New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.

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.

Detection of Aspergillus and Penicillium extracellular polysaccharides (EPS) by ELISA: using antibodies raised against acid hydrolysed EPS.

Species of the fungal genera Aspergillus and Penicillium produce immunologically active extracellular polysaccharides (EPS) in which galactofuranose residues are immunodominant. The antigenic determinant of the EPS of A. fumigatus, A. niger and P. digitatum could be removed by acid hydrolysis. Due to the hydrolysis of the EPS the immunological reaction between IgG anti-native EPS and hydrolysed EPS disappeared. Antibodies raised in rabbits against the acid hydrolysed EPS revealed new antigenic determinants that were exposed as a result of the acid hydrolysis. Immunological inhibitory experiments showed that the antibodies were no longer directed to galactofuranose residues. Enzyme Linked Immunosorbent Assay, carried out with antibodies raised against the acid hydrolysed EPS showed that the antibodies against the acid hydrolysed EPS were more species specific in comparison with the antibodies against the native EPS.

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.

A Rapid and Reliable Method for the Detection of Molds in Foods: Using the Latex Agglutination Assay.

An agglutination test by using latex beads (0.8 µm diameter) coated with IgG from the antibodies against extra-cellular polysaccharide of Penicillium digitatum has been developed. As low as 5 to 10 ng/ml of the purified extra-cellular polysaccharide of the same species can be detected by this preparation. Analysis of culture filtrates from 25 different molds showed that the positive reactivity was obtained only with the species of genera Penicillium and Aspergillus . The application of this test was confirmed in testing the samples of spices and nuts. Further, the reliability could be enhanced by including specific blocker in the assay, the synthetic epitopes consisting of four ß (1-5)-linked D-galactofuranosyl residues.