Is intraspecific variability of growth and mycotoxin production dependent on environmental conditions? A study with Aspergillus carbonarius isolates

doi:10.1016/j.ijfoodmicro.2010.10.030

International Journal of Food Microbiology

Volume 144, Issue 3, 5 January 2011, Pages 432-439

https://doi.org/10.1016/j.ijfoodmicro.2010.10.030 Get rights and content

Abstract

The aim of this study was to assess the impact of suboptimal environmental conditions on the intraspecific variability of A. carbonarius growth and OTA production using thirty isolates of A. carbonarius.

Three a_w/temperature conditions were tested, one optimal (0.98a_w/25 °C) and two suboptimal: 0.90a_w/25 °C and 0.98a_w/37 °C as suboptimal water activity and temperature, respectively, which might take place through over ripening and dehydration of grapes. For each condition, 12 Petri dishes were inoculated, and colony growth and OTA production were measured over time.

ANOVA revealed significant differences among μ and λ within the 30 assayed isolates. Coefficients of variation (CV%) revealed a wider dispersion of growth rates at 0.90a_w/25 °C compared to 0.98a_w/25 °C, and a more than 4-fold higher CV at 0.98a_w/37 °C compared to 0.98a_w/25 °C. However, dispersion of lag phases was similar at 0.98a_w/25 °C and 0.90a_w/25 °C and wider at 0.98a_w/37 °C.

There were significant differences (p < 0.05) among OTA levels (ng/mm²) for the different conditions, values being lower under marginal conditions, and particularly at 0.98a_w/37 °C. Coefficients of variation (CV%) revealed a wider dispersion of OTA production at 0.90a_w/25 °C compared to 0.98a_w/25 °C, while CV at 0.98a_w/37 °C was similar to that at 0.98a_w/25 °C.

In order to address the strain variability in growth initiation and prove the well-established notion of reducing OTA in foods by preventing fungal growth, a greater number of strains should be included when developing models for conditions that are suboptimal both for a_w for OTA production and temperature levels for growth.

Research Highlights

► This work is an interesting research concerning the effect of marginal conditions on intraspecific variability on growth and ochratoxin A production by Aspergillus carbonarius isolates. Besides, we studied ecophysiology of this mould and intraspecific variability among the isolates, with 30 isolates, which is a high number. Many studies exist with a few isolates, but their results might not be representative for the majority of the strains.

Introduction

Mycotoxin contamination of food products is an important health hazard of growing interest around the world. Aspergillus carbonarius has been considered the main species responsible for ochratoxin A (OTA) accumulation in grapes, wine, dried vine fruits and probably in coffee (Cabañes et al., 2002, Abarca et al., 2003). OTA is a toxic fungal secondary metabolite which poses a risk for the human and animal health. The toxicological profile includes teratogenesis, nephrotoxicity and immunotoxicity (Kanisawa, 1984, Bendele et al., 1985, Krogh, 1987, Patel et al., 1997) and it is classified as a possible human renal carcinogen (group 2B) (International Agency for Research on Cancer, 1993).

A. carbonarius has been found in a great variety of substrates for human and animal consumption in regions with warm and tropical climates. Strains of this mould have been isolated and identified in several Mediterranean wine producing countries, including Spain, Italy, Israel, Portugal and Greece (Serra et al., 2003, Battilani et al., 2004, Bellí et al., 2004a, Mitchell et al., 2004, Tjamos et al., 2004, Tjamos et al., 2006).

There are many factors that influence mould growth and mycotoxin contamination such as temperature, substrate aeration, water activity, inoculum concentration, microbial interactions, physiological state of mould, etc. Many studies have been published on the effects of some of these factors on growth of one to eleven mycotoxigenic strains. Some authors reported a high intraspecific variability on mould growth and mycotoxin production when several strains were included in the studies (Bellí et al., 2004b, Parra and Magan, 2004, Pardo et al., 2004, Pardo et al., 2005a, Arroyo et al., 2005, Astoreca et al., 2007, Astoreca et al., 2010, Romero et al., 2007, Tassou et al., 2009). Others did not find differences among isolates of the same species (Bellí et al., 2004b, Pardo et al., 2005b).

The use of cocktail inocula of different isolates to minimize the variation that might be expected among different isolates of the same species has been proposed (e.g. Hocking and Miscamble, 1995, Patriarca et al., 2001, Pose et al., 2009, Romero et al., 2007, Romero et al., 2010). This concept was introduced for physiological studies on foodborne bacterial pathogens, particularly in acquisition of data for predictive modelling studies, as a way of determining the extremes of growth limits for particular species (Gibson et al., 1987, Buchanan et al., 1993).

Generally, foods and feeds are stored at marginal conditions for mould growth. Different environmental factors could influence the variability of responses of different isolates of the same species in their growth patterns and mycotoxin production, and this might be increased when marginal conditions exist. This point requires clarification for further development of predictive mycology.

The aim of this study was to assess the impact of suboptimal environmental conditions for growth on the intraspecific variability of A. carbonarius growth and OTA production using thirty isolates of A. carbonarius.

Section snippets

Fungal isolates and preparation of inoculum

This work was carried out on thirty isolates of A. carbonarius previously isolated from grapes of Spain (Catalonia, Andalucía, La Rioja, Valencia). Isolates are maintained in the culture collection of the Food Technology Department of Lleida University. The isolates were sub-cultured on malt extract agar (MEA) plates and incubated at 25 °C for 7 days to enable significant sporulation. After incubation, a sterile inoculation loop was used to remove the conidia from MEA plates and they were

Effect of marginal conditions on intraspecific growth differences

All isolates of A. carbonarius grew under the three conditions tested. Analysis of variance revealed significant differences in growth among isolates and among incubation conditions; moreover, differences among isolates depended on environmental conditions (p < 0.05). Table 1 shows mean values of growth rate (μ, cm/day) and lag phase (λ, days) of all isolates for the conditions studied and their dispersion. As expected, there were significant differences (p < 0.05) among estimated μ and λ levels

Discussion

Individual strains exhibit differences in their growth and in the amount of mycotoxin produced (Romero et al., 2010). These differences can be attributed to isolate geographical origin, substrate, environmental conditions, molecular characteristics, etc.

In this research we compared growth parameters and OTA production of thirty isolates of A. carbonarius incubated at three different conditions. All isolates grew under all conditions studied. However, some isolates showed an exceptional response

Acknowledgement

The authors are grateful to the Spanish Government (CICYT, Comisión Interministerial de Ciencia y Tecnología, project AGL 2007-66416-C05-03/ALI and AGL2010-22182-C04-04), Comissionat per a Universitats i Recerca d'Innovació, Universitats I Empresa de la Generalitat de Catalunya (AGAUR) and European Social Fund for the financial support.

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Is intraspecific variability of growth and mycotoxin production dependent on environmental conditions? A study with Aspergillus carbonarius isolates

Abstract

Research Highlights

Introduction

Section snippets

Fungal isolates and preparation of inoculum

Effect of marginal conditions on intraspecific growth differences

Discussion

Acknowledgement

Journal of Food Protection

International Journal of Food Microbiology

The Science of the Total Environment

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Mycological Research

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