Ota standard

The rice sample (Sadri Hashemi variety) was purchased from retail markets in Mazandaran. OTA standard was obtained from Sigma (St. Louis, MO). Acetonitrile and HPLC‐grade methanol were purchased from Merck (Darmstadt, Germany). OchraTest clean‐up step immunoaffinity chromatography columns (IACs) were supplied by Vicam (Neogen Europe, Scotland, UK). Phosphate‐buffered saline (PBS) was obtained from Panreac (Panreac Química SA, Spain).

OTA quantification was performed in Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) equipment coupled to a fluorescence (FLD) detector (Agilent Technologies, Santa Clara, CA, USA). The column Zorbax SB C18 (2.1 mm × 50 cm × 1.8 µm; Agilent Technologies, Santa Clara, CA, USA) was used. The mobile phase was acetonitrile:water:acetic acid (50:100:1 v/v/v) with a flow rate of 0.1 mL/min. FLD detection was performed using 330 and 450 nm excitation and emission wavelengths, respectively. The calibration curve of OTA by HPLC-FLD revealed a linear relationship (r² ≥ 0.99) between detector response and OTA standard (Sigma-Aldrich Co., San Luis, MO, USA) amounts between 0.5 and 50 ng/mL. The limit of detection (LOD) obtained in this study was 0.58 µg/kg, and the quantification limit (LOQ) was 1.94 µg/kg.

The OTA standard and the U-[¹³C₂₀]-OTA standard (internal standard, IS) used to prepare standard solutions for the validation of the analytical method were purchased from Sigma Aldrich (St Louis, MO, USA) and Or Sell (Limidi di Soliera (MO), Italy), respectively. Acetonitrile (LC-MS grade) and formic acid (LC-MS grade), methanol (LC-MS grade), n-hexane (analytical grade), and acetic acid (glacial) were purchased from Carlo Erba Reagents (Cornaredo, MI, Italy), Honeywell (Charlotte, NC, USA), Panreac Applichem (Barcellona, Spain), and Sigma Aldrich (St Louis, MO, USA), respectively. Ultrapure water used throughout the experiments was produced by an Evoqua Water Technologies system (Pittsburgh, PA, USA). Ochraprep^® immunoaffinity columns from R-Biopharm AG (Darmstadt, Germany) were used for samples purification.

The chemicals and solvents used for OTA extraction and for HPLC analysis were analytical grade or HPLC grade. The OTA standard used to prepare standard solutions for the validation of the applied methodology was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Immunoaffinity columns used for samples purification (OchraTest™ WB) were purchased from Vicam® (Milford, MA, USA). PBS buffer pH 7.0 used during sample clean up procedure was prepared by dissolving sodium chloride (8.0 g), sodium phosphate dibasic (1.2 g), potassium phosphate monobasic (0.2 g), and potassium chloride (0.2 g) in purified water so as to obtain 1 litre of buffer. Water, acetonitrile, isopropyl alcohol, dichloromethane, and methanol were purchased from Mallinckrodt Baker B.V. (Deventer, The Netherlands); citric acid, phosphoric acid, sodium bicarbonate, sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and potassium chloride were purchased from Carlo Erba Reagents (Cornaredo, MI, Italy); acetic acid and ethyl acetate were obtained from Sigma-Aldrich Co. (St Louis, MO, USA).

In order to determine the levels of OTA produced by the strains, three pieces of the grape-based medium were removed from the internal, middle and external areas of each colony during 21 days of incubation at 25 °C (day 3, 6, 15 and 21). The reagents used were all of high purity or HPLC grade (99.9%). For extraction, 1 mL of methanol (Sigma-Aldrich) was added, followed by vortex homogenization for 5 seconds and incubation at room temperature for 60 minutes. The extracts were filtered in PVDF - Polyvinylidene Fluoride (0.22 μm) filter units (Millipore), according to the methodology proposed by Bragulat, Abarca, and Cabañes^{60 (link)} and then submitted for quantification. The standard curve was prepared using a stock solution previously prepared by dissolving the commercial OTA standard (Sigma-Aldrich) in methanol (1 μg/mL). Subsequently, standard solutions with concentrations of 3.75; 15.0; 30.0; 105.0 and 135.0 ng/mL were prepared by dilution. For the recovery assay, the grape-based culture medium was spiked at three levels with concentrations equal to 1.0 μg/g; 3.0 μg/g and 6.0 μg/g, in triplicates. The results of the recovery trials were 98.32% (±12.29), 97.23% (±7.07), and 97.47% % (±7.82), respectively.

The isolate LAMAI 31 was grown on Yeast extract 15 % sucrose agar (YESA) at 25 °C for 7 days and evaluated for the production of OTA by the agar plug technique, which tests small samples from Petri dishes by thin layer chromatography (TLC) (Filtenborg et al. 1983 (link)). The TLC plates were developed in toluene/ethyl acetate/formic acid (5:4:1) and visualized under UV light at 365 nm. An OTA standard (Sigma Chemical Co., St Louis, USA) and A. carbonarius producing OTA were used for comparison.