Extracellular polysaccharides as target compounds for the immunological detection of Aspergillus and Penicillium in food (1992)

This thesis is devoted to the immunological detection of Aspergillus and Penicillium in food products. More specifically, the immunogenicity, antigenicity, production and structure of the water-soluble extracellular polysaccharides (EPS) of these moulds have been studied, and a latex-agglutination assay, based on the detection of EPS has been developed.For the detection of moulds many methods are available, each of them with specific advantages and disadvantages, mostly related to reliability and applicability ( Chapter 2 ).An overview of the immunogenicity and antigenicity of EPS produced by moulds is presented in Chapter 3 . The role of β(1,5)-galactofuranoside sequences as epitopes of galactomannans from Aspergillus and Penicillium is documented. Antigenically specific polyclonal antibodies raised against P.digitatum EPS are directed towards β(1,5)-linked galactofuranosyl residues. These antibodies react specifically with EPS from Aspergillus and Penicillium.Synthetic tetramers and heptamers of β(1,5)-linked galactofuranosides are conjugated to tetanus toxoid and polyclonal antibodies are raised in rabbits against these synthetic immunogens ( Chapter 4 ). Antibodies obtained after immunisation with the heptamer conjugate possess the same genus specific antigenicity as the antibodies raised against P.digitatum EPS. No reactions are observed with the Penicillium subgenus biverticillium species and species belonging to genera other than Aspergillus and Penicillium . In contrast, antibodies raised against the tetramer conjugate reacted only with six out of 24 tested Aspergillus and Penicillium strains.From Glucanex, a Trichoderma harzianum enzyme preparation, an exo-β-D- galactofuranosidase is purified. This enzyme is used to hydrolyse specifically the immunodominant β(1,5)-linked galactofuranosyl residues from Aspergillus and Penicillium EPS. This enzyme alleviates the antigenicity of the EPS completely ( Chapter 5 ).Additionally, the reductive cleavage method for determination of the glycosidic bonds revealed that the β(1,5)-linked galactofuranosyl side chains in P.digitatum EPS carry side-chains of β(1,6)-1inked galactofuranosyl residues. These results allowed to propose a new structural model for the antigenically active galactofuranoside side chains of Penicillium galactomannans.In Chapter 6 , the production of antigenic EPS by P. aurantiogriseum and P. digitatum has been described under various growth conditions. Antigenic EPS was produced under almost all conditions investigated. However, both P.aurantiogriseum and P.digitatum do not produce antigenic EPS on lactate as the carbon source. Also, P.camemberti isolated from a mould fermented cheese (Camembert) does not produce antigenic EPS on lactate, althought, P.camemberti produces antigenic EPS on other substrates. The monosaccharide composition of the EPS produced by P.aurantiogriseum and P.digitatum under various conditions varies considerably.Immunopotent acid-labile β(1,5)-linked galactofuranosyl residues of Aspergillus fumigatus, Aspergillus niger and Penicillium digitatum EPS were acid-hydrolysed ( Chapter 7 ). Antibodies are raised against these acid-treated extracellular polysaccharides. It was supposed that these acid-treated EPS preparations would elicit antibodies with a broader specificity, making them useful in the detection of nearly all or all moulds occurring in food products. However, the antibodies obtained are more species specific and are generally directed to glucosyl and/or mannosyl residues of the EPS.Antibodies raised against P.digitatum EPS are used for the development of a rapid and reliable latex-agglutination assay for the detection of Aspergillus and Penicillium in food and feed ( Chapter 8 ). The reliability of the assay is enhanced by using a synthetic epitope, a tetramer of β(1,5)-Iinked galactofuranosides. With this tetramer false-positive results can easily be recognised.Finally, in Chapter 9 the applicability of the developed latex-agglutination assay is tested in both comparative and collaborative studies. The significance of extracellular polysaccharides produced by moulds for the detection of moulds in food and feed is discussed.