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Bread shelf life is generally compromised by fungi mainly belonging to Aspergillus and Penicillium genera, which colonise the surface of the product within few days from the production. The aim of this study was to select a Lactobacillus brevis-based bioingredient (LbBio) able to inhibit the growth of Aspergillus niger ITEM5132 on pan bread in order to prolong its shelf life. Four LbBio formulations, obtained by growing a selected L. brevis strain in a flour-based medium containing different carbon sources or acid precursors (fructose, LbBio1; fructose and maltose, LbBio2; α-chetoglutaric acid, LbBio3; short-chain fructooligosaccharides, LbBio4), were evaluated for their content of organic acids (lactic, acetic, propionic, phenyllactic, 4-hydroxy-phenyllactic, valeric, isovaleric acids). The LbBio formulations were applied in yeast-leavened bread during bread-making trials and the resulting products were inoculated after baking with A. niger spore’s suspension and the fungal growth was monitored during storage (25°C for 6 days). The formulation showing the highest inhibitory activity was separated by ultra-filtration method, and whole and fractions obtained were evaluated for their in vitro activity. The fraction showing the highest activity was further separated by gel-filtration and the resulting products were investigated for their protein content and in vitro inhibition. The results from the bread-making trials performed using different formulations of LbBio showed a delay in fungal growth (1 day) respect to the bread not containing the bioingredient (control) or including calcium propionate (0.3% w/w). The formulation LbBio2, prepared with fructose and maltose 1% (w/vol), contained the highest amount of total organic acids, including phenyllactic and hydroxyl-phenyllactic acids, and reduced the visual spoilage of bread. This formulation was separated by ultra-filtration and fractions containing metabolites with molecular weight higher than 30 kDa showed high inhibitory effect in the in vitro assay. In particular, the microfluidic analysis highlighted the presence of a protein with a molecular weight of 56 kDa only in the active fraction. Further studies have to be done in order to identify the protein involved in the antifungal activity.
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