Techniques For Metabolite Profiling

Metabolite profiling for identification in its simplest form consists of three elements: getting hold of the metabolites (e.g., extraction), determining the compounds (the profile) (e.g., analysis), and data processing (e.g., chemometrics). This will include all relevant metabolites needed for reliable identification of a fungus, and as discussed in the previous section, profiles of metabolites rather than single metabolites should be used for identification. It is very important to note that metabolite profiles are strongly influenced by the analytical scheme used, thus full metabolite profiles can only be compared if they are produced by the same analytical protocol. Over the years, many specific techniques have been developed to determine a few selected metabolites, mostly known mycotoxins, from cultures and from complex samples such as food and feed. Some of these methods can be expanded to include a broad spectrum of metabolites, but dedicated profiling methods are required to efficiently obtain the best possible metabolite profiles and allow reliable identification.

Developments in chromatography and mass spectrometry (MS) have greatly increased their resolution, sensitivity and productivity in analytical chemistry. There is, therefore, a set of tools available that allows fast metabolite screening from a very small amount of sample. As a result, a broad range of metabolites can now be determined in one analysis with high selectivity. Furthermore, several of the newer techniques have shown their potential as general rapid profiling methodology working directly on raw samples or extracts. These include MS, nuclear magnetic resonance, and FT-IR (including NIR), which eliminate a time consuming chromatographic step. However, currently it is not wise to fully eliminate chromatography and one should rather use systems based on complementary methods. In planning an identification from an analytical approach the following considerations must be made: (a) A priori knowledge about metabolites produced by the genus, (b) whether the general chemical classes are alkaloids, acids, neutrals, volatiles, as well as large or small molecules, (c) sample matrix or growth medium composition and interference, (d) number of samples to handle, (e) expected biological variation, and if large or small chemical diversity is expected, (f) sensitivity of analytical instrumentation, and (g) cost.

Table 1 Recommended media for microscopy and chemotaxonomy of important fungal genera

Metabolite

Genus

Teleomorphic state

Microscopy

profiling

Penicillium subgenus: Furcatum,

Eupenicillium

MEA

YES, CYA

Penicillium, Aspergilloides

Penicillium subgenus

Talaromyces

MEA

OAT, MEA, YES

Biverticillium

Aspergillus

Eurotium, Emericella, Neocarpenteles, Neosartorya,

MEA

YES, CYA

Petromyces, Neopetromyces, Chaetosartorya, Sclerocleista,

Hemicarpenteles, Fennellia

Paecilomyces

Byssochlamys

MEA

YES, PD

Stachybotrys

Melanopsamma

CMA

PD, ALK

Trichoderma

Hypocrea

OAT, CMA

YES, PD

Fusarium

Gibberella, Nectria, Cosmospora, Haematonectria

SNA

YES, PD

Alternaria

Lewia

GAK

DRYES

Media recipes, see text or our website http://www.biocentrum.dtu.dk/mycology/analysis/.

Media recipes, see text or our website http://www.biocentrum.dtu.dk/mycology/analysis/.

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