Growth of Aspergillus ochraceus, A. carbonarius and A. niger on culture media at different water activities and temperatures

Crescimento de Aspergillus ochraceus, A. carbonarius e A. niger em meios de cultura com diferentes atividades de água e temperaturas

MEDICAL MICROBIOLOGY

Growth of Aspergillus ochraceus, A. carbonarius and A. niger on culture media at different water activities and temperatures

Crescimento de Aspergillus ochraceus, A. carbonarius e A. niger em meios de cultura com diferentes atividades de água e temperaturas

Héctor Palacios-Cabrera^I; Marta Hiromi TaniwakiI,^*; Jorge Minoru Hashimoto^I; Hilary Castle de Menezes^II

^IInstituto de Tecnologia de Alimentos, Campinas, SP, Brasil

^IIFaculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, Brasil

ABSTRACT

The objective of this paper was to determine the influence of three culture media with different water activities, times of incubation and temperatures on the growth of A. ochraceus, A. carbonarius and A. niger. Spores of A. ochraceus, A. carbonarius and A. niger were inoculated onto three culture media: Czapeck Yeast extract Agar (CYA), Dichloran 18% Glycerol Agar (DG18) and Malt Yeast extract 40% Glucose Agar (MY40G). The plates were incubated at five different temperatures (8, 25, 30, 35 and 41ºC). The growth of fungi was evaluated every 24h, measuring the colony diameter (mm). None of the species grew at 8ºC in any culture media. For A. carbonarius, 30ºC was the best temperature for growth while for A. niger temperatures above 30ºC were better in all culture media. A. ochraceus presented good growth at 25 and 30ºC in all culture media, while at 35ºC, growth was slower, especially on CYA. At 41ºC, A. ochraceus did not grow in any culture media and A. carbonarius was significantly inhibited. A. niger grew at 41ºC and was shown to be the most xerophilic fungi when compared to A. carbonarius and A. ochraceus.

Key words:Aspergillus ochraceus, Aspergillus carbonarius, Aspergillus niger, growth measurement, water activity, temperature

RESUMO

Esporos de A. ochraceus, A. carbonarius e A. niger foram inoculados em três meios de cultura: Agar extrato de levedura Czapeck (CYA), Agar Glicerol 18% com dicloran (DG18), Agar Extrato de levedura e malte com 40% de glicose (MY40G). As placas foram incubadas em 5 temperaturas diferentes: 8, 25, 30, 35 e 41ºC. O crescimento dos fungos foi avaliado medindo o diâmetro da colônia a cada 24h. O objetivo deste trabalho foi determinar a influência de três meios de cultura com atividade de água diferente tempo de incubação e temperaturas, sobre o crescimento de A. ochraceus, A. carbonarius and A. niger. Nenhuma das espécies cresceu à 8ºC em nenhum dos meios de cultura. Para A. carbonarius, 30ºC foi a melhor temperatura para o seu crescimento enquanto para A. niger temperaturas acima de 30ºC foram melhores em todos os meios de cultura. A. ochraceus apresentou bom crescimento a 25 e 30ºC em todos os meios de cultura, enquanto seu crescimento à 35ºC foi mais lento, especialmente no meio CYA. A 41ºC, A. ochraceus não cresceu em nenhum dos meios de cultura estudados e A. carbonarius foi significantemente inibido. A. niger cresceu à temperatura de 41ºC e apresentou-se como o fungo mais xerofílico, comparado com A. carbonarius e A. ochraceus.

Palavras-chave:Aspergillus ochraceus, Aspergillus carbonarius, Aspergillus niger, medida de crescimento, atividade de água, temperatura

INTRODUCTION

Fungi are significant environmental microrganisms especially in foods where they are responsible for spoilage, production of mycotoxins and, in some cases desirable bioconversions. Consequently, it is important to know their requirements for water, temperature, nutrients, oxygen and other factors for their growth. The most common genera of fungi in food are Aspergillus, Penicillium and Fusarium. Several species of these genera are able to produce mycotoxins, which are of concern to public health. Among them, ochratoxin A is nefrotoxic and carcinogenic to some animals (20,25) and has been detected in different types of foods such as cocoa and cocoa products (14), coffee (11,22,23), dried fruits (12,13), cereals (9), wines (15,26,27), beer (5) and others (2). Ochratoxin A (OA) is believed to be produced in nature by three main species of fungi, Aspergillus ochraceus, A. carbonarius and Penicillium verrucosum, with a minor contribution by A. niger (8). P. verrucosum is believed to occur only in cool temperate climates, and is mainly associated with cereals (18,19). A. carbonarius and A. niger were described as sources of OA (1,6,24). A. ochraceus has been isolated from several green coffee samples originating from coffee producing countries (4,23).

Several data of growth conditions for A. niger and A. ochraceus have been recorded. However, little has been published about A. carbonarius. Most information about its physiology is assumption based on A. niger, because of its similarity. It may be closely related, but it differs from A. niger most notably in the production of larger spores (10). The importance of studying A. carbonarius is because of its ability to produce ochratoxin A, which is much greater than that of A. niger. A. niger has been reported to grow optimally at relatively high temperatures, with a maximum of 45 to 47ºC and optimal conditions from 35 to 37ºC. This species has been reported as a xerophile with germination reported at 0.77 a_w at 35ºC (19). A. ochraceus has been described as a mesophilic xerophile. Growth occurs between 8 and 37ºC, with the optimum from 24 to 31ºC (16,19,21). Previous studies suggest that the a_w minima for A. ochraceus varies from 0.76 to 0.88 depending on the substrate (3) and the optimum from 0.98 to 0.96 (21). The objective of this work was to study the effect of temperature, culture media and water activity (a_w) on the growth of three OA producing fungi: A. niger, A. carbonarius and A. ochraceus.

