Food poisoning, causing around 420,000 deaths annually according to the WHO, is a significant global health concern. *Staphylococcus aureus*, a prevalent foodborne pathogen, poses a substantial risk due to its ability to survive in diverse environments. The use of synthetic antimicrobials raises ecological and health concerns, prompting research into natural alternatives. Plant extracts, particularly those rich in phenols and flavonoids, show promise as safe and effective antimicrobial agents. Chinese vine tea (*Ampelopsis grossedentata*) and its major flavonoid, dihydromyricetin (DMY), have demonstrated antibacterial, antioxidant, and antihypertensive effects. While previous studies have shown their antibacterial activity against *S. aureus*, a comprehensive understanding of their mechanism of action remains elusive. This study aims to elucidate the antibacterial mechanism of VTE and DMY against *S. aureus*, providing a theoretical foundation for their application as food preservatives.

Numerous studies highlight the antibacterial activities of plant extracts, particularly phenols and flavonoids. The use of plant-derived compounds as natural antimicrobials is gaining traction due to concerns about synthetic alternatives. Chinese vine tea (*Ampelopsis grossedentata*) has been recognized for its various health benefits, including antibacterial properties. Dihydromyricetin (DMY), a major component of vine tea, has been shown to possess various biological activities, and previous research, notably by Xiao et al. (2019), indicated the antibacterial effect of DMY against foodborne bacteria. However, detailed mechanistic studies are lacking, hindering wider application. This research builds upon existing knowledge of VTE and DMY's antibacterial properties by systematically investigating their mechanism of action against *S. aureus*.

This study employed a multi-faceted approach to investigate the antibacterial mechanism of VTE and DMY against *Staphylococcus aureus* ATCC 6538. VTE was prepared by extracting dried vine tea leaves with deionized water at 100°C. DMY (purity ≥90%) was sourced commercially. The minimum inhibitory concentration (MIC) of VTE and DMY was determined using a broth dilution method. Bacterial growth curves were established by measuring optical density (OD540) at various time points. Morphological changes were assessed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane integrity was evaluated by measuring the leakage of alkaline phosphatase (AKPase) and β-galactosidase. Intracellular protein expression was analyzed using SDS-PAGE, and the activities of key energy metabolism enzymes (succinate dehydrogenase, malate dehydrogenase, and total ATPase) were measured. DNA binding was studied using UV and fluorescence spectroscopy, and competitive binding assays were performed using ethidium bromide (EB) and 4',6-diamidino-2-phenylindole (DAPI). Finally, the antibacterial efficacy of VTE and DMY was assessed in Chinese cabbage and barley food systems over nine days. Statistical analysis was performed using one-way ANOVA.

The MIC of VTE and DMY against *S. aureus* were determined to be 6.3 mg/mL and 1.25 mg/mL, respectively. SEM and TEM analyses revealed significant morphological changes in *S. aureus* cells treated with VTE and DMY, including surface irregularities, cell wall damage, and cytoplasmic alterations. Leakage of AKPase (VTE: +21.8%, DMY: +10.3%) and β-galactosidase (VTE: +90.2%, DMY: +57.4%) indicated disruption of membrane integrity. Both VTE and DMY significantly decreased total protein expression (VTE: -15.5%, DMY: -9.9%) and reduced the activities of key energy metabolism enzymes: MDH (VTE: -12.7%, DMY: -4.7%), SDH (VTE: -65.8%, DMY: -16.7%), and total ATPase (VTE: -31.5%, DMY: -15.9%). UV and fluorescence spectroscopy studies demonstrated interactions between VTE/DMY and DNA through both intercalation and groove binding. Competitive binding assays confirmed that VTE and DMY competed with EB and DAPI for DNA binding sites. In food model systems, both VTE and DMY effectively inhibited *S. aureus* growth, with complete inhibition observed at 1xMIC within six days.

This study demonstrates that both VTE and DMY effectively inhibit the growth of *S. aureus* through a multifaceted mechanism. The observed morphological changes, membrane disruption, protein synthesis inhibition, and effects on energy metabolism enzymes suggest that VTE and DMY target multiple bacterial cellular processes. The interaction with DNA further suggests a mechanism interfering with genetic processes. The stronger effect of VTE compared to DMY might be attributed to the presence of other bioactive compounds in VTE besides DMY. The findings corroborate previous research on the antibacterial effects of flavonoids, particularly the role of hydroxyl groups in forming hydrogen bonds with DNA bases. The efficacy of VTE and DMY in food model systems suggests their significant potential as natural food preservatives.

This research has successfully elucidated the mechanism of antibacterial action of vine tea extract (VTE) and dihydromyricetin (DMY) against *Staphylococcus aureus*. Both substances exhibit similar mechanisms, targeting cell wall and membrane integrity, protein synthesis, energy metabolism, and DNA. VTE displays a more pronounced effect on membrane integrity and energy metabolism compared to DMY, potentially due to the presence of other compounds. The study supports the potential of VTE and DMY as safe and effective food preservatives. Further research could explore their application in other food products and examine the synergistic effects with other natural preservatives.

This study primarily focused on *in vitro* analyses. Further *in vivo* studies are necessary to confirm the findings and assess the safety and efficacy in real-world food preservation scenarios. The specific composition of VTE beyond DMY warrants further investigation to fully characterize its antibacterial effects. The study used a limited number of food models. Future research should consider a wider range of food matrices to assess the generalizability of the findings.