Letters in Applied Microbiology 1992, 15, 20-22

Influence of age on ergosterol content in mycelium of Aspergillus ochraceus

M . TORRES, R . V I L A D R I C H * , V . S A N C H I S & R . CANELA* Department of Food Technology, Universitat Polittcnica de Catalunya, and *Area of Crop Protection, Centre R & D de Lleida (UPC-IRTA), Rovira Roure 177,2.5006-Lleida, Spain

MOMIO5.5: received 13 February 1992 and accepted 14 March 1992

TORRES, M . , V I L A D R I C H , R . , SANCHIS, V . & CANELA, R . 1992. Influence of age on ergosterol content in mycelium of Aspergillus ochraceus. Letters in Applied Microbiology 15, 20-22.

Ergosterol has been suggested as a parameter for studying the content and evolu- tion of fungal infestation in different products. Important differences in this param- eter have been described for different genera and species. A study of the relationship between ergosterol content and dry mass in a strain of Aspergillus ochraceus, which is able to produce an antifungal agent, has therefore been carried out in order to use this parameter to accurately evaluate the fungal mass of this strain. We report herein our research work to determine the ergosterol content of the mycelial mass of our mould and how it changes with the fungal age.

Many of the foods and feeds consumed by humans and farm animals, such as cereals, oil seeds and fruits, are susceptible to fungal con- tamination. The degree of fungal infestation varies with the kind of substrate and the environmental conditions. This kind of infesta- tion leads to deterioration of foods and feeds, and often to contamination of the substrate by mycotoxins. Many attempts have been made to evaluate fungal contamination on solid sub- strates. One of these makes use of the ergosterol content as an index of fungal contamination based on its presence in fungal membranes and its usual absence from vegetable and animal tissues. After Seitz et al. (1977) proposed this parameter as a measure of fungal contamination in grains, different authors have used ergosterol as a useful parameter for estimating fungal con- tamination in different materials (Seitz et al. 1983; Newell et al., 1987, and references therein; Jambunathan et al. 1991).

Although those results were very promising, different authors have also found that ergosterol content is influenced by such variables as fungal species, oxygen availability, temperature and growth substrate (Safe 1973; Weete 1981; Seitz

et al. 1979), and it is also influenced by the ana- lytical method (Newell et al. 1988).

Since we are working on the bioproduction of aspyrone, an antifungal metabolite produced by a strain of A. ochraceus, our interest has been focused on the study of the relationship between mould dry weight and ergosterol concentration in our strain. Moreover, although mould age does not seem to have a great influence on the variation of this parameter (Seitz et al. 1979; Evans & Gealt 1985; Elliot et al. 1974), a study of this relationship has also been made.

Materials and Methods

O R G A N I S M A N D C U L T U R E C O N D I T I O N S

Aspergillus ochraceus, strain 1/89/M, originally isolated from environmental samples, was incu- bated on potato dextrose agar (PDA) for 7 d at 28°C. One millilitre of spore suspension (2.106 spores/ml) was inoculated into 500 ml Erlen- meyer flasks containing 100 ml of an aqueous medium made up from potassium dihydrogen phosphate (0.1 %), magnesium sulfate heptahy- drate (@05%0), potassium chloride (0.05Y0), urea

Ergosterol content

I 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 4 8 12 16 20 24 26

Days of incubation

Fig. 1. Changes in ergosterol content with age. Each point is the mean of two replicates.

(0.07%) and dextrose (75%). The pH was adjusted to 5.5 before autoclaving. The flasks were incubated in static culture at 28°C.

Triplicate flasks were harvested every 2 d for 22 d and finally at 25 d. Mycelia were recovered by filtration, rinsed with distilled water, freeze-

I I I I

H 0 4 8 12 16 20 24 26

Days of incubation

Fig. 2. Mycelial dry mass versus age.

in A. ochraceus 21 dried and weighed. Weighed mycelia were stored at -21°C until ergosterol analysis.