MATERIALS AND METHODS

Fungal species

Three species of fungi producers of ochratoxin A isolated from green coffee from São Paulo State, Brazil by Taniwaki et al. (23) were studied: A. niger ITAL 704, A.carbonarius ITAL 170 and A. ochraceus ITAL 118.

Culture media

The following media were used: Czapeck Yeast Extract Agar (CYA), a_w 0.997 (17); Dichloran 18% Glycerol Agar (DG18), a_w 0.955 (7) and Malt Yeast 40% Glucose Agar (MY40G), a_w 0.897 (19).

Cultivation

Cultures were grown on each medium in standard size (85mm) plastic Petri dishes. Inocula were prepared from 5 day old cultures grown on Malt Extract Agar (MEA) (17). A suspension of spores was prepared in a phosphate buffer (pH 7.2) with 0.1% of Tween 80. The spores were counted in a haemacytometer giving 10⁴ spores/ml. A drop of suspension of each fungus was inoculated, separately, on the centre of each culture medium. Plates were incubated upright at temperatures of: 8, 25, 30, 35 and 41ºC. All experiments were performed in 6 replicates.

Growth Measurements

Fungal growth was measured at intervals of 24h after the third day of incubation. The reverse side of the colonies was measured in millimetres with a ruler.

Statistical analysis

Co-variance was used to analyse colony diameter so that the effects of one or more factors (a_w, temperature, species and time) could be assessed separately, for statistically significant differences. The SAS System (version 6.12, SAS Institute, Cary, NC 27513, USA) statistical package was used.

RESULTS

The growth of A. ochraceus, A. carbonarius and A. niger on different culture media, time and temperatures is shown in Figs. 1 to 3, respectively.

The results represent an average of 6 replicates. At 8ºC there was no growth of any of these three species on the three culture media. As shown in Fig. 1, the temperatures of 25 and 30ºC favoured fast colony diameter growth for A. ochraceus. On the other hand, slower growth can be observed at 35ºC and total inhibition at 41ºC on all of the culture media. At 35ºC, A. ochraceus grew better on DG18 and MY40G than on CYA. A more xerophilic character may be attributed to this fungus at 35ºC. For A. carbonarius, 30ºC was shown to be the best temperature for its growth on CYA, DG18 and MY40G. A. carbonarius grew poorly at 41ºC reaching 12, 33 and 40 mm on CYA, DG18 and MY40G, respectively, and with no more growth after 6 to 8 days (Fig. 2). In general, growth of A. niger was faster at 35ºC on all culture media. This shows that A. niger prefers to grow at temperatures higher than 30ºC. At 41ºC, this species spread over the whole plate (85 mm) on CYA, DG18 and MY50G after 8, 9 and 10 days, respectively (Fig. 3). At the temperature of 41ºC, A. carbonarius was significantly inhibited in all culture media. The range of temperatures between 25 to 41ºC was not a limiting factor for growth of A. niger. A. carbonarius grew faster than A. ochraceus at temperatures above 30ºC, especially at 35ºC. Significant differences in a_w and temperature were observed for the growth of all the species tested.

Statistical analyses showed significant differences between species (P < 0.001) due to a_w, temperature, species, and two-and three-way interactions (Table 1).

Analysing the data for species as it is shown in Table 2 a similar tendency can be observed from the data of Table 1, in relation to the single effect. The differences appeared when the interactions of the effects of a_w X temperature were compared. However, they were only significant for A. ochraceus. Another fact which should be emphasized is the interaction of time X a_w, which was only statistically significant for A. niger (Table 2).

DISCUSSION

The temperature of 8ºC was inhibitory for A. ochraceus, A. carbonarius and A. niger. Pitt and Hocking (19), have recorded minimum growth of A. niger and A. ochraceus at 6 and 8ºC, respectively, which differs from the data obtained in the present experiment. This may be due to the differences among the strains isolated from different origins around the world. A. carbonarius although similar to A. niger, has its own distinct growth character, especially at temperatures higher than 35ºC. While A. niger grew very well at 41ºC on all media, A. carbonarius did so poorly. A. ochraceus did not grow at all at 41ºC in all culture media. The media with reduced a_w such as DG18 and MY40G, were not a limiting factor for these three species. On MY40G (a_w 0.897), the three species were able to grow at temperatures from 25 to 35ºC. A. carbonarius grew better at MY40G than CYA at 41ºC. These observations may suggest that the substrate may influence the thermotrophic behaviour of the fungi. MY40G has a higher sugar concentration than CYA. Although DG18 is not a suitable medium to study the kinetic of fungal growth, it was also included because it is commonly used to isolate fungi from food and it was important to evaluate the behaviour of these three species on this medium. Studies on culture media with different compositions and water activities at the laboratorial level are useful in order to follow the kinetic of fungal growth under different conditions.

ACKNOWLEDGMENTS

The authors wish to thank Consórcio Brasileiro de Pesquisa e Desenvolvimento do Café for the financial support, to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for funding the PhD program for H.P. and Mr. S. Shaw for reviewing the manuscript.

Submitted: March 10, 2004; Approved: February 01, 2005

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Publication Dates

• Publication in this collection
  12 Sept 2005
• Date of issue
  Mar 2005

History

• Accepted
  01 Feb 2005
• Received
  10 Mar 2004