R.A. Samson

Important mycotoxins and the fungi which produce them

The assessment of the relationship between species and mycotoxins production has proven to be very difficult. The modern literature is cluttered with examples of species purported to make particular mycotoxins, but where the association is incorrect. In some cases, mycotoxins have even been named based on an erroneous association with a particular species: verruculogen, viridicatumtoxin and rubratoxin come to mind. As time has gone on, and more and more compounds have been described, lists of species-mycotoxin associations have become so large, and the inaccuracies in them so widespread in acceptance, that determining true associations has become very difficult. It does not need to be emphasised how important it is that these associations be known accurately. The possible presence of mycotoxigenic fungi in foods, and rational decisions on the status of foods suspected to contain mycotoxins, are ever present problems in the food industry around the world. In defining mycotoxins, we exclude fungal metabolites which are active against bacteria, protozoa, and lower animals including insects.

Enumeration and identification of airborne viable mould propagules in houses. A field comparison of selected techniques.

A number of techniques for the enumeration and identification of viable mould propagules in the indoor air of houses were evaluated in order to document to what extent different results are obtained when different methods are used. A comparison was made between the results obtained with five commercially available air sampling devices (Slit‐to agar sampler, N6‐Andersen sampler, Surface Air System sampler, Reuter Centrifugal Air sampler. Gelatine Filter sampler) and a non‐volumetric sampler (the Open Petri Dish), in combination with four culture media (malt extract agar, dichloran glycerol‐18 agar, oxytetracycline glucose yeast extract agar and dichloran rose bengal chloramphenicol agar). The coefficients of variation were high (generally < 20%) for all combinations. Statistical analysis showed that the Slit sampler and the N6‐Andersen sampler in combination with DG18 and MEA gave the best precision and the highest yield in terms of colony forming units per square cubic meter of air (CFU/m3) and number of species isolated.

Presence of viable mould propagules in indoor air in relation to house damp and outdoor air.

The presence of viable mould propagules in indoor air was investigated using the N6‐Andersen sampler in combination with DG18‐agar, in relation to house damp (characterized with a checklist) and in relation to the presence of moulds in outdoor air. The first part of the study was conducted in 46 houses in the autumn of 1987, the second part in 84 houses in May 1989. Further, in the second part, the results obtained with settlement plates (OPD) were compared with those obtained with the N6‐Andersen sampler. The number of CFU/m3 in the indoor and outdoor air varied widely. A large variety of mould genera and species was isolated. Species of Cladosporium, Penicillium and Wallemia predominated. The variability in time was high and the reproducibility of the measurements in terms of CFU/m3 and of species isolated was only moderate. The low predictive value of these measurements limits their use in epidemiological studies of the relationship between exposure to moulds and respiratory symptoms. Overall, the geometric mean concentration was somewhat higher outdoors than indoors. However, the clear differences found between the number of CFU/m3 belonging to different mould species in in‐ and outdoor air show that the presence of viable mould propagules in indoor air is not simply a reflection of the presence of moulds in outdoor air. The presence of moulds in indoor air was only weakly related to house damp as characterized by the checklist. High, statistically significant correlations were found between the CFU yield obtained with the OPD and the CFU/m3 yield obtained with the N6‐Andersen sampler. However, the number of species isolated with the OPD was significantly lower than that obtained with the N6‐Andersen sampler.

Fungal propagules in house dust. I: Comparison of analytic methods and their value as estimators of potential exposure

The presence of viable mold propagules in house dust was investigated by 10 different analytic methods, in order to determine to what extent different results are obtained when different analytic methods are used. Moreover, the value of this measurement as an estimator of the potential exposure to fungi in epidemiologic studies was assessed. Floor and mattress dust was sampled in 60 homes in The Netherlands during autumn 1990. For investigation of the variability in time, sampling was repeated in 20 homes after 6 weeks. Each analytic method is characterized by a unique combination of culture medium, suspension medium, and dilution step. The highest mean number of colony‐forming units (CFU)/g dust was obtained by suspension of at least 100 mg dust in a peptone or sucrose solution in a ratio of 1:50 (w/w), followed by 10‐fold dilution and plating on DG18 agar (geometric mean (GM) approximately 60000 CFU/g dust). The lowest mean number of CFU/g dust was obtained by direct plating of 30 mg dust on V8 agar (GM approximately 5300 CFU/g dust). The mean coefficient of variation of duplicate analyses varied from 11%, for suspension in sucrose and plating on DG18 agar, to 27%, for suspension and dilution in sucrose in combination with V8 agar. The highest mean number of species isolated was obtained by direct plating of 30 mg dust on DG18 agar (mean number of species: 17). Suspension and dilution on DG18 or V8 agars yielded an average of approximately six species. In duplicate analyses, the mean percentage of agreement for the species isolated varied from approximately 35%, for suspension and dilution, to 60%, for direct plating. The reproducibility of the number of CFU/g dust in time was better for mattress dust than for floor dust; however, also for mattress dust, the predictive value of a single measurement was rather low. The variability in time in species isolated was substantial, both for mattress dust and floor dust. We concluded that results of measurements of viable mold propagules in house dust, both quantitatively and qualitatively, depend greatly on the analytic methods used. Furthermore, a single measurement of fungal propagules in settled house dust does not provide a reliable measure of potential exposure to fungi in indoor environments.

