Antibacterial activity and multi-target mechanism of naringenin against Alicyclobacillus acidoterrestris and its application in apple juice

Alicyclobacillus acidoterrestris (A. acidoterrestris) poses a major spoilage risk in pasteurized fruit juices, owing to its thermophilicity and acidophilicity. Naringenin, a natural flavonoid, has been investigated for its potential activity to inhibit A. acidoterrestris. The inhibitory efficacy of naringenin was systematically characterized by evaluating its effect on both vegetative cells and spores in AAM medium and apple juice, and the underlying mechanisms-alongside its application in apple juice-were investigated. In AAM, the minimum inhibitory and bactericidal concentrations (MIC and MBC) against vegetative cells were 0.15 mg/mL; in apple juice, they decreased to 0.05 mg/mL. For spores, the MIC was 0.15 mg/mL, but the MBC exceeded 1 mg/mL in both matrices. Naringenin inhibited vegetative cell growth in a concentration- and matrix-dependent manner and suppressed spore germination at 1× MIC. Mechanistic studies revealed that naringenin compromised cell integrity, reducing intracellular ATP and pH, hyperpolarizing the membrane, and causing cellular leakage. Scanning electron microscopy confirmed morphological damage. Molecular docking indicated that naringenin formed complexes with multiple key proteins, supporting the observed multifaceted damage. Reactive Oxygen Species levels increased, whereas intracellular proteins and DNA showed degradation. RT-qPCR confirmed the upregulation of related genes. Moreover, naringenin did not significantly alter physicochemical properties and antioxidant capacity of apple juice during storage at 4 °C and 25 °C. Sensory evaluation showed that low concentrations of naringenin improved the flavor profile of apple juice. These results indicated that naringenin serves as a natural preservative for fruit juices.