Mechanism and antibacterial activity of vine tea extract and dihydromyricetin against *Staphylococcus aureus*

Foodborne illness remains a global health issue, with Staphylococcus aureus being a major foodborne pathogen capable of surviving in diverse, often harsh environments. To mitigate contamination, safe and effective antimicrobials are needed, with growing interest in natural plant-derived agents due to concerns about synthetic preservatives. Vine tea (Ampelopsis grossedentata) is widely used in China, and its extracts (VTE) and major flavonoid dihydromyricetin (DMY) have been reported to possess antibacterial, antioxidant, and other bioactivities. Prior work suggested that DMY can extend food shelf life in combination with traditional preservatives, and that VTE/DMY have inhibitory effects against S. aureus, but their antibacterial mechanisms are not clearly defined. This study aims to systematically evaluate the antibacterial mechanisms of VTE and DMY against S. aureus, including effects on growth dynamics, cell morphology, membrane/wall integrity, protein synthesis and metabolic enzymes, and interactions with bacterial genomic DNA, as well as their antibacterial performance in model food systems.

The study situates VTE and DMY within the broader context of plant-derived antimicrobials, particularly flavonoids, which have documented antibacterial activity (e.g., apigenin against S. aureus). DMY is highlighted as a predominant flavonoid in vine tea (content ranging from ~20% to 93.1% depending on extraction) with reported antibacterial effects and synergy with conventional preservatives. Prior studies indicated DMY can damage bacterial cells (e.g., Vibrio parahaemolyticus) and bind DNA grooves in S. aureus, suggesting potential multi-target antibacterial mechanisms relevant to food preservation.

Materials: S. aureus ATCC 6538 was used. Vine tea leaves (Ampelopsis grossedentata) were collected (Zhangjiajie, Hunan, China; May 2019), fermented, dried, and extracted with deionized water (10 g in 100 mL at 100 °C for 1 h); filtrates were combined, concentrated to 20 mL, and stored at 4 °C. DMY (≥90% purity) was sourced commercially. Standard media and assay kits (AKPase, enzyme activity) were used. HPLC analysis: Agilent 1260 Infinity with C18 column (4.6×250 mm, 5 µm). Mobile phase methanol/water (0.03% phosphoric acid) 35:65, 0.5 mL/min, 40 °C, 5 µL injection, detection at 292 nm. DMY standard (90% purity) used for calibration. MIC determination: Broth dilution method. Log-phase S. aureus inoculated to 1×10^6 CFU/mL in nutrient broth with serial concentrations of VTE or DMY (0.16–50 mg/mL). Controls had no inhibitor. After incubation (37 °C, 24 h), aliquots plated; MIC defined as lowest concentration preventing growth. Growth curves: Cultures with VTE or DMY at 1/4×MIC, 1/2×MIC, 3/4×MIC; control without inhibitor. Incubated at 37 °C, 120 rpm; OD540 measured at multiple time points up to 30 h. Antibacterial activity in food systems: Cabbage suspension (50% juice in water) and barley soup (5% w/v) sterilized; 0.1% Tween 80 added. S. aureus inoculated to 1×10^6 CFU/mL; VTE or DMY added at 1/2×MIC or 1×MIC. Incubated at 37 °C up to 9 days. Viable counts by plating serial dilutions on nutrient agar (37 °C, 24 h). Membrane integrity assays: After treatment with 1×MIC VTE or DMY for 12 h, cells washed in PBS and processed. Extracellular AKPase quantified using a commercial kit. Extracellular β-galactosidase release measured using nitrobenzene β-D-galactoside substrate; supernatants collected at 0–4 h and absorbance at 416 nm recorded. Electron microscopy: SEM and TEM conducted after 12–24 h treatments with 1×MIC VTE or DMY. Cells fixed with 3% glutaraldehyde, post-fixed with osmium acid, dehydrated through graded ethanol, and prepared for imaging; isoamyl acetate (SEM) or anhydrous acetone (TEM) used as intermediate solvents. Protein and enzyme assays: Total intracellular proteins quantified per established protocols. SDS-PAGE performed with 4% stacking and 10% resolving gels; samples from cultures treated with 1×MIC VTE or DMY for 6 and 24 h. Activities of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), and total ATPase measured using commercial kits. DNA binding assays: UV–vis spectroscopy of VTE or DMY (0.6 mg/mL) with increasing genomic DNA concentrations (0–0.6 mg/mL), scanning 200–450 nm after 1 h incubation at 37 °C. Fluorescence spectroscopy with excitation at 437 nm, emission 400–650 nm. Competitive binding assessed by monitoring changes in fluorescence of ethidium bromide (EB)–DNA and DAPI–DNA complexes upon addition of VTE or DMY across concentrations. Statistics: All experiments performed in triplicate. Data presented as mean ± SD. One-way ANOVA (SPSS) with significance at P<0.05.

