Abstract:
In recent years, consumer perceptions are that they would like minimum levels of
preservatives or even preservative free food. However, this leads to higher risks of
microbial spoilage problems, especially due to growth of spoilage fungi, which are
capable of growth at intermediate environmental conditions. Studies have been
carried out to evaluate the effect of different preservatives at optimal and sub-optimal
concentrations on growth, biosynthesis of mycotoxin production at a molecular and
phenotypic level for Fusarium graminearum (Tri5) and trichothecene production and
Penicillium verrusocum (otapksPv) and ochratoxin A (OTA) production. These were
complimented by studies on development of RNAi approaches to inhibit key regulatory
genes in the biosynthetic pathways for mycotoxins in these two species. Additional
studies were carried out to develop a rapid technique for RNA extraction from fungal
biomass.
Initial liquid media based studies identified the growth boundaries of a range of 20
spoilage fungi including 3 mycotoxigenic species in relation to preservatives and pH.
This showed that up to the legal allowable concentrations of sorbic and benzoic salts at
pH 3.0 all strains were capable of growth after 24h. With the exception of F.
graminearum all the other species and strains of spoilage fungi were able to grow in
these conditions. The use of a mixture of preservatives, a common practise in the food
industry, proved effective at inhibiting growth of most spoilage fungi for 21 days at the
EU legal limits. Over the EU legal limit of 250 ppm of potassium sorbate mixed with
150 ppm of sodium benzoate only Aspergillus niger had observable growth. Mixtures
of weak organic acids, fumaric and malic acid, with the preservative potassium sorbate
was shown to be effective at inhibiting growth below the legal limits of use of these
food additives, even though the presence of potassium sorbate appears to be
fundamental to the inhibition effect, of these natural food additives. Moreover the
presence of fumaric acid stimulated growth of Aspergillus flavus. The extraction of high
quality total RNA from low amounts of mycelium showed that up to 3 times higher
yields can be achieved while improving RNA integrity and overall quality. This
development also reduced the time required to extract fungal RNA and the risk of cross-contamination showing the potential use in high throughoutput gene expression
studies.
In vitro and In situ studies demonstrated the risk of using single sub-optimal antifungal
compounds to inhibit growth and mycotoxin production. For F. graminearum, while
growth was reduced, the Tri5 gene expression and trichothecenes type B production
were stimulated in the presence of thyme essential oil, Prochloraz and BHA. This was
also shown with P. verrucosum where otapksPv gene expression and OTA production
were stimulated at different water conditions by the presence of sub-optimum
concentrations of thyme essential oil and Prochloraz. The antioxidant BHA was able to
reduce both otapksPv expression and OTA production in P. verrucosum.
The use of siRNA oligonucleotides to silence Tri5 and otapksPv demonstrated that both
F. graminearum and P. verrucosum possess the RNAi pathway machinery. In both
mycotoxigenic fungi the expression of the target key biosynthetic pathway was
knocked down. The optimum levels of the designed siRNA molecules were of between
10 and 25 nM for the molecules targeting P. verrucosum otapksPv. Even though gene
silencing using siRNA molecules is transient the effect on otapksPv was still observable
after 15 days. This lead to a 3 to 5 times a reduction in the amount of OTA. On the
other hand, the silencing of Tri5 in F. graminearum was only detectable in the first 6
days after transfection.