Spoilage Investigation of Pasteurized Apple Juice with Visual Defects Identifies a Potentially Novel Acetobacter Species As the Primary Spoilage Agent

Jellified materials were observed in spoiled pasteurized apple juice that contained dimethyl dicarbonate (DMDC). Microbiological analysis showed a high microbial load (4-5 log CFU/mL) in the sample. Acetobacter spp. was identified as the spoilage microorganism by 16S rRNA gene sequencing. Metataxonomic analysis showed Acetobacter represented 99 % of the bacterial community. Three Acetobacter isolates (LX5, LX9 and LX16) were selected for whole genome sequencing and characterized for their susceptibility to DMDC. Genome-based phylogeny supported the species-level classification of LX5 as A. fabarum. It also suggested LX9 and LX16 are the same microorganisms from a potentially novel species closely related to A. lovaniensis. The minimum inhibitory concentrations (MICs) of DMDC for Acetobacter isolates in sterile apple juice (pH ∼3) at 30 °C were 46 ppm and 329 ppm for A. fabarum LX5 and Acetobacter LX9/LX16, respectively. The minimum bactericidal concentrations (MBCs) were 250 and 500 ppm for A. fabarum LX5 and Acetobacter LX9/LX16, respectively. The inoculum concentration for the MIC assay was approximately 6 log CFU/mL, representing the "worst-case" scenario. When the contamination level was reduced to 500 CFU/mL per US federal regulation (21 CFR 172.133) and the apple juice was refrigerated, Acetobacter isolates did not grow and were completely inhibited by 238 ppm DMDC. Pangenome analysis identified gene clusters that potentially play a role in biofilm development, carbohydrate metabolism, and oxidative stress tolerance, but it also ruled out the involvement of Acetobacter in apple juice gel formation. The investigation concluded that post-pasteurization contamination, high microbial load and ambient storage were factors leading to this spoilage incident.