Fungal propagules in house dust. II: Relation with residential characteristics and respiratory symptoms

As part of a case‐control study on the relation between home dampness and respiratory symptoms of children, house‐dust samples were collected from bedroom floors and mattresses in 60 homes in The Netherlands. The house‐dust samples were analyzed for the presence of fungal propagules by plating 30 mg of dust directly onto DG18 agar. A checklist and questionnaire were used to obtain information on the home characteristics and occupant behavior that may have an effect on the presence of fungal propagules in house dust. The geometric mean (GM) number of colony‐forming units (CFU)/g dust collected from the floors was 8990. The number of CFU/g dust was significantly higher in dust from carpeted floors than in dust from smooth floors (GM, respectively, 12880 CFU/g dust and 3530 CFU/g dust). The GM number of CFU/g dust collected from mattresses was 6760. Overall, the mean numbers of CFU/g dust collected from floors and mattresses were higher in bedrooms where damp spots mold growth, or both were observed. However, these differences were not statistically significant. The relation between home characteristics and the number of CFU/g dust of the most frequently isolated mold species (n= 17), including Alternaria alternata, Cladosporium cladosporioides, Penicillium brevicompactum, and Scopulariopsis brevicaulis, was also investigated. Only the type of flooring had a significant and consistent effect on the number of CFU/g floor dust of the different mold species. For P. brevicompactum, the number of CFU/g floor dust was significantly higher in bedrooms where damp spots were observed. The number of CFU/g mattress dust of S. brevicaulis was also significantly higher for bedrooms where damp spots were observed. However, in view of the large number of statistical comparisons made, these two significant relationships might have been caused by chance alone. The total numbers of CFU/g mattress and floor dust were not related to the average relative indoor humidity measured over 6 weeks. Furthermore, there was no association between the presence of fungi in house dust and respiratory symptoms. We conclude that there was only a very weak relationship between the home characteristics and occupant behavior, as determined by checklist and questionnaire, and the presence of fungal propagules in floor dust and mattress dust. Only the type of flooring had a substantial and statistically significant effect on the presence of fungal propagules in floor dust. Therefore, the presence of fungal propagules in house dust cannot be predicted reliably by home characteristics.

Advances in Food Mycology

Understanding the fungi producing important mycotoxins.- Important mycotoxins and the fungi which produce them.- Recommendations concerning the chronic problem of misidentification of mycotoxigenic fungi associated with foods and feeds.- Media and method development in food mycology.- Comparison of hyphal length, ergosterol, mycelium dry weight, and colony diameter for quantifying growth of fungi from foods.- Evaluation of molecular methods for the analysis of yeasts in foods and beverages.- Standardization of methods for detecting heat resistant fungi.- Physiology and ecology of mycotoxigenic fungi.- Ecophysiology of fumonisin producers in Fusarium section Liseola.- Ecophysiology of Fusarium culmorum and mycotoxin production.- Food-borne fungi in fruit and cereals and their production of mycotoxins.- Black Aspergillus species in Australian vineyards: from soil to ochratoxin A in wine.- Ochratoxin A producing fungi from Spanish vineyards.- Fungi producing ochratoxin in dried fruits.- An update on ochratoxigenic fungi and ochratoxin A in coffee.- Mycobiota, mycotoxigenic fungi, and citrinin production in black olives.- Byssochlamys: significance of heat resistance and mycotoxin production.- Effect of water activity and temperature on production of aflatoxin and cyclopiazonic acid by Aspergillus flavus in peanuts.- Control of fungi and mycotoxins in foods.- Inactivation of fruit spoilage yeasts and moulds using high pressure processing.- Activation of ascospores by novel food preservation techniques.- Mixtures of natural and synthetic antifungal agents.- Probabilistic modelling of Aspergillus growth.- Antifungal activity of sourdough bread cultures.- Prevention of ochratoxin A in cereals in Europe.- Recommended methods for food mycology.

