Vicente Sanchis

Biological control of major postharvest pathogens on apple with Candida sake

Epiphytic microorganisms isolated from apples, pears and the surfaces of apple leaves were screened for antagonistic activity against Penicillium expansum (blue-mold), Botrytis cinerea (gray-mold) and Rhizopus nigricans (Rhizopus rot) on apple (Malus domestica). A total of 933 bacteria and yeasts were tested in primary screening against P. expansum. Ninety-two strains reduced the lesion size on apples by more than 50%, 72 of which were isolated from the surface of apples. For secondary screening against P. expansum, B. cinerea and R. nigricans, 31 strains were selected. The most promising isolate, CPA-1, was identified as Candida sake. This yeast, isolated from apples in storage season was very effective against all three diseases. Wounded Golden Delicious apples protected with the yeast suspension at a concentration of 2.6 x 10(6) CFU/ml and inoculated with conidia of B. cinerea and R. nigricans of 10(5) and 10(4) conidia/ml, respectively, did not develop rot. Complete control of P. expansum was obtained at the same concentration of the antagonist with a pathogen inoculum concentration of 10(3) conidia/ml. This strain, also provided excellent control of rot development under cold storage conditions. The strain of Candida sake can grow actively in aerobic conditions. In drop-inoculated wounds of apples, the populations of C. sake increased by more than 50-fold during the first 24 h at 20 degrees C. The maximum population of C. sake on apple wounds was the same at 20 as at 1 degrees C and was recovered after three and twenty days, respectively.

Predicting mycotoxins in foods: a review.

The need to ensure the microbiological quality and safety of food products has stimulated interest in the use of mathematical models for quantifying and predicting microbial behaviour. For 20 years, predictive microbiology has been developed for predicting the occurrence of food-borne pathogens, although these tools are dedicated to bacteria. Recently, the situation has changed and a growing number of studies are available in the literature dealing with the predictive modelling approach of fungi. To our knowledge the present one is the first review focussed on predictive mycology and food safety, including mycotoxins; existing kinetic and probability models applied to mycotoxigenic fungi germination and growth, and mycotoxin production are reviewed.

Environmental factors, in vitro interactions, and niche overlap between Fusarium moniliforme, F. proliferatum, and F. graminearum, Aspergillus and Penicillium species from maize grain

The effects of temperature and water availability on growth and interactions between fumonisin-producing isolates of Fusarium moniliforme and F. proliferatum and seven other fungi from maize grain were determined in vitro . The type of interaction and index dominance ( I d ) between species were markedly influenced by temperature and a w . Generally, F. moniliforme and F. proliferatum were very competitive and dominant against the Penicillium spp. and A. flavus . They were in turn dominated by A. niger , but mutually antagonistic when paired with F. graminearum and A. ochraceus . Under slightly drier conditions ( a w ) A. ochraceus became more competitive and dominant over the fumonisin-producing species. A. flavus was dominant only at 30°C and a w . F. moniliforme and F. proliferatum demonstrated dominance against all species over a range of temperatures and 0.994 to 0.96 a w . At lower a w levels they were less competitive. The growth rate of the two fumonisin-producing species was significantly reduced by F. graminearum , regardless of a w . F. moniliforme and F. proliferatum reduced growth of Penicillium and Aspergillus spp., especially at > 0.96 a w . At a w , growth of these species was unaffected. Using Biolog plates the effect of a w and temperature on utilization patterns of carbon sources in maize were evaluated for the first time. The niche overlap indices relative to F. moniliforme and F. proliferatum were determined and compared with that of each interacting species. NOIs for F. moniliforme and F. proliferatum were > 0.90 at > 0.96 a w and 25 and 30°, indicative of co-existence with other species. Most of species had NOIs > 0.90, except in some cases when paired with F. moniliforme , where NOIs d and NOI methods both suggested that the niche overlap between species was in a state of flux and significantly influenced by both temperature and water availability. This suggests that interpretation of I d NOIs carried out under one set of environmental conditions may be misleading when considering interactions between species and also where screening for biocontrol potential is being considered.

Fumonisin-producing strains of Fusarium: a review of their ecophysiology.

This review focuses on the fumonisin-producing Fusarium species and the ecophysiology of these species. The effects of environmental biotic and abiotic factors on germination, growth, and fumonisin B1 production by Fusarium verticillioides and Fusarium proliferatum have been investigated under laboratory, field, and storage conditions. An understanding of the factors involved in production of fumonisins is the first step in preventing accumulation of these toxins.

