Ambra Prelle

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.

A new method for detection of five alternaria toxins in food matrices based on LC–APCI-MS

A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and tenuazonic acid (TeA), five alternaria toxins (ATs) was developed by liquid chromatography-triple quadrupole mass spectrometry equipped with atmospheric pressure chemical ionisation (APCI). A single extraction was used to recover the five ATs by apple juices, beers, tomato sauces, olives and dried basil. Different Solid Phase Extractions (SPE) and clean-up were selected to optimise the purification step for each food matrix. Limits of detection and quantification were, respectively, in the range 0.16-12.31 and 0.54-41.04 ng g(-1). Recovery rates were generally above 70%, except for dried basil and olives. Thirty out of 70 samples analysed (7 apple juices, 14 beers and 9 tomato sauces) resulted positive to at least one alternaria toxin investigated. AOH was the most common AT (14 samples), followed by ALT (10 samples). The highest concentration of ATs was found in commercial apple juices (35.33 ng g(-1)).

Efficacy of plant essential oils on postharvest control of rots caused by fungi on different stone fruits in vivo

The antifungal activity of plant essential oils was evaluated as postharvest treatment on stone fruit against brown rot and grey mold rot of stone fruit caused by Monilinia laxa and Botrytis cinerea, respectively. The essential oils from basil (Ocimum basilicum), fennel (Foeniculum sativum), lavender (Lavandula officinalis), marjoram (Origanum majorana), oregano (Origanum vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), savory (Satureja montana), thyme (Thymus vulgaris), and wild mint (Mentha arvensis) were tested at two different concentrations on apricots (cv. Kyoto and cv. Tonda di Costigliole), nectarines (cv. Big Top and cv. Nectaross) and plums (cv. Italia and cv. TC Sun). The volatile composition of the essential oils tested was determined by gas chromatography-mass spectrometry analysis. The treatments containing essential oils from oregano, savory, and thyme at 1% (vol/vol) controlled both B. cinerea and M. laxa growing on apricots cv. Tonda di Costigliole and plums cv. Italia and cv. TC Sun; however, the same treatments were phytotoxic for the carposphere of nectarines cv. Big Top and cv. Nectaross. Treatments with 10% (vol/vol) essential oils were highly phytotoxic, notwithstanding their efficacy against the pathogens tested. The essential oils containing as major components α-pinene, p-cymene, carvacrol, and thymol showed similar results on stone fruit, so their antimicrobial activity and the phytotoxicity produced could be based on the concentration of their principal compounds and their synergistic activity. The efficacy of the essential oil treatments on control of fungal pathogens in postharvest depended on the fruit cultivar, the composition and concentration of the essential oil applied, and the length of storage.

High performance ion chromatography of haloacetic acids on macrocyclic cryptand anion exchanger

A new high performance ion chromatographic method has been developed for the separation of the nine chlorinated-brominated haloacetic acids (HAAs) that are the disinfection by-products of chlorination of drinking water, using a macrocycle-based adjustable-capacity anion-exchange separator column (IonPac Cryptand A1). A gradient method based on theoretical and experimental considerations has been optimized in which 10 mM NaOH-LiOH step gradient was performed at the third minute of the analysis. The optimized method allowed us to separate the nine HAAs and seven possibly interfering inorganic anions in less than 25 min with acceptable resolution. The minimum concentrations detectable for HAAs were between 8.0 (MBA) and 210 (TBA) microg L(-1), with linearity included between 0.9947 (TBA) and 0.9998 (MBA). To increase sensitivity, a 25-fold preconcentration step on a reversed phase substrate (LiChrolut EN) has been coupled. Application of this method to the analysis of haloacetic acids in real tap water samples is illustrated.

