Unveiling the Potential of <i>Lentilactobacillus hilgardii</i> in Malolactic Fermentation: Comparative Genomics and Fermentation Dynamics

This study aimed to assess the potential of Lentilactobacillus hilgardii as a novel candidate for malolactic fermentation (MLF) in winemaking, through comparative genomics and experimental validation, in direct comparison with Oenococcus oeni. We performed a pangenome analysis on 16 L. hilgardii and 7 O. oeni strains to explore their genetic diversity, focusing on wine-related traits. Functional predictions were generated using genome-scale metabolic models (ModelSEED/KBase), including in silico co-inoculation with Saccharomyces cerevisiae EC1118 and post-alcoholic fermentation simulations. The reference strains L. hilgardii DSM 20176 and O. oeni DSM 20252 were experimentally tested for MLF performance in a synthetic wine-like medium at 25°C and 10°C. Core-genome comparison revealed that 67.9% of the malolactic enzyme sequence is conserved between the two species, with comparable docking affinity to L-malic acid. L. hilgardii harboured unique enzymes with potential oenological interest (phenolic acid decarboxylase, mannitol dehydrogenase, glucosidase) and distinctive stress-related proteins (YaaA, HrcA, ASP23), suggesting improved tolerance to oxidative, temperature, and alkaline stresses. Notably, L. hilgardii showed genomic potential to degrade putrescine, arginine, and ornithine, precursors of ethyl carbamate. Experimentally, L. hilgardii reduced L-malic acid from 2.5 g/L to < 0.1 g/L within 12 days at 10°C, while O. oeni showed no MLF activity at this temperature. At 25°C, both strains completed MLF within 6-7 days. L. hilgardii also consumed > 80% of residual fructose at 10°C, whereas O. oeni showed minimal utilisation. Our results demonstrate that L. hilgardii combines a favourable genomic repertoire for wine adaptation with superior MLF performance at low temperature, suggesting its potential as an alternative to O. oeni in cool-climate winemaking. This work provides the first genome-scale comparative and functional evaluation of L. hilgardii in the winemaking context, highlighting its technological promise to improve fermentation reliability, reduce spoilage risk, and expand the biodiversity of malolactic starters.