Distribution of fumonisins and aflatoxins in corn fractions during industrial cornflake processing

The aim of this study was to investigate the distribution of fumonisins (B(1) B(2,) and B(3)) and total aflatoxins (B(1), B(2), G(1), and G(2)) in various corn processed fractions. 92 batches of whole corn and derived dry-milled fractions (animal feed flour, flaking grits, corn flour and corn meal) and cooked and roasted cornflakes fractions were industrially obtained. Samples were analyzed for both groups of mycotoxins by enzyme-linked immunosorbent assay (ELISA). Dry milling of corn led to a heterogeneous distribution of the two groups of mycotoxins in the different parts of the grain, with increased levels in fractions processed from outer layers (animal feed flour and corn flour) and decreased levels in fractions processed from inner portions, such as corn meal and flaking grits. Levels of fumonisins in cornflakes were lower than 400 microg/kg, the maximum tolerable limit set by the EU. By contrast, three samples of final product were found to exceed the aflatoxin maximum tolerable limit of 4 microg/kg. Animal feed flour showed concentration factors of 317 and 288% for fumonisins and aflatoxins, respectively. Food traceability system was used by the industrial companies which processed corn into breakfast cereals. Nevertheless, even though the use of food traceability, which is defined as the ability to trace any food, feed, food-producing animal or substance that will be used for consumption through all stages of production, processing and distribution, only initial fumonisin contamination of whole corn and contamination of animal feed flour and corn flour were found to be correlated.

Colonization of Maize Grain by Fusarium moniliforme and Fusarium proliferatum in the Presence of Competing Fungi and Their Impact on Fumonisin Production

This study was carried out to determine the effect of water activity (aw) and temperature on the patterns of colonization of maize grain by isolates of Fusarium moniliforme and F. proliferatum in the presence of interacting spoilage fungi, such as Aspergillus flavus, A. niger, A. ochraceus, and Penicillium implicatum, over 4-week incubation periods. The impact that such interactions have on Fusarium infection of maize grain and populations and on the production of fumonisins were all evaluated. At 0.93 and 0.95 aw, interactions of the two Fusarium spp. with other species to a large extent resulted in mutual inhibition on contact or overgrowth by the Aspergillus and Penicillium spp., regardless of temperature and growth rates of the species. Seed infection by the Fusarium spp. at 25 degrees C was influenced mainly by A. flavus and A. niger, with a reduction of infection by up to 45 and 30%, respectively, after 2 weeks. At 15 degrees C and lowered aw levels, infection was reduced, and sometimes completely inhibited, by A. niger, A. ochraceus, and P. implicatum. By contrast, populations of the Fusarium spp. (CFU per gram grain) were reduced mainly by A. flavus and P. implicatum. A. niger reduced populations of the Fusarium spp. at 0.93 and 0.95 aw, particularly after 4 weeks of incubation. Interaction with A. niger, A. ochraceus, and A. flavus at 15 and 25 degrees C and 0.98 aw resulted in a significant stimulation in fumonisin production by both Fusarium spp. Interaction with P. implicatum resulted in a decrease in fumonisin production by F. moniliforme. However, interaction with F. proliferatum resulted in an enhanced production at both temperatures and 0.95 and 0.98 aw. This study has shown that fungal interactions may act as an additional control factor in Fusarium spp. development on grain. However, although interactions lead to a decreased colonization by Fusarium, mainly at lower aw, a decrease in fumonisin production accumulation does not occur at the same time. Knowledge of these interactions is essential for improving effective control procedures for preventing growth and mycotoxin production by such fungi.

Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain).

BACKGROUND Aflatoxin M₁ (AFM₁) is the main monohydroxylated derivative of aflatoxin B₁ (AFB₁) formed in liver and excreted into milk. Although AFM₁ is less toxic than AFB₁, it has been classified as a possible human carcinogen, Group 2B agent by International Agency for Research on Cancer (IARC). OBJECTIVES The objectives of this study were (i) to determine the occurrence of AFM₁ in the main dairy products consumed in Catalonia region (Spain), and (ii) to assess the exposure of Catalonian population to aflatoxin M₁ through deterministic and probabilistic method. METHODS Occurrence of Aflatoxin M₁ (AFM₁) was determined in 72 composites of milk, 72 composites of cheese and 72 composites of yoghurt from Catalonia. AFM₁ content was analysed using an Enzyme-Linked ImmunoSorbent Assay commercial kit. Three approaches to exposure assessment were conducted: one deterministic method and two probabilistic models with Monte Carlo simulations. RESULTS AFM₁ was detected in 94.4% (68/72) of whole UHT milk samples, in 2.8% (2/72) of yoghurt samples and not detected in cheese. The maximum level was detected in one yoghurt sample with 51.58 ng/kg, only this sample being over the legal EU limit of 50 ng/kg. Milk, cheese and yoghurt mean concentrations were 9.29±2.61, <12.5 and 13.22±4.82 ng/kg, respectively. CONCLUSIONS According to these values, it should be expected Catalonian population is not exposed to a significant risk from aflatoxin M₁ including average and high consumers.

