Diversity and detection of microfungi and their mycotoxins in silage

Ensiling maize represents a very important agricultural activity leading to prolonged preservation of the crop. However, when the (anaerobic and low pH) conditions necessary for obtaining maize silage of an excellent quality are disrupted, mould growth can occur. The presence of moulds will lead to a reduction of the nutritional value of this feed commodity, but can also lead to the production of secondary metabolites or mycotoxins. These toxins can be produced during post-harvest stages as well as in the field (pre-harvest). Mycotoxins can cause both profound and hidden symptoms influencing growth, production, fertility, digestion, the immune system and other. As a result, they can negatively influence animal production/welfare/health. Silage commodities are mostly contaminated with several mycotoxins, resulting in a possible increased (synergetic) health risk. The presence of moulds and mycotoxins in silage commodities has important economic implications due to for example feed ration loss, decreased animal production and veterinary costs. In some cases, certain of these toxins or their metabolites can be transmitted into animal-derived products such as meat and milk. This research focused on the development of a methodology for the detection, identification and quantification of moulds and mycotoxins contaminating maize silage. Within this framework, finding a suitable medium for isolating and enumerating fungal contaminants in silage was aimed at. Because conventional plating techniques are often time-consuming and expertise-demanding, more objective, genotype-based methods were also used for molecularly identifying and characterizing the mould species in different Belgian silage samples. As information on the mycotoxin production of a fungal isolate can be used as an additional confirmation of its identity, an Ultra High Performance Liquid Chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed for the simultaneous detection of over twenty mycotoxins from Yeast Extract Sucrose (YES) agar, a growth medium suitable for inducing secondary metabolite production by fungal isolates. Finally, an UHPLC-MS/MS method for the simultaneous detection of 27 mycotoxins from maize silage was developed and validated. In conclusion, combining different techniques available for mould and mycotoxin detection/identification, i.e., a multidisciplinary or polyphasic research approach, will result in more reliable results which are important in view of protecting animal health/welfare

Ultrahigh-performance liquid chromatographic-tandem mass spectrometric multimycotoxin method for quantitating 26 mycotoxins in maize silage

A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5-348 and 11-695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3-45 and 5-49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well

Optimization and validation of a method without alkaline clean-up for patulin analysis on apple puree agar medium (APAM) and apple products

A sensitive high-performance liquid chromatography-UV (HPLC-UV) method, based on the Association of Analytical Communities (AOAC) Official method 2000.02, was developed and validated for the high-throughput analysis of patulin in in vitro experiments on apple puree agar medium (APAM). The importance of repeating the ethyl acetate extraction step at liquid-liquid extraction (LLE) was examined, as well as the extent of patulin degradation during the sodium carbonate clean-up. In addition to this alkaline clean-up, the efficiency of using an Oasis HLB or C-18 cartridge as solid-phase extraction (SPE) clean-up was compared. This resulted in a two-step ethyl acetate LLE, followed by an Oasis HLB SPE clean-up, without alkaline clean-up conditions. The method was fully validated for APAM, cloudy apple juice, and apple puree. Average patulin recoveries at levels of 100, 500, and 1000 mu g kg(-1) of APAM varied between 95 and 113 % over 3 independent days, with an interday precision (RSDR) of 5 to 10 %. Recovery experiments carried out with the spiked apple juice (at 50 mu g kg(-1)) and apple puree (10 mu g kg(-1)) showed average recovery rates laying between 80-101 % (RSDR = 12 %) and 77-100 % (RSDR = 9 %), respectively. This method offered a detection limit of 3-4 mu g kg(-1) and a quantification limit of 5-8 mu g kg(-1) for APAM, apple juice, and puree