Synergistic inhibition effect of anisaldehyde and cinnamaldehyde (SAC) against Aspergillus species (A. flavus & A. niger) in vitro and on bread

To delay the spoilage of bread and reduce higher costs associated with using plant-derived compounds as natural bacteriostatic agents, synergistic antifungal effects were studied against the main bread spoilage fungi, Aspergillus niger and Aspergillus flavus. Among six combinations tested using a checkerboard method with four natural agents, only one combination of anisaldehyde and cinnamaldehyde (SAC) exhibits synergistic antifungal effects against both fungi. The combined minimum inhibitory concentration of SAC decreased to 75% compared to the individual minimum inhibitory concentration of both essential oil's component. SAC inhibits spore germination by up to 99% and 98% against both fungi, respectively, at 2 MIC. At this concentration, SAC completely inhibits the mycelium growth of both fungi, resulting in severe morphological alternations observed via SEM. Moreover, SAC disrupts cell membrane integrity by increasing permeability, intracellular leakage, fluidity, and saturated fatty acid content. It also reduces intracellular protein concentration and ergosterol content, affecting cell membrane composition. SAC also disrupted energy metabolism by decreasing ATP concentration and ATPase activity. Additionally, SAC decreases the activity of chitin synthase and Beta 1,3-glucan synthase, compromising cell wall integrity. In vivo application of SAC at 2 MIC significantly increased the shelf life of bread by 15 days, demonstrating its potential as a natural additive against bread spoilage caused by A. flavus and A. niger.