Survey: Ochratoxin A in European special wines

The occurrence of Ochratoxin A (OTA) was examined in 121 special wines made using different winemaking techniques and from many European origins. The wine groups with the highest OTA content and occurrence, above 90%, were those were the must was fortified before fermentation (mean: 4.48μg/l) and those made from grapes dried by means of sun exposure (mean: 2.77μg/l). Fortified wines with long aging in wooden casks were about 50% contaminated, with OTA levels below 1.00μg/l. Wines affected by noble rot, late harvest wines and ice wines did not contain OTA. Overall, 19.8% of the wines studied contained OTA levels above the maximum permissible limit for the European Union (2μg/kg) in wine (excluding liqueur wines).

Determination of aflatoxins, deoxynivalenol, ochratoxin A and zearalenone in wheat and oat based bran supplements sold in the Spanish market.

The aflatoxins (AFs), deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based raw materials. Usually, the higher mycotoxins concentrations in cereals are found in the external layers of the grain (bran). Nowadays bran is increasingly consumed for its high fibre concentration. The objectives of this study were determining the concentration of these mycotoxins in bran samples intended for direct human consumption and to study the influence of some characteristics of the samples that may affect the mycotoxins content, there are not studies about fibre for direct human consumption. 67 bran samples from shops and supermarkets from two different Spanish cities were analyzed, being 37 samples of wheat bran and the remaining of oat bran. The results showed a major presence of DON in the analyzed samples, with levels above the EU legislation in some samples. Presence of DON was more frequent in wheat samples, compared to oats ones (p<0.05). Extruded or toasted samples, subjected to a heat treatment during processing, presented a significantly lower concentration of OTA, and differences between the organically and conventionally produced samples were also detected in OTA, which showed higher levels in the organic samples. Co-occurrence was frequently found between the Fusarium mycotoxins (ZEA and DON). Due to the high levels of DON in the analyzed samples, a calculation of DON intake has been made and it has been demonstrated that bran can account for an important percentage of DON exposure in the total diet.

Modelling of growth of aflatoxigenic A. flavus isolates from red chilli powder as a function of water availability

The aim of the present work was to apply mathematical models for the prediction of growth of aflatoxigenic moulds in powdered Capsicum fruits as a function of its water availability. As prevention of fungal growth effectively conduces to prevention of mycotoxin accumulation, the development of models for prediction of growth of mycotoxigenic fungi becomes a key step in risk management. Two aflatoxigenic A. flavus from chilli powder were grown on 3% chilli powder extract agar at different water activity levels and their growth was evaluated over time in terms of colony sizes and ergosterol accumulation. Both variables were modelled over time, and the resulting parameters (growth rates and lag phases) were modelled as a function of water availability using the Rosso cardinal model. Linear logistic regression was also applied to predict the probability of growth over storage time. Both isolates showed a similar pattern of behaviour, with decreasing growth rates and increasing lag phases with decreasing water activity level. While estimation of optimum a(w) for growth was consistently around 0.97-0.99, the minimum estimated a(w) varied from 0.82 to 0.88 depending on the isolate and on the parameters used for predictions. Comparing growth rates obtained for colony size and ergosterol accumulation, a linear relationship between them could be observed. The rate of root square ergosterol/colony diameter/unit of time was 0.25-0.27. Probabilities of growth before 10 days over 90% were estimated at a(w) 0.91, while the safe period could be extended to more than 20 days (22-29 days) if water activity was decreased to an a(w)=0.87. Finally, the probability of growth is always under 50% when water availability is under a(w) 0.85, and almost null for A. flavus UdLTA 3.147. It was concluded that for safe production, storage and transport, chillies and chilli powder must be kept under 31% mc (db) (probability of growth <50%). However, growth is unlikely to occur if chilli is kept at approx. 34% for less than 10 days, or at approx. 33% for less than 20 days. Careful hazard analysis and critical control point (HACCP) techniques during raw material production and the subsequent stages of drying, transportation, elaboration and storage are indispensable.