Combinatorial effects of bovicin HC5 and epsilon-polylysine against Alicyclobacillus acidoterrestris in orange juice
Authors:   , Iduu Nneka Vivian 
Published: September 2023

Alicyclobacillus acidoterrestris is a thermo-acidophilic spore-forming bacterium that can survive pasteurization procedures and cause spoilage of fruit juices and acidic beverages in the food industry. Antimicrobial peptides (AMPs) are considered an alternative strategy to control the growth of this spoilage microorganism in acidic beverages. AMPs can be used alone or in combination with other antimicrobials to enhance the shelf-life of foods. In this study, the antibacterial effect of bovicin HC5 and ɛ-polylysine were evaluated individually and in combination against vegetative cells and germinated endospores of A. acidoterrestris DSMZ 2498 grown in AAM broth and orange juice. A significant decrease (P < 0.05) in the microbial growth and specific growth rate, and an increase in lag phase (P < 0.05) of vegetative cells and germinated endospores of A. acidoterrestris DSMZ 2498 were observed with the addition of increasing concentrations of both antimicrobial peptides in AAM broth. Both AMPs exhibited strong inhibition of the target bacterium with a minimum inhibitory concentration (MIC) of 2.50 and 5.00 µg/mL of bovicin HC5, and 7.81 and 7.81 µg/mL of ɛ-polylysine against vegetative cells and germinated endospores of A. acidoterrestris DSMZ 2498, respectively. Further studies showed that bovicin HC5 possessed bactericidal properties having a minimum bactericidal concentration (MBC) of 5.00 µg/mL against vegetative cells and germinated endospores of A. acidoterrestris DSMZ 2498. The antibacterial activity of both AMPs in combination was evaluated using the checkerboard assay. A fractional inhibitory concentration (FIC) index of 1 was obtained against the vegetative cells and germinated endospores of A. acidoterrestris DSMZ 2498 indicating an additive effect between the two AMPs. The AMPs alone and in combination reduced (P < 0.05) the viability of A. acidoterrestris DSMZ 2498 germinated endospores that were inoculated (10 6 CFU/mL) in concentrated orange juice (11 oBx). However, the antimicrobial effect was more pronounced with bovicin HC5 as viable cells and germinated endospores reduced below the detection limit after 36 h of treatment. The exposure time required to kill 90 % of the initial microbial population at 95 oC (D95oC) was used to assess the effect of the AMPs in the thermal reduction of A. acidoterrestris DSMZ 2498 endospores. Concentrations of 80, 160 µg/mL of bovicin HC5, 125, and 250 µg/mL of ɛ-polylysine and their combination respectively were evaluated. Viable spores were evaluated by spread-plating aliquots taken at different time intervals in AAM medium (pH 4). The results showed that the mean D-value without AMPs was 7.68 min at 95 oC. With the addition of ɛ-polylysine into the juice, no significant difference was observed (P > 0.05) while treatment with bovicin HC5 reduced the D-value (P < 0.05) compared to control. This effect was observed with bovicin HC5 alone and in combination with ɛ-polylysine with a D-value reduction of 71.48 % and 68.36 %, respectively. Additionally, the effect of the AMPs on the morphology and cell structure of A. acidoterrestris DSMZ 2498 was evaluated by atomic force microscopy (AFM). The AFM topographical images and cross-sectional images revealed structural changes in the cell morphology of vegetative cells upon a 24 h treatment with 1X and 2X MIC of the AMPs alone and in combination. The findings in this study revealed the potential application of bovicin HC5 and ɛ-polylysine as natural preservatives in hurdle technologies to control A. acidoterrestris and improve the microbiological stability and safety of fruit juices. Keywords: Antimicrobial peptides. Orange juice. Alicyclobacillus acidoterrestris. Thermoacidophilic. Spoilage. Fruit juice preservation