- HPLC: DMY constituted approximately 65.29% of VTE (calibration: y = 66478x + 7.3095, R² = 0.9991). - MICs against S. aureus ATCC 6538: VTE 6.3 mg/mL; DMY 1.25 mg/mL. - Growth curves: VTE and DMY significantly reduced culture OD540, prolonged lag phase, and suppressed logarithmic growth in a concentration-dependent manner (1/4–3/4×MIC). - Morphology (SEM/TEM): Both agents caused surface deformation and lysis; VTE produced more pronounced external morphological disruption. TEM showed severe cell wall/membrane damage, blurred cell boundaries, cytoplasmic lightening, and altered nuclear regions, especially with VTE; cell division appeared impaired after both treatments. - Membrane/wall integrity: After 12 h at 1×MIC, extracellular AKPase increased by 21.8% (VTE) and 10.3% (DMY), indicating wall damage. Extracellular β-galactosidase increased by 90.2% (VTE) and 57.4% (DMY), indicating increased membrane permeability. - Protein synthesis: Total intracellular protein decreased; by 12 h, total protein content reduced by 15.5% (VTE) and 9.9% (DMY) vs control (P<0.05). SDS-PAGE bands (notably ~85 kDa and 55–33 kDa regions) faded with time; changes more pronounced for VTE, especially at 24 h, with disappearance/appearance of some bands. - Energy metabolism enzymes (12 h): Activities reduced versus control: with VTE, MDH −12.7%, SDH −65.8%, total ATPase −31.5%; with DMY, MDH −4.7%, SDH −16.7%, total ATPase −15.9%. VTE exerted stronger inhibition, particularly on SDH. - DNA interactions: UV–vis spectra showed red-shifts (approx. 290→325 nm) and hyperchromicity upon DNA addition, indicating interaction; beyond ~0.2 mg/mL DNA, absorbance plateaued, suggesting multiple binding modes. Fluorescence scans showed red-shifts and enhanced fluorescence of VTE/DMY in presence of DNA (hyperchromic effect), consistent with intercalation. Competitive assays showed VTE and DMY quenched EB–DNA fluorescence (to <60% at 0.2 mg/mL for VTE and 0.1 mg/mL for DMY) and significantly quenched DAPI–DNA fluorescence (at 0.1 mg/mL, decreases of 38.5% for VTE and 64.4% for DMY), indicating both intercalative and minor-groove binding; DMY showed stronger groove-binding competition. - Food model systems (cabbage suspension and barley soup): At 1×MIC, both VTE and DMY reduced S. aureus counts to undetectable levels within 6–9 days; at 1/2×MIC, both markedly reduced counts by day 6. Higher concentrations led to faster colony count reductions; performance was similar in both food matrices.

The study addressed how VTE and DMY inhibit S. aureus by examining multiple cellular targets. Both agents compromised cell wall integrity and increased membrane permeability, leading to leakage of periplasmic and cytoplasmic enzymes. They reduced total protein synthesis and inhibited key energy metabolism enzymes, thereby suppressing bacterial growth and metabolic activity. Spectroscopic and competitive binding experiments demonstrated that both compounds interact directly with bacterial genomic DNA via intercalation and minor-groove binding, with DMY exhibiting relatively stronger DNA interactions. VTE, a complex extract rich in DMY (~65%) and other flavonoids, exerted broader and stronger effects on membranes, protein synthesis, and metabolic enzymes than DMY alone, suggesting synergistic or additive contributions from additional constituents (e.g., myricetin, rutin, quercetin). The observed multi-target actions translate to effective suppression of S. aureus in model food systems, supporting potential preservative applications. These findings clarify the antibacterial mechanisms and support the hypothesis that VTE and DMY can serve as natural antimicrobial agents for food safety.

Vine tea extract (VTE) and dihydromyricetin (DMY) exhibit significant antibacterial activity against Staphylococcus aureus. They disrupt cell wall integrity and membrane permeability, decrease total protein synthesis, inhibit key metabolic enzymes (MDH, SDH, ATPase), and bind bacterial DNA through both intercalative and minor-groove modes. While DMY is the major component of VTE and shows strong DNA interactions, VTE has greater overall impacts on membranes, protein synthesis, and enzyme activities, likely due to additional bioactive constituents. Both agents effectively reduced S. aureus in cabbage and barley model food systems, highlighting their potential as natural food preservatives. Future work could delineate contributions of individual VTE components, assess synergy, and evaluate efficacy and safety in diverse food matrices and real processing conditions.