Aspergillus niger, the predominant species within the Aspergillus genus, is ubiquitously distributed in the environment (Li et al., 2013). It is a well-known agent of food spoilage and deterioration in industrial and agricultural products, resulting in substantial economic losses (Sun et al., 2020; Wu et al., 2014). To address fungal contamination issues, synthetic chemical fungicides have traditionally been utilized in the food industry to control spoilage and inhibit microbial growth. Nevertheless, these synthetic fungicides exhibit several drawbacks, including the emergence of drug-resistant strains, carcinogenic potential, and the capacity for environmental pollution (Al-Maqtari et al., 2022). In light of growing consumer concerns regarding food safety, there is a growing interest in investigating natural fungicides as an alternative solution to conventional synthetic counterparts (Rao et al., 2019; Wang et al., 2022; Zhang et al., 2016a).

Cinnamaldehyde, the principal component of cinnamon essential oil derived from cinnamon bark, stands out for its remarkable antifungal properties, surpassing other constituents in efficacy (Huang et al., 2021; Wang et al., 2017). Notably, it has earned GRAS status and received approval from the US Food and Drug Administration (FDA) for use both as a food additive and an antimicrobial agent (Friedman, 2017b; Sun et al., 2020). Numerous studies have demonstrated cinnamaldehyde's favorable inhibitory effect on various fungi, including Aspergillus and Penicillium (Huang et al., 2021; Shreaz et al., 2016). Despite these findings, the precise molecular mechanisms governing cinnamaldehyde's impact on fungal cells remain only partially understood. Specifically, there exists a significant knowledge gap concerning the detailed molecular interactions between cinnamaldehyde and its target proteins (Rao et al., 2019; Yang et al., 2022). Hence, it is imperative to further elucidate the comprehensive molecular mechanism underlying cinnamaldehyde's antifungal activity against Aspergillus niger.

Activity-based protein profiling (ABPP) represents a chemoproteomics methodology leveraging active site-directed covalent probes to identify target proteins of bioactive natural products in complex proteomes. This approach unravels the intricate interaction mechanisms between bioactive natural products and their target proteins (Chen et al., 2021; Deng et al., 2020; Zhang et al., 2021). Cinnamaldehyde, a molecule bearing two electrophilic reaction sites, namely the conjugated double bond and the aldehyde group, readily interacts with nucleophilic residues in proteins, particularly cysteine (Friedman, 2017b). Notably, the acrolein moiety (α, β-unsaturated carbonyl portion) plays a pivotal role in cinnamaldehyde's antifungal activity (Shreaz et al., 2016; Vasconcelos et al., 2018). Our working hypothesis posited that cinnamaldehyde primarily exerts its inhibitory effect through covalent binding (Michael addition), involving the β‑carbon on the conjugated double bond and nucleophilic residues of the target protein.

To investigate this hypothesis, a cinnamaldehyde-derived activity probe named Cin-P was designed and synthesized. Subsequently, we employed a diverse array of proteomic techniques to profile the target proteins of cinnamaldehyde in Aspergillus niger cells. Furthermore, our investigation delved into cinnamaldehyde's impact on the cell membrane, mitochondrial malate dehydrogenase activity, and intracellular ATP levels within Aspergillus niger cells. This study represents a pivotal step towards unraveling the molecular-level antifungal mechanism of cinnamaldehyde using chemoproteomics approaches. These findings offer valuable insights that can be harnessed for the continued development and strategic utilization of cinnamaldehyde in the realm of food spoilage prevention and mitigation.