c) AF (A. flavus in the absence of yeasts, control batch). The three batches have been stored at 25 C, and sampling was carried out at 3, 7, 9, 10, 11, 12, 15 and 21 days of incubation. Growth parameters, aflR gene expression and aflatoxin production had been determined on every single sampling day. The assay was carried out twice, and three replicates had been performed for each repetition. 4.four. Evaluation of Volatile Compounds Extraction and analysis of VOCs produced by the two yeast strains in the presence and absence from the filamentous fungus were conducted as described by Ruiz-Moyano et al. [41]. These volatile compounds have been extracted by utilizing a 10-mm long, 75- thick fiber coated with carboxen/polydimethylsiloxane in the space of every single DDS by solid-phase microextraction (SPME) (Supelco, Bellefonte, PA, USA). The origin of volatile compounds from PDA in addition to a. flavus was assigned by extraction and analyses of batch AF. Following volatile compound extraction, analyses were conducted by gas chromatography mass Trk Storage & Stability spectrometry (GC/MS) using an Agilent 6890 GC-5973 MS program (Agilent Technologies, Tiny Falls, DE, USA) MMP Purity & Documentation equipped using a 5 phenyl-95 polydimethylsiloxane column (30 m 0.32 mm inner diameter, 1.05 film thickness, Hewlett-Packard). The Kovats index of the compounds was calculated by analysis of n-alkanes (R-8769, Sigma Chemical Co., St. Louis, MO, USA) run below the same conditions as the samples. The NIST/EPA/NIH mass spectrum library (comparison top quality 90 ) and Kovats index were utilized to recognize the volatile compounds developed by the two yeast strains. In addition, the identity of particular compounds was confirmed by a comparison with the retention time and MS spectra, utilizing a laboratory-built MS spectral database, obtained from chromatographic runs of pure compounds performed below exactly the same experimental situations by using the identical equipment. Quantitative data were obtained in the total ion current chromatograms by integration on the GC peak locations. The volatile compounds connected with yeast strains in batches AF + L479 and AF + L793 had been determined by comparison of volatile compounds identified in such batches with these encountered in a PDA manage with no yeast inocula and in batch AF (batch manage inoculated only using a. flavus). The production of those volatile compounds that were not detected in each handle PDA and PDA inoculated having a. flavus (batch AF), or those whose relative abundances have been drastically decrease than those encountered in yeast-inoculated batches (AF + L479 and AF + L793), was exclusively linked to the strains H. opuntiae L479 and H. uvarum L793 based on the methodology described in Ruiz-Moyano et al. [41]. 4.5. Determination of Growth Parameters of Aspergillus Flavus The diameter from the A. flavus colony was measured in two perpendicular directions and recorded on every sampling day. Development curves were obtained by graphical representation from the mycelium diameter (mm) against the incubation occasions (days). Data plots showed, right after a lag phase, a linear trend with time; consequently, a linear model was applied. The development rate ( mm/day) was determined in the slope with the growth curve throughout the linear phase of development. The lag phase (; days) was determined from the linear regression equation equaling the regression line formula to the original inoculum size (diameter, mm) in accordance with Le et al. [55].Toxins 2021, 13,13 of4.six. Relative Quantification in the Expression in the aflR Gene 4.6.1. Sample Preparation Soon after every single incubat
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