Association of D-limonene and nisin: the action on vegetative cells and spores of Alicyclobacillus spp. in processed orange juice

Abstract Contamination by Alicyclobacillus spp. is a challenge in the juice and acidic beverage industry, since it can cause spoilage and, consequently, economic losses. Natural preservatives are interesting strategies as an alternative search for microbial growth control in food, in particular the use of D-limonene (DL) and nisin (Nis). This study aimed to evaluate the antibacterial activity of the DL combination and Nis against five strains of Alicyclobacillus spp. A. acidoterrestris (CBMAI 0244 T ); A. herbarius (CBMAI 0246 T ); A. acidiphilus (CBMAI 0247 T ); A. hesperidum (CBMAI 0298 T ) and A. sendaiensis (KCTC 3843 T ). The interaction among the compounds was initially determined in culture medium by the checkerboard method, and the efficacy was evaluated by disk diffusion tests, Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC). Subsequently, the combination was evaluated in orange juice to determine the Minimum Sporicidal Concentration (MSC). The checkerboard results against A. acidoterrestris revealed synergism, with a Fractional Inhibitory Concentration Index (FICI) of 0.07. The combination (DL + Nis) demonstrated larger inhibition halos (24–26 mm) compared to DL (10–11 mm) and Nis (20–24 mm) alone. The MIC values of the combination were reduced, ranging from 0.12 to 3.90 µg/mL which means it was bactericidal for all strains. In reconstituted orange juice, the combination was effective, with a MSC of 0.48 µg/mL against A. acidoterrestris spores. Electron microscopy revealed significant morphological damage and signs of cell lysis in treatments with the combination, suggesting a loss of membrane integrity consistent with the observed synergistic effect. Additionally, DL showed antioxidant activity of 0.23 µmol Trolox/100 g (ABTS), 91.67 µmol Trolox/100 g (FRAP), and electronic nose (E-nose) analysis demonstrated that the combination did not significantly alter the volatile profile of the juice. Therefore, the combination of D-limonene and nisin represents a natural alternative with potential for the control of Alicyclobacillus spp. in commercial orange juice.