The prevention of fungal spoilage of bread without addition of chemical preservatives remains an unsolved issue. This work aimed to identify alternative strategies to chemical preservatives in order to prevent or delay fungal spoilage of bread. The minimum inhibitory concentration (MIC) of bacterial metabolites and chemical preservatives was evaluated in vitro and in situ in baking experiments with addition of preservatives or metabolites. Calcium propionate, sorbic acid, phenyl lactate, ricinoleic acid, and acetic acid were tested both individually and in combination at their MIC values against A. niger and P. roqueforti (12, 0.4, 50, 3.4, 25 mM, respectively). The combination of acetic acid with propionate and sorbate decreased the MIC values against the tested moulds. For these reasons, we introduced sourdough fermentation with defined strains of lactobacilli in order to generate natural acetate in bread. A combination of Lactobacillus hammesii and propionate reduced propionate concentration required for shelf life extension of wheat bread 7 fold. Flaxseed sourdough bread fermented with L. hammesii, showed a shelf life 2 days longer than the control bread. The organic acid quantification indicated a higher production of acetic acid (33.81 \ub1 4.43 mM) when compared to other sourdough breads. When 4% of sucrose was added to sourdough fermentation with L. brevis, the longest mould free shelf-life (4 days longer then the control) was obtained. In conclusion, the in vitro MIC of bacterial metabolites and preservatives matches the in situ antifungal effect. Of the different bacterial metabolites evaluated, acetic acid has the most prominent and consistent antifungal activity and the use of sourdough fermentation with selected strains able to produce acetic acid allows reduced use of chemical preservatives
