Effect of sulfur dioxide and ethanol concentration on fungal profile and ochratoxin a production by Aspergillus carbonarius during wine making

Fungal profiles and ochratoxin A (OTA) accumulation during wine making were investigated using five different wine grape cultivars, Cabernet Sauvignon, Pinot Noir, Merlot, Syrah and Petit Verdot and the intrinsic influences caused by sulfur dioxide, ethanol and combine effect of ethanol and reducing sugar were analyzed using Cabernet Sauvignon and inoculation of Aspergillus carbonarius. Aspergillus spp. and Penicillium spp. were found as the major fungi in all winemaking processes and were highly correlated with OTA accumulation in wine. Most fungi died and OTA production decreased after 48 h of alcoholic fermentation, being consistent with the period when ethanol accumulation increased. The addition of SO₂ significantly inhibited the growth and OTA production of A. carbonarius with complete inhibition at 500 mg/L. When the ethanol concentration in the must increased to the range of 2–4%, growth and OTA production of A. carbonarius were significantly inhibited. Reducing sugar concentration had no significant effect on the growth and OTA production of A. carbonarius within the levels changing during the winemaking. Therefore, the increase of ethanol concentration played an important role in causing the decrease of fungal contamination and OTA accumulation during winemaking.     

The incidence and distribution of ochratoxin A in Daqu,a Chinese traditional fermentation starter

Daqu, a traditional starter culture mainly used to produce Chinese liquor and vinegar, is spontaneously fermented by diverse bacteria, yeasts and filamentous fungi under thermophilic condition. Therefore, mycotoxins may exist in Daqu, resulting in the contamination of end-foods. Ochratoxin A (OTA), a mycotoxin produced by certain species of Aspergillus and Penicillium, is not known whether existing in Daqu. However, specific method to detect OTA as well as OTA occurrence in Daqu has not been reported so far. With this in mind, a new method was developed to detect OTA in Daqu by the combination of ultrasound-assisted solid-liquid extraction (USLE), solid phase extraction (SPE) cleanup and UPLC-MS/MS. The USLE conditions of OTA from Daqu were optimized using Plackett-Burman (PB) design coupled with Box-Behnken (BB) design. Under the optimized conditions, no matrix effects were found, and the external standard method can be used to determine OTA in Daqu. The recoveries for spiked samples were 87–106% with the relative standard deviations (RSD) < 15%. The limits of detection and quantification were respectively 0.33 and 0.41 ppb. This approach was then applied to analyze 133 Daqu samples from different geographical regions in China, including 26 low temperature-, 33 medium temperature- and 74 high temperature-type Daqu. The results showed that OTA was detected in 66 samples with a maximum concentration of 28.87 ppb in low temperature Daqu, and the OTA incidence was on increase in the order of high temperature-, medium temperature- and low temperature-type Daqu. This implied that fermentation temperature is the key factor influencing OTA occurrence in Daqu. Moreover, there may be some fungi possessing the biosynthesis ability of OTA under high temperature environment (more than 45 °C).     

Identification of antifungal peptides produced by Lactobacillus plantarum IS10 grown in the MRS broth

The use of secondary metabolites of lactic acid bacteria for preservation of foods is increasingly gaining interest to the food industry to replace synthetic preservatives. In this study, the cell free supernatant containing peptides obtained from Lactobacillus plantarum IS10 was fractionated by size exclusion chromatography using sephadex G-25, and tested against Aspergillus flavus MD3, Penicillium roqueforti MD4 and Eurotium rubrum MD5. Among the fractions, fraction number 10 showed 60% antifungal activity at a concentration of 0.02 mg peptide/mL. Four novel peptides out of twenty peptides obtained from fraction 10 were identified and determined by de novo sequencing. Peptide FPSHTGMSVPPP with a net charge +1, hydrophobicity ratio 58% and molecular weight of 1253 was further studied. The selected peptide showed a good activity at a concentration of 5 mg/mL against selected fungi and poor activity at low concentrations. This work indicates that L. plantarum IS10 has the capability of producing peptides which are affective against spoilage fungi.     

Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution

The immunoaffinity column cleanup method coupled with HPLC separation and fluorescence detection is still one of the most frequently used quantitative methods for routine analyses of AFM1. In this study, aflatoxin M1 (AFM1) was quantified with a chromatography column packed with 2.6 μm core-shell particles on a conventional HPLC. A large volume of solvent (100 μL) was injected into the highly efficient column without any noticeable reduction in separation performance with the help of stepwise gradient elution. The instrumental conditions were optimized by response surface analysis methodology (RSM) with a three-level three-factor Box–Behnken design.The use of core-shell columns on conventional HPLC for AFM1 analysis under the optimized instrumental conditions leads to increased analytical performance compared with traditional totally porous columns without the heavy costs associated ultra-high-pressure instruments. Moreover, the improved instrumental sensitivity enables simplified sample preparation by avoiding any solvent replacement. The method could be easily applied to enhance the sensitivity of HPLC-FLD for AFM1 analysis that is based on isocratic elution and is more widely used. The method was successfully applied to 40 raw milk samples collected in summer from 20 cattle ranches located in two different provinces in southwestern China.     

Co-occurrence of aflatoxins and ochratoxin A in cereal flours commercialised in Turkey

In this study, we aim to determine co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in cereal flours commercialised in Corum, Turkey. One hundred cereal flours were checked for target fungal metabolites between the years 2011 and 2013. The samples were analysed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after immunoaffinity column (IAC) clean-up procedure. The method was successfully validated in accordance to European Union guidelines acceptance criteria for specificity, linearity, sensitivity, trueness and repeatability. All the results are well below the maximum limits specified in the EU legislation. AFs were detected neither wheat flour nor rice flour samples, while 66.7% of maize flours contained AFs with maximum concentration of 1.12 μg kg⁻¹. OTA was present in 26.7% of wheat flour, 41.7% of maize flour and 18.8% of rice flour samples, with mean levels of 0.247, 0.218 and 0.154 μg kg⁻¹, respectively. The co-occurence of AFs and OTA was found in 9 maize flour samples.