Wild Australian niches as a source of novel lactic acid bacteria and malolactic fermentation candