Development of a microcantilever-based immunosensing method for mycotoxin detection.

Mycotoxins, such as aflatoxins and ochratoxin A, are presently considered as the most important chronic dietary risk factor, more than food additives or pesticide residues. Therefore, the serious health and economic consequences of mycotoxin contamination have created the need for rapid, sensitive, and reliable techniques to detect such dangerous molecules within foodstuffs. We here report on the development of an innovative immunosensing method for mycotoxin detection, based on antibody-immobilized microcantilever resonators, a promising label free biosensing technique. A considerable part of the work is devoted to show the effect on microcantilever resonance frequency of the composition of the incubation buffer, as well as of the washing and drying procedure. We show the feasibility of using microcantilever resonator arrays to effectively identify total aflatoxins and ochratoxin A, at low concentrations (3 ng/mL and less than 6 ng/mL, respectively), with relatively low uncertainty (about 10%) and good reproducibility for the same target concentration. Furthermore, the developed immunosensing method shows a limited cross-reactivity to different mycotoxins, paving the way to a highly specific technique, able to identify different mycotoxins in the sample. To our knowledge, this work represents the first example in literature of successfully immunodetection of low concentrations of multiple mycotoxins by microcantilever resonator arrays.

Ochratoxigenic black species of Aspergilli in grape fruits of northern Italy identified by an improved PCR-RFLP procedure.

A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP) and tested for ochratoxin A (OTA) production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%), while A. carbonarius was the major species during 2007 (46.6%). All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES), as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7%) isolates produced OTA ranging from 0.05 to 3.0 µg mL(-1). Most of the ochratoxigenic isolates were A. carbonarius (46) in both years, but also some strains of A. tubingensis (11) and A. japonicus (6) produced lower amounts of OTA.

Potential of patulin production by Penicillium expansum strains on various fruits.

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100-13,200 µg/kg and nine strains ranging from 100-12,100 µg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 µg/kg of rotten fruit) and on apples (12,500 µg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 µg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 µg/kg) at 25°C and 10,300 µg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 µg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.

Low levels of ochratocin A in wines from Piedmont.

Ochratoxin A (OTA) is a mycotoxin mainly produced by a number of species of Aspergillus, commonly found in warm and tropical climates. OTA poses risks for the human health because of its nephrotoxic, teratogenic, immunotoxic and neurotoxic activity. The mycotoxin, classified as possible human carcinogen (Group 2B) by the IARC, naturally occurs in a wide range of foods, including wine, where the main producer is A. carbonarius. The aim of this work was the validation of a procedure for the analysis of OTA in Piedmontese red and white wines produced after vintage 2003 and 2004, in relationship with the limit of 2.0 microg l(-1) introduced by European Union for wine, must or grape juice (Regulation CE N. 123/2005). An analytical method based on immunoaffinity column (IAC) for clean-up and liquid chromatography with fluorescence detection (LC-FLD) was used to determine the occurrence of OTA in wines. Detection limit (LOD) and quantification Limit (LOQ) were 7.18 pg/ml and 9.31 pg/ml based on statistical method (IUPAC). Average recoveries of OTA from wine samples spiked at levels from 0.1 to 10 ng/ml ranged from 90.8% to 92.4%, with relative standard deviations (RSDs) between 2.64 and 2.71%. Repeatability limit was 8.73 pg/ml for samples spiked with 0.1 ng/ml of OTA. Ninety-one Denomination of Controlled Origin (DOC) wines were analysed, including 41 Barbera (red), 38 Dolcetto (red), and 16 white wines, such as Erbaluce, Cortese and Roero Arneis. The study focused on wines commercialized in Italian supermarkets and wine shops. The white wines resulted, as expected, less contaminated than the red ones. Wines produced after vintage 2003, a season particularly conducive to the growth of A. carbonorius, contained higher levels of OTA than the wines produced in 2004. The samples, resulting positive, contained a concentration of OTA highly inferior to the threshold limits introduced by the European Union. The sample of the highest level of OTA was a Dolcetto produced in 2004, with 1.10 ng/ml of mycotoxin.