E R G O S T E R O L A N A L Y S I S

The ergosterol assay was based on that described previously by Seitz et al. (1979), with some modifications. Half a gram of each dried mycelial sample was blended with 30 ml of methanol for 2 min. The blended mixture was centrifuged for 5 min at 800 g. The supernatant was poured off and the solid resuspended in 30ml of methanol, shaken, placed in an ultra- sound bath for about 30 s, and centrifuged again. The two supernatants were combined, mixed with 15 g KOH and 37.5 ml of ethanol, and refluxed for 60 min. The cooled saponified mixture was diluted with 30 ml of doubly dis- tilled water and extracted with 100 ml, then 50 ml, and finally 50ml of petroleum ether. The three organic extracts were combined and evaporated to dryness. The residue was dis- solved in 6 ml of dichloromethane-isopropanol (99: 1, v/v) and transferred to a vial. Before high-performance liquid chromatography analysis, the solution was evaporated to com- plete dryness. The residue was again dissolved in 1 ml of chloromethane-isopropanol (99 : 1, v/v), filtered using a HV 45 pm filter (Millipore) and 20 p1 were injected into a Waters liquid chromatograph using a reverse phase column (Spherisorb ODs-2, 10 pm, 4 mm by 25 cm) and methanol as the mobile phase with a flux of 1.7 ml/min. Ergosterol was monitored at 282 nm. The analysis was performed in duplicate.

Results and Discussion

Figure 1 shows the variation in specific ergos- terol content of mycelium with time, each point being the mean of two replicates, and it can be seen that the ergosterol content per gram dry weight varies over a threefold range. This is a much greater variation than that reported by other authors. Thus Matcham et nl. (1985), Newel1 et al. (1987) and Seitz et al. (1979) reported variations of 1.2-fold, or even less, in the ergosterol content of mycelium of different ages.

Our results show that, even though the varia- tion of the above mentioned parameter is very similar to that found by other authors after 10 d

22 M . Torres et al. of incubation (Seitz et al. 1979; Newel1 et al. 1987; Matcham et al. 1985), during the first days the parameter value is between 1/3 and 2/3 lower (1.5 at day 2 and 2.6 at day 6). As the resulting growth curve is a very usual one (Fig. 2), the ergosterol variations do not seem to be due to any abnormal growth.

From our results we can conclude that ergos- terol can be used as an indicator of fungal pres- ence, although it is necessary to be very careful in using it as a quantitative measure. Further- more, when it is used to study the evolution of mould infestation in crops or other kinds of substrate, a misinterpretation about the fungal presence during the first days may result because ergosterol content in younger moulds seems to be lower than when mould gets older.

This research was supported by CAYCIT (grant PA86-0291). We wish to acknowledge the assist- ance of Dolors Morlans of the Department of Crop Science, Universitat Politecnica de Cata- lunya, Spain.

References ELLIOT, C.G., KNIGHTS, B.A. & FREELAND, J.A. 1974

Sterols of Neurospora crassa and the pattern of their binding during the growth cycle. Biochimica et Biophysica Acta 360,339-347.

EVANS, L.E. & GEALT, M.A. 1985 The sterols of growth and stationary phases of Aspergillus nidu-

lans cultures. Journal of General Microbiology 131,

JAMBUNATHAN, R., KHERDEKAR, M.S. & VAIDYA, P. 1991 Ergosterol concentration in mold-susceptible and mold-resistant sorghum at different stages of grain development and its relationship to flaven-4- 01s. Journal of Agricultural Food Chemistry 39,

MATCHAM, S.E., JORDAN, B.R. & WOOD, D.A. 1985 Estimation of fungal biomass in a solid substrate by three independent methods. Applied Microbiology and Biotechnology 21, 108-1 12.

NEWELL, S.Y., MILLER, J.D. & FALLON, R.D. 1987 Ergosterol content of salt-marsh fungi: effect of growth conditions and mycelial age. Mycologia 79,

NEWELL, S.Y., ARSUFFI, T.L. & FALLON, R.D. 1988 Fundamental procedures for determining ergosterol content of decaying plant material by liquid chro- matography. Applied and Environmental Micro- biology 54, 1876-1879.

SAFE, S. 1973 The effect of environment on the free and hydrosoluble sterols of Mucor rouxii. Bio- chimica et Biophysica Acta 326.471-475.

SEITZ, L.M., MOHR, H.E., BURROUGHS, R. & SAUER, D.B. 1977 Ergosterol as an indicator of fungal inva- sion in grains. Cereal Chemistry 54, 1207-1217.

SEITZ, L.M., BURROUGHS, R., SAUER, D.B., MOHR, H.E. & HUBBARD, J.D. 1979 Ergosterol as a measure of fungal growth. Phytopathology 1979, 1202-1203.

SEITZ, L.M., MOHR, H.E., BURROUGHS, R. & GLUECK, J.A. 1983 Preharvest fungal invasion of sorghum grain. Cereal Chemistry 60, 127-130.

WEETE, J.D. 1981 Lipids in fungal growth and repro- duction. In The Fungal Spore: Morphoyenetic Con- trols ed. Turian, G. & Hohl, H.R. pp. 463-485. New York: Academic Press.

279-284.

1866-1870.

688-695.