Abstract

Tocosh, an ancestral fermented potato product, relies on spontaneous processes near freshwater springs under extreme high-altitude conditions and represents an underexplored reservoir of microbial diversity with significant potential for the discovery of probiotics. This study provides, for the first time, a comprehensive probiogenomic characterization of 19 lactic acid bacteria (LAB) isolated from tocosh, in the Peruvian Andes, at three distinct altitudes-2992, 3882, and 4451 m above sea level (m.a.s.l.)-using whole genome sequencing (WGS) and bioinformatic profiling. A total of six species were identified: Lactiplantibacillus plantarum and Levilactobacillus brevis at all three study sites, Lacticaseibacillus paracasei and Lentilactobacillus buchneri at the lowest altitude (2992 m.a.s.l.), and Latilactobacillus curvatus and Latilactobacillus sakei at the highest altitudes (3882 and 4451 m.a.s.l.). Our results reveal that the extreme Andean environment is associated with stability in L. plantarum (genome sizes from 3.36 to 3.38 Mb) across all altitudinal levels. Functional analysis using CAZymes determined that L. brevis and L. buchneri act as primary degraders (high percentage of glycosyl hydrolases/carbohydrate binding) while L. curvatus and L. sakei function as primary builders through exopolysaccharide biosynthesis, likely a cryoprotective adaptation preventing cell damage during cold temperatures at high altitudes. Additionally, L. sakei and L. plantarum exhibited unique auxiliary activity (AA) enzymes, suggesting an oxidative mechanism to breach recalcitrant starch surfaces. All isolates were confirmed as genomically safe, lacking transferable antibiotic resistance genes and virulence factors. Pathogenic risk potential scores (PPRS) were consistently <= 2.0, fulfilling qualified presumption of safety (QPS) criteria. These findings provide the first genomic characterization of tocosh-associated LAB, establishing a basis for tocosh standardization, enabling the rational design of starter cultures that preserve ancestral traits and ensure microbiological safety in modern food applications.