Comparative Assessment of Antibacterial Activity of Haskap Berry (Lonicera caerulea L.) Extract, Gallic Acid, and Combination Against Alicyclobacillus acidoterrestris for Application in Orange Juice.

Alicyclobacillus acidoterrestris, a thermo-acidophilic, spore-forming bacterium, is a major cause of spoilage in fruit juices, leading to substantial economic losses. This study presents a comparative evaluation of gallic acid (GA), Lonicera caerulea L. ethanol extract (LCEE), and LCEE + GA as natural preservatives against this spoilage agent. The assessment included the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Notably, the MBC of LCEE alone could not be definitively established under tested conditions due to bacterial regrowth at concentrations above 12.5 mg/mL. Antimicrobial mechanisms were analyzed via membrane integrity and function assays (ATP levels, membrane potential, SEM, FCM, confocal laser scanning microscopy [CLSM]), and antioxidant capacity was measured using DPPH and ABTS methods. Practical efficacy was further tested in a fruit juice model through physicochemical. LCEE showed stronger bacteriostatic activity, whereas GA demonstrated superior bactericidal efficacy. Their combination revealed significant synergy (fractional inhibitory concentration [FIC] indices: 0.5 for cells, 0.38 for spores), drastically reducing the effective MIC and MBC of both components and accelerating cell death. Mechanistically, all treatments induced membrane damage, hyperpolarization, and ATP depletion. The antioxidant capacity ranked as LCEE + GA combination > GA > LCEE. In juice, all formulations inhibited microbial growth and enhanced antioxidant activity. Notably, GA and the combination more effectively increased total phenolic content, whereas LCEE uniquely enriched specific phenolic acids. Critically, the synergistic combination showed markedly less adverse impact on key juice quality parameters compared to LCEE alone. This study presents two natural antibacterial agents (LCEE and GA) along with their synergistic combination; provides a comparative analysis of their efficacy; and offers novel formulations to control Alicyclobacillus-mediated spoilage in fruit juice production.