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Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese

https://doi.org/10.1016/S0168-1605(01)00429-9Get rights and content

Abstract

In the present work we studied the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum (NRRL 2274 and NRRL 2269) in culture media and in different Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón). The rate of growth was assessed by measuring colony diameters and the production of citrinin was measured using a rapid semi-quantitative fluorometric technique confirmed by RP-HPLC. A stronger inhibitory effect of eugenol than thymol was evident. 200 μg/ml of eugenol in solid culture medium increased the lag time of growth up to 9 days, and decreased the rate of colony growth. In liquid medium, a complete inhibition of fungal growth was observed. By contrast, thymol in the liquid culture medium only affected the growth rate. In Arzúa-Ulloa cheese, 200 μg/ml of eugenol fully inhibited fungal growth, while in Cebreiro cheese no effect was observed for this compound. Regarding the capacity to inhibit mycotoxin production 100 μg/ml eugenol delayed citrinin production until the sixth day, after which a limiting effect persisted. In Arzúa-Ulloa cheese, no citrinin was detected at a concentration of 150 μg/ml of eugenol, but citrinin was detected after 5 days in the case of thymol at the same concentration. In Cebreiro cheese, neither eugenol nor thymol prevented the production of citrinin at the concentrations applied.

Introduction

Penicillium spp. are able to produce toxins such as citrinin in dairy products Jarvis, 1983, Chapman et al., 1983. Citrinin attracted great attention when it was isolated in human fluids from people in Eastern Europe suffering from renal disease with symptoms similar to pork nephropathy Krogh et al., 1974, Betina, 1984. A technique that has been used since ancient times in foods such as cheese to prevent fungal growth, involves physically rubbing the product with certain herbs or spices, or their oils, with well-known antimicrobial properties (Leung, 1978). Over 30,000 different components have been isolated from these plant oils, compounds containing phenol groups being those most used in the food industry (Meeker and Linke, 1988). In this sense, eugenol (the main component of clove oil) and thymol (from thyme oil) are two of the most important representatives and their antibacterial and antifungal properties are well known Buchanan and Shepherd, 1981, Moleyar and Narasimham, 1992, Kim et al., 1995, Paster et al., 1995, Mansour et al., 1996, Outtara et al., 1997.

The aim of the present study was to determine the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum in culture media and in three types of Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón).

Section snippets

Microorganisms

Two strains of P. citrinum NRRL 2274 and P. citrinum NRRL 2269, both from the Colección Española de Cultivos Tipo (Burjasot, Valencia, Spain), and 10 citrinin-producing strains, isolated and also classified as P. citrinum in our laboratory (Vázquez et al., 1995), from Spanish cheeses were used. The strains were maintained on potato dextrose agar (PDA, Oxoid, Hampshire, England) slopes and stored in a refrigerator.

Cheeses

Studies were carried out on three types of Galician cheese (Northwest Spain) with

Inhibitory effects of eugenol and thymol on growth of P. citrinum

Fig. 1a shows the effects of different concentrations of eugenol on P. citrinum (NRRL 2274) growth on YES agar. It is seen that at a concentration of 200 μg/ml fungal growth was delayed for up to 9 days, after which slow growth was observed. This result is consistent with the findings of other authors Martini et al., 1996, Hitokoto et al., 1980, Mansour et al., 1996, Karapinar, 1990. At low concentrations (50 and 100 μg/ml), an initial antifungal activity could be seen but later, when the

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