Diversity and Functional Genome Analysis of Lactic Acid Bacteria Isolated from Trás-os-Montes Artisanal Alheira

Alheira is a traditionally smoked, fermented, non-ready-to-eat meat sausage produced in the Trás-os-Montes region of Portugal, where lactic acid bacteria (LAB) are major determinants of product safety and quality. This study used whole-genome sequencing to characterize 59 LAB isolates collected from artisanal alheira produced in six municipalities. Eight species belonging to six genera were identified, and average nucleotide identity analysis resolved 24 non-redundant strain groups. Functional annotation revealed broad repertoires of carbohydrate-active enzymes, proteases, lipases, transport systems, and genes associated with tolerance to acid, oxidative, bile, heat, and salt stress. Genotype–phenotype inference was performed with a subset of 22 strain groups represented by unflagged genome assemblies. After false-discovery-rate correction, carbon-metabolism, transport, energy-metabolism, total functional-gene, acid-stress, bile-stress, and total-stress counts were positively associated with total pH decline. A mixed-effects model showed a positive association between standardized total functional-gene count and acidification ($b=0.137\pm 0.059$, $p=0.049$; marginal $R^2=0.266$, conditional $R^2=0.582$), but within–between decomposition indicated that the association was detectable between species rather than within species. Regularized regression, Random Forest, AICc model comparison, and partial least-squares regression converged on carbon metabolism, transport, energy metabolism, and acid- or bile-stress functions as the principal exploratory signals; however, sparse LASSO regression for variable selection was unstable, and prediction of unobserved species was limited. None of the five targeted pathways: acetaldehyde, citrate, diacetyl, exopolysaccharide, and lactose metabolism, remained significant after multiple-testing correction. These findings provide a genomic and phenotypic basis for selecting native starter-culture candidates while emphasizing the need for independent functional and safety validation.