Lieu and S. A. Seier Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde and eugenol. 1. Food Sci. 42: ulierman, L.., L. L. Schroeder and K. Park Formation and control of mycotoxins in food. J. Food Prot. 47: hristensen,. M. and D.. Sauer Microflora. pp In. M. hristensen (ed.). Storage of cereal grains and their products. American Association of ereal hemists, Inc., St. Paul, MN. 11. Hitokoto, H., S. Morozumi, T. Wauke, S. Sakai and H. Kurata. 19. Inhibitory effects of spices on growth and toxin production of toxigenic fungi. Appl. Environ. Microbiol. 39: Llewellyn, G.., M. L. urkett and T. Eadie Potential mold growth, aflatoxin production and antimycotic activity of selected natural spices and herbs. 1. Assoc. Off. Anal. hern. 64: Miller, R. G Simultaneous statistical inference. McGraw - Hill, New York, NY. 14. Moleyer, V. and P. Narashimam Antifungal activity of some essential oil components. Food Microbiol. 3: Paster, N.,. J. Juven, E. Shaaya, M. Menasherov, R. Nitzan, H. Weisselowicz and U. Ravid Inhibitory effect of oregano and thyme essential oils on molds and foodbome bacteria. Lett. Appl. Microbiol. 11: Pixton, S. W Moisturecontent - its significance and measurement in stored products. J. Stored Prod. Res. 3: Pixton, S. W. and S. Warburton Moisture content relative humidity equilibrium, at different temperatures of some oil seeds of economic importance. J. Stored Prod. Res. 7: Ravid, U. and E. Putievsky Essential oils of Israeli wild species of Labiatae. pp I. In. Svendsen and J. J.. Scheffer (eds.). Essential oils and aromatic plants. Martinus Nijhoff, Dordrecht, the Netherlands. 19. Rusal, G. and E. H. Marth Food additives and plant components control growth and aflatoxin production by toxigenic Aspergilli: A review. Mycopathologia 101: Shelef, L. A Antimicrobial effects of spices. J. Food Safety 6: I. Thompson, D. P Fungitoxic activity of essential oil components on food storage fungi. Mycologia 81: