Ramon Canela

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities (aw, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B1 (FB1) and B2 (FB2) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing aw and best at 30°C. All three isolates produced more FB1 than FB2 regardless of aw or temperature. Very little FB1 and FB2 were produced at 0.925 aw, with maximum produced at 0.956 and 0.968 aw at both temperatures tested. Most FB1 and FB2 were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all aw levels and both temperatures there was an increase in FB1 and FB2 concentration with time. Statistical analyses of aw, temperature, time, two‐ and three‐way interactions showed some significant differences between isolates and FB1 and FB2 production.

Two-dimensional profiles of fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain.

This study has examined in detail the effect of temperature (7-37 degrees C) and water availability (water activity, a(w), 0.89-0.97) on fumonisin B1 (FB1) production by an isolate of Fusarium moniliforme and F. proliferatum on irradiated maize grain after incubation for 28 days. The optimum conditions for F. moniliforme and F. proliferatum were 30 degrees C at 0.97 a(w) and 15 degrees C at 0.97 a(w), respectively. The maximum concentrations were 2861 mg kg(-1) and 17,628 mg kg(-1) dry wt. maize grain, respectively. At marginal a(w)/temperature conditions for growth (e.g. 0.89-0.91 a(w)) no FB1 was detected (<0.1 mg kg(-1)). A high variability was found between replicates for F. moniliforme, but not for F. proliferatum. These data were used to construct two-dimensional diagrams of all the a(w) x temperature conditions favourable for FB1 production for the first time. The data were also subjected to a polynomical regression, which demonstrated that there was a very good fit for the 15-30 degrees C range of temperature and at 0.97 a(w). However, at marginal environmental conditions this was not possible. This suggests that it may be possible to predict within a limited environmental range the potential for significant FB1 production.

Determination of carotenoids by liquid chromatography/mass spectrometry: effect of several dopants

Various carotenoids were analyzed by ultra-high-pressure liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). Three different techniques to ionize the carotenoids were compared: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). For all of the carotenoids tested, it was possible to obtain characteristic transitions for their unequivocal identification using each ionization technique. APCI was shown to be a more powerful technique to ionize the carotenoids than ESI or APPI. Transitions to differentiate carotenoids that coelute were determined to distinguish antheraxanthin from astaxanthin and lutein from zeaxanthin. In addition, four dopants were evaluated to improve ionization and enhance the carotenoid signal strength in APPI. These dopants were acetone, toluene, anisole, and chlorobenzene. Carotenoids improved their response in almost all cases when a dopant was used. The use of dopants allowed the enhancement of the carotenoid signals strength up to 178-fold.

Fumonisins B1 and B2 and toxigenic Fusarium strains in feeds from the Spanish market

Natural occurrence of fumonisins B1 and B2, incidence of Fusarium species, and capacity to produce fumonisins by Fusarium isolates, were investigated in 50 corn-based samples from Spain destined for animal consumption. Forty-four samples (88%) were found to be contaminated with fumonisins. The levels of contamination were very low, with a mean of 400 ng/g in the samples. We investigated the capacity of 11 isolates of Fusarium moniliforme and 19 isolates of F. proliferatum to produce fumonisins. All F. proliferatum isolates and 8 out of the 11 F. moniliforme isolates assayed produced fumonisins on a corn medium. The FB1/FB2 ratio in the isolates ranged from 1.1 to 3.5.

Control of growth and fumonisin B1 production by Fusarium verticillioides and Fusarium proliferatum isolates in moist maize with propionate preservatives

The effect of propionic acid, its sodium salt or a commercial formulation of propionates (0.03, 0.05 and 0.07%), on growth and fumonisin B1 production by Fusarium verticillioides and F. proliferatum isolated was evaluated on irradiated maize at different water activities (aw, 0.93, 0.95, 0.98) and temperatures (15, 25 degrees C). The four isolates grew at all aw x temperature treatments in the absence of propionates. At the highest propionate concentration tested (0.07%), however, growth was restricted to 0.98 aw, for F. proliferatum isolates but not for those of F. verticillioides. Inhibition of growth was maximum when propionates were added in the acid form. In the presence of low propionate concentrations (0.03%), growth was sometimes enhanced probably due to assimilation of these compounds by the fungus. Water activity, temperature, concentration and source of propionate, as well as most two-, three-, four-, and five-way interactions had a significant influence on growth of Fusarium isolates. None of the assayed treatments had any effect on fumonisin B1 production by F. verticillioides isolates. For F. proliferatum, higher fumonisin B1 production occurred in the absence of propionates, and in general concentration decreased with increasing doses of preservatives. Single factors (aw, propionate concentrations and temperature) and temperature x aw and propionate concentration x temperature interactions had a significant effect on fumonisin production (p < 0.01). Moreover, propionate concentration was the single most important factor, besides temperature, which affected fumonisin B1 production.

Selective effect of propionates and water activity on maize mycoflora and impact on fumonisin B1 accumulation.

The effect of a commercial mixture of propionates at two different doses (0.05% and 0.1%) on fungal spoilage of natural maize stored at 0.85, 0.90 and 0.95 water activity (aw) was investigated. Parallel treatments with added inoculum of Fusarium Liseola section isolates (Fusarium moniliforme and F. proliferatum) were carried out in order to determine the effect of fungal interactions on the development of fumonisin-producers on maize in relation to preservative efficacy. Fungal colonisation of grain was measured as fungal counts (CFUs g−1 maize). In general, no differences were found between inoculated and uninoculated samples. Besides the selective effect of aw on maize mycoflora, it was demonstrated that most genera which colonise maize remained unaffected by the preservative concentrations applied. However, Penicillium populations (CFUs g−1 maize) counts decreased significantly. As they represent a major component of the total fungal counts, an overall control of total mycoflora was observed. Furthermore, there was a significant statistical interaction between preservative and aw levels, with the preservative activity enhanced at low aw. The concentrations of fumonisin B1 were unaffected by treatment with no significant differences in concentrations found. This suggests that the natural mycoflora of maize may act as an inhibitor of Fusarium development, and consequently of fumonisin biosynthesis.

Influence of Sample Processing on the Analysis of Carotenoids in Maize

We performed a number of tests with the aim to develop an effective extraction method for the analysis of carotenoid content in maize seed. Mixtures of methanol–ethyl acetate (6:4, v/v) and methanol–tetrahydrofuran (1:1, v/v) were the most effective solvent systems for carotenoid extraction from maize endosperm under the conditions assayed. In addition, we also addressed sample preparation prior to the analysis of carotenoids by liquid chromatography (LC). The LC response of extracted carotenoids and standards in several solvents was evaluated and results were related to the degree of solubility of these pigments. Three key factors were found to be important when selecting a suitable injection solvent: compatibility between the mobile phase and injection solvent, carotenoid polarity and content in the matrix.

Juvenile hormone and diapause in the Mediterranean corn borer, Sesamia nonagrioides.

Juvenile hormone content of Sesamia nonagrioides larvae reared under different environmental conditions that induce diapause was studied using a bioassay on newly-emerged Tribolium confusum pupae. Two analytical methods were also used to measure JHIII content. Extracts from larvae that developed under different conditions of photoperiod and temperature caused different effects in T. confusum pupae. This can be related to different diapause intensities. Extracts from diapausing larvae produced a higher juvenilizing effect than the expected, considering their JHIII titer calculated by the chromatographic analysis. This indicates that hormones other than JHIII must be present in extracts of diapausing larvae of S. nonagrioides. The analytical procedures confirm that diapausing larvae have a higher JHIII titer in the haemolymph than non-diapausing larvae. This shows that JH is involved in the maintenance of diapause in this species.

Analysis of underivatizated patulin by a GC-MS technique.

An alternative approach based on the use of gas chromatography-mass spectrometry (GC-MS) is used to confirm the presence of patulin in apple juice. In the gas chromatography (GC) methods previously described, derivatization of patulin was always necessary in order to achieve good chromatographic detection. The use of electronic pressure control (EPC) and on-column injection avoids the need for patulin derivatization and allows a sensitive analysis of patulin. A detection limit of 4 microg/liter in apple juice can be attributed to the method.

Determination of patulin by reversed-phase high-performance liquid chromatography with extraction by diphasic dialysis

A simple and economical method has been developed for the determination of patulin in apple juice. The sample is extracted with ethyl acetate in a diphasic dialysis system, and the extract is cleaned up by elution from a Sep-Pak cartridge. Patulin is detected and determined by reversed-phase high-performance liquid chromatography using a Novapak C18 column and an ultraviolet detector. The lower detection limit is 1 microgram l-1 and the recovery is 85% at the 20 micrograms l-1 level.