The Penicillium Funiculosum Complex — Well Defined Species and Problematic Taxa

The morphology and production of secondary metabolites of 96 isolates of Penicillium funiculosum and related taxa have been examined. On basis of both morphology and chemistry P. pinophilum and P. minioluteum are kept separate. P. varians placed in synonymy with P. funiculosum by Pitt (1979), proved to be morphologically distinct from P. funiculosum by its strongly pigmented stipes, cylindrical conidia and by its secondary metabolite production. Isolates FRR 1714 and CBS 642.68 both ex type of P. minioluteum proved to be different. Both isolates were believed to represent the original Biourge isolate no. 60. CBS 642.68 fully fits the original description of the species, but FRR 1714 resembles Penicillium rubrum sensu Raper and Thorn. P. allahabadense is kept as a separate species and not included in P. pinophilum, because it differs from P. pinophilum in growth rate at 37°C and the shape of conidia. P. diversum is characterized by its poor growth on Czapek agars. The species accepted are shortly described and illustrated, while isolates of P. funiculosum from major culture collections were reidentified. A list of mycotoxins and other secondary metabolites specific for each species is given.

Detection and identification of moulds in Dutch houses and non-industrial working environments.

Abstract The mycoflora of indoor non-industrial environments is reported from “case” studies in The Netherlands. Both air sampling by a RCS-Reutcr centrifugal air sampler and surface sampling by swabs and cellotape preparations were carried out in homes, archives and libraries, musea, offices and schools. Common species encountered in these indoor environments are Aspergillus versicolor, Penicillium brevicompactum, P. chrysogenum, Cladosporium spp. and the xerophilic fungi Eurotium spp. and Wallemia sebi. Aspergillus fumigatus, Scopulariopsis spp. and Stachybotrys chartarum were occasionally isolated. It is not always possible to detect the mycoflora growing on surfaces by air sampling. Therefore direct microscopical examination and sampling from surfaces in addition to air sampling is strongly recommended for the detection of viable moulds in indoor environments. Selection of the most suitable media for isolation of fungi is discussed.

Standardization of methods for detecting heat resistant fungi

Heat resistant fungi can be defined as those capable of surviving temperatures at or above 75°C for 30 or more minutes. The fungal structures which can survive these temperatures are ascospores, and sometimes chlamydospores, thick walled hyphae or sclerotia (Scholte et al., 2000). During the last three years, spoilage incidents involving heat resistant fungi occurred increasingly in various products examined in our laboratory. Paecilomyces variotii, Fusarium oxysporum, Byssochlamys fulva, B. nivea, Talaromyces trachyspermus and Neosartorya species were often encountered in pasteurized fruit, dairy products and soft drinks. A questionnaire sent to many laboratories showed that inappropriate methods were used for the detection of heat resistant fungi, or that sometimes there was no special protocol at all. The use of inappropriate media, such as Sabouraud agar, wrong incubation conditions and the analysis of inadequately sized samples were often encountered. In addition, accurate identification of the isolated colonies to species level often was not performed. In the literature many methods are described for the detection of heat resistant fungi (Murdock and Hatcher, 1978; Beuchat and Rice, 1979; Beuchat and Pitt, 1992). Beuchat and Pitt (1992) described two methods: the Petri dish method or plating method and the direct incubation method. In the first method, test tubes are used for the heating of the sample. Subsequently, the sample is poured into large Petri

Mycological condition of maize products

Maize and maize-related products were investigated in a collaborative study for viable moulds and antigenic extracellular polysaccharides (EPS) produced by Aspergillus and Penicillium species. In addition, the samples were tested for the presence of aflatoxin B1. All maize products, with the exception of the heat processed products, contained viable moulds on an average of (log10 values) 3.3 +/- 0.7 colony-forming units per gram. In most samples a mixed mould flora was present. Species of the genus Fusarium were dominant, followed by Aspergillus, Eurotium and Penicillium. The mould colony count correlated positively with the presence of antigenic extracellular polysaccharides produced by species of Aspergillus and Penicillium. Gamma irradiation did not affect the detection of antigenic extracellular polysaccharides. Aflatoxin B1 was detected in two out of 35 samples; these contained 0.6 and 0.8 microgram/kg. From one of these aflatoxin B1-containing samples, Aspergillus flavus was isolated.