Comparison of Clean-Up Methods for Ochratoxin A on Wine, Beer, Roasted Coffee and Chili Commercialized in Italy

The most common technique used to detect ochratoxin A (OTA) in food matrices is based on extraction, clean-up, and chromatography detection. Different clean-up cartridges, such as immunoaffinity columns (IAC), molecular imprinting polymers (MIP), Mycosep™ 229, Mycospin™, and Oasis® HLB (Hydrophilic Lipophilic balance) as solid phase extraction were tested to optimize the purification for red wine, beer, roasted coffee and chili. Recovery, reproducibility, reproducibility, limit of detection (LOD) and limit of quantification (LOQ) were calculated for each clean-up method. IAC demonstrated to be suitable for OTA analysis in wine and beer with recovery rate >90%, as well as Mycosep™ for wine and chili. On the contrary, MIP columns were the most appropriate to clean up coffee. A total of 120 samples (30 wines, 30 beers, 30 roasted coffee, 30 chili) marketed in Italy were analyzed, by applying the developed clean-up methods. Twenty-seven out of 120 samples analyzed (22.7%: two wines, five beers, eight coffees, and 12 chili) resulted positive to OTA. A higher incidence of OTA was found in chili (40.0%) more than wine (6.6%), beers (16.6%) and coffee (26.6%). Moreover, OTA concentration in chili was the highest detected, reaching 47.8 µg/kg. Furthermore, three samples (2.5%), two wines and one chili, exceeded the European threshold.

Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N2, 0.1% O2 and 1% O2, 21% O2), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB1 of over 70% was obtained. Aflatoxins B1 and G1 were more sensitive to plasma treatments compared to aflatoxins B2 and G2, respectively. Under plasma treatment, aflatoxin B1 was more sensitive compared to aflatoxin G1. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.

Light affects fumonisin production in strains of Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides isolated from rice

Three Fusarium species associated with bakanae disease of rice (Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides) were investigated for their ability to produce fumonisins (FB1 and FB2) under different light conditions, and for pathogenicity. Compared to darkness, the conditions that highly stimulated fumonisin production were yellow and green light in F. verticillioides strains; white and blue light, and light/dark alternation in F. fujikuroi and F. proliferatum strains. In general, all light conditions positively influenced fumonisin production with respect to the dark. Expression of the FUM1 gene, which is necessary for the initiation of fumonisin production, was in accordance with the fumonisin biosynthetic profile. High and low fumonisin-producing F. fujikuroi strains showed typical symptoms of bakanae disease, abundant fumonisin-producing F. verticillioides strains exhibited chlorosis and stunting of rice plants, while fumonisin-producing F. proliferatum strains were asymptomatic on rice. We report that F. fujikuroi might be an abundant fumonisin producer with levels comparable to that of F. verticillioides and F. proliferatum, highlighting the need of deeper mycotoxicological analyses on rice isolates of F. fujikuroi. Our results showed for the first time the influence of light on fumonisin production in isolates of F. fujikuroi, F. proliferatum, and F. verticillioides from rice.

Aflatoxin monitoring in Italian hazelnut products by LC-MS

Hazelnut samples of different origin were collected in stores in Northern Italy and analysed for aflatoxin (AF) contamination by a sensitive chromatographic method on the basis of a tandem mass spectrometer with electrospray ionisation. The effects of two extracting solvent mixtures (methanol/water and acetonitrile/water) and different extraction times were tested and compared in terms of recovery. Analysis showed that 35 out of 93 samples (37.6%) were contaminated by AFs. The incidence of positive samples was higher in Turkish (66.7%) than in Italian samples (35.9%). Mean AF contamination was higher (p = 0.045, Kruskal–Wallis test) in the Turkish samples (0.33 µg kg–1) compared to the Italian (0.14 µg kg–1) ones. In both cases, however, a low level of total AF contamination was found in the positive samples (0.64 µg kg–1), both being far below the maximum limit set in European legislation.

Microcantilever Resonators for Ochratoxin A Detection in Food and Drinks

Mycotoxins food contamination represents a serious risk for consumers health. They are 12 secondary metabolites of fungi that can be present in a wide range of foodstuffs. Ochratoxin A 13 (OTA) is one of the most toxic compound and it is classified as a possible carcinogenic molecule. 14 The harmful effects of OTA on human and animal health lead to a big boost to develop and 15 optimize highly sensitive and accurate methods for OTA detection. An innovative and rapid 16 detection method based on microcantilever resonators for ochratoxin A identification in food 17 matrix has been developed. This work demonstrates the possibility to apply microcantilever 18 technology in food safety field, showing for the first time in literature the successful detection of 19 one of the most dangerous mycotoxin in different food matrixes both solids and liquids, such as 20 green coffee, grape juice and wine. Sensing performances are discussed in terms of calibration plot 21 and limit of detection. 22