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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. References

Inhibitory effects of essential oils of oregano (Origanum vulgare), mint (Menta arvensis), basil (Ocimum basilicum), sage (Salvia officinalis) and coriander (Coriandrum sativum), on the mycelial growth and ochratoxin A production by Aspergillus ochraceus NRRL 3174 were studied. Cultures were incubated on yeast extract-sucrose (YES) broth, at concentrations of 0, 500, 750 and 1000 p.p.m. of essentials oils during 7, 14 and 21 d at 25 °C. At 1000 p.p.m., oregano and mint completely inhibited the fungal growth and ochratoxin A production up to 21 d, while basil was only effective up to 7 d. At 750 p.p.m., oregano was completely effective up to 14 d, whereas mint allowed fungal growth but no ochratoxin A production up to 14 d. At 500 p.p.m., no evident inhibition could be observed with any of the essential oils under analysis. Sage and coriander showed no important effect at any of the concentrations studied. These inhibitory effects are interesting in connection with the prevention of mycotoxin contamination in many foods and they could be used instead of synthetic antifungal products.

Ochratoxins are produced by a variety of fungal species included in the genera Aspergillus and Penicillium ( Pitt & Hocking 1997). While members of Aspergillus Section Circundati (A. ochraceus group) are the main producers in the aspergilli, their production has also been reported by species of other sections ( Abarca et al. 1997 ). At present, it is accepted that Penicillium verrucosum is the main producer of ochratoxin A (OA) ( Pitt & Hocking 1997).

The highest incidence of OA contamination has been found in foodstuffs and animal feeds of cereal origins ( Basílico 1995; Höhler 1998). OA has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunosuppressive ( Höhler 1998).

Storage fungi are commonly controlled using synthetic preservatives, but most of these substances produce several side-effects as carcinogenicity, teratogenicity and residual toxicity. Thus, in the last few years, research has focused on the use of natural preservatives ( Foegeding & Busta 1991). The antimicrobial properties of essential oils and of their constituents have been the subject of many investigations during the past decades and many authors have mentioned their antifungal and antitoxigenic action ( Maruzzella & Henry 1958; Bullerman 1974).

Although many of the spices and herbal essential oils which have been tested have an antagonistic effect against aflatoxigenic Aspergillus strains ( Bullerman et al. 1977 ; Batt et al. 1980 ; Buchanan & Shepherd 1981; Mahmoud 1994), few studies have been performed on OA production ( Hitokoto et al. 1977 ; Hitokoto et al. 1980 ).

The object of this study is to determine the inhibitory effects of five spice essential oils (SEO) on the growth and OA production by A. ochraceus NRRL 3174.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. References

Microorganism

Aspergillus ochraceus NRLL 3174 was obtained from the Northern Regional Research Laboratory (USDA, Peoria, IL). This strain was checked for purity and identity ( Pitt & Hocking 1997). The lyophilized mould was cultured on slants of agar-malt medium (MEA) and maintained at 2–4 °C on MEA ( Pitt & Hocking 1997).

Preparation of spore suspension

A. ochraceus was grown on MEA slants at 25 °C until well sporulated (7–10 d). Spores were harvested by adding sterilized Tween 80 solution (0,01%, v/v) and they were filtered through several layers of sterilized cheese cloth. The spore concentration was determined by plate count on MEA. Peptone-water (0,1%, wt/vol) was used as the diluent. Plates were incubated at 25 °C for 3 d. The suspension was adjusted with peptone-water (0,1%, wt/vol) to contain ≈106 viable spores per ml.

Spice essential oils

Spice essential oils (SEO) were kindly provided by Dr H. Elder from ‘Programa Plantas Aromaticas y Medicinales’ (Gobierno Provincia Santa Fe, Argentina). SEO were extracted from the following plants: oregano (Origanum vulgare), mint (Menta arvensis), basil (Ocimum basilicum), sage (Salvia officinalis) and coriander (Coriandrum sativum). The extractions were carried out by the method described by Helder et al. (1998) ; basically, plant materials (100 g) were cut into small pieces and placed in a flask (2 l) with double distilled water (1,5 l). A steam distillation continuous extraction head was attached to the flask. After steam distillation (3 h), the oils were isolated and dried over anhydrous sodium sulphate.

Growth media and conditions

Yeast-extract-sucrose medium (YES) was used as a basal medium for growth and OA production in stationary cultures ( Abarca et al. 1997 ). Fractions of 50 ml of YES medium were added to 250 ml Erlenmeyer flasks, and they were autoclaved at 121 °C for 15 min and cooled to room temperature. SEO were aseptically added to each Erlenmeyer flask (VAISI LAB, Buenos Aires, Argentina) used to give concentrations of 0, 500, 750 and 1000 p.p.m. Thereafter, they were inoculated with 1 ml of 106 spore suspension. Cultures were incubated statically at 25 °C for 7, 14 and 21 d. Three flasks per essential oil, concentration and incubation time were analysed for toxin and mycelium production.

Determination of fungal growth

Mycelial mats were collected on dried, pre-weighed filter paper (n°300210, Schleicher & Schuell, Dassel, Germany) after OA extraction. They were then washed with distilled water and dried at 100 °C up to constant weight. The dry weight of mycelial mats was used as the measurement of fungal growth.

Analysis of OA

At the end of each incubation period OA was extracted with hot chloroform ( Hitokoto et al. 1977 ). Each extract was evaporated to dryness in a rotatory vacuum evaporator (Buchi, Switzerland), and then redissolved in benzene-acetic acid (99 : 1). Mycotoxin assays were carried out by thin-layer chromatography on silica gel 60 (n°1·05553, Merck, Germany) in the presence of OA standard (Sigma, USA). The developing solvent was toluene – ethyl acetate – formic acid (60 : 30 : 10). OA was quantified by CHR-Scan Densitometer (Engineering Co., Budapest).

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. References

The average OA production in controls of A. ochraceus NRLL 3174, was 35 ± 3; 40 ± 7 and 233 ± 11 μg/50 ml of YES broth, while dried mycelium production was 458 ± 15; 795 ± 20 and 854 ± 25 mg at 7, 14 and 21 d, respectively. These results were similar to others ( Munimbazi et al. 1997 ), although the authors could not observe OA production on the 7th d.

SEO obtained from oregano and mint completely inhibited fungal growth for more than 21 d at a concentration of 1000 p.p.m. At the same concentration, basil was completely effective initially (7 d) but permitted mould growth afterwards. Sage exerted a slight inhibition but its effectiveness decreased with time. Since no evident reduction in mycelial growth was observed at any coriander concentration, data are not shown. At 750 p.p.m., oregano was completely effective up to 14 d, but caused only partial inhibition of fungal growth on the 21st d. At this concentration, no complete inhibition was observed with mint. The results also indicated that at 500 p.p.m. no significant growth inhibition was observed. ( Fig. 1).

image

Figure 1. Inhibitory effects of essential oils, (O) oregano, (M) mint, (B) basil and (S) sage, □ at 500, ▒ at 750 and ▪ at 1000 ppm on fungal growth of A. ochraceus NRRL 3174 incubated on YES media, during 7, 14 and 21 d, respectively. Since coriander produced no inhibitory action at any concentration, data are not shown. Each bar represents the mean of 3 assays.

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Figure 2 shows the effects of SEO on the inhibition of OA production after incubation times of 7, 14 and 21 d at 750 and 1000 p.p.m. in YES media. No effects of inhibition of OA production were recorded at 500 p.p.m. (data not shown). Oregano at 750 p.p.m. – 21 d, where only partial inhibition of fungal growth was observed (34%), produced a small stimulating effect on OA production (278 ± 14 vs. 233 ± 11 μg/50 ml of YES broth).

image

Figure 2. Inhibitory effects of essential oils, (O) oregano, (M) mint, (B) basil and (S) sage, ▒ at 750 and ▪ at 1000 ppm on fungal growth of A. ochraceus NRRL 3174 incubated on YES media, during 7, 14 and 21 d, respectively. Since no or negligible inhibition action was observed at 500 ppm and coriander produced no inhibitory action at any concentration, data are not shown. Each bar represents the mean of 3 assays.

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The results indicate that only oregano, mint and basil produced inhibitory effects. However, if the preservative effect is only partial, or its effectiveness decreases with time, mould can grow with a possible stimulation on OA synthesis.

Each of the SEO under study is characterized by having a different major compound, i.e. thymol for oregano, menthol for mint and methyl-chavicol for basil, while thuyone and linalol are the main compounds of sage and coriander, respectively ( Guenther 1952).Farag et al. (1989) reported that the inhibitory effect of spice oils was mainly due to the most abundant component and not to the other associated substances. Thymol had been demonstrated to have a high microbiocide and anti-aflatoxigenic action due to the presence of a phenolic-OH group ( Buchanan & Shepherd 1981; Farag et al. 1989 ).

In summary, these inhibitory effects are interesting in connection with the prevention of mycotoxin contamination in many foods and they could be used instead of synthetic antifungal products.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. References
  • Abarca, M.L., Bragulat, M.R., Castellá, G et al. 1997 New ochratoxigenic species in the genus Aspergillus. Journal of Food Protection., 60, 1880 1582.
  • Basílico, J.C. 1995 Micotoxinas en alimentos: el riesgo sobre la mesa. Centro de Publicaciones, Universidad Nacional del Litoral, Santa Fe. Argentina.
  • Batt, C., Solverg, M., Ceponis, M. 1980 Inhibition of aflatoxin production by carrot root extract. Journal of Food Science, 45, 1210 1213.
  • Buchanan, R.L. & Shepherd, A.J. 1981 Inhibition of Aspergillus parasiticus by thymol. Journal of Food Science, 46, 976 977.
  • Bullerman, L.B. 1974 Inhibition of aflatoxin production by cinnamon. Journal of Food Science, 39, 1163 1165.
  • Bullerman, L.B., Lien, Y., Seier, S.A. 1977 Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde and eugenol. Journal of Food Science, 42, 1107 1109.
  • Farag, R.S., Daw, Z.Y., Abo-Raya, S.H. 1989 Influence of some spice essential oils on Aspergillus parasiticus growth and production of aflatoxins in a synthetic medium. Journal of Food Science, 54, 74 76.
  • Foegeding, P.M. & Busta, F.F. 1991 Chemical food preservatives. In: Disinfection, Sterilisation, and Preservation (ed. S.S.Block), p. 802 832. Lea & Febiger, Malvern, Pennsylvania.
  • Guenther, E. 1952 The essential oils, 3 and 4. D. van Nostrand Co. New York.
  • Helder, H., Spekuljak, V., Monella, H. 1998 Extractor de aceites esenciales de elevada eficiencia térmica. LACTYM. Argentina.
  • Hitokoto, H., Morozumi, S., Wauke, T et al. 1977 Mycotoxin production of fungi on commercial foods. In: Mycotoxins (ed. J.V.Rodricks), p. 479 487. Human and animal health. Pathotox Publishers, Inc., Chicago.
  • Hitokoto, H., Morozumi, S., Wauke, T et al. 1980 Inhibitory effects of spices on growth and toxin production of toxicogenic fungi. Applied and Environmental Microbiolology, 39, 818 822.
  • Höhler, D. 1998 Ochratoxin A in food and feed: occurrence, legislation and mode of action. Z Ernährungswiss, 37, 2 12.
  • Mahmoud, L.E. 1994 Antifungal action and antiaflatoxicogenic properties of some essential oils constituents. Letters in Applied Microbiology, 19, 110 113.
  • Maruzzella, J.C. & Henry, P.A. 1958 The in vitro antibacterial activity of essential oils and oil combination. Journal Am. Pharm. Assoc. Sci., eds. 47, 294 296.
  • Munimbazi, C., Saxena, J., Tsai, W.Y.J., Bullerman, L.B. 1997 Inhibition of production of cyclopiazonic acid and ochratoxin A by the fungicide Iprodione. Journal of Food Protection, 60, 849 852.
  • Pitt, J.I. & Hocking, A.D. 1997 Fungi and food spoilage. Blackie Academic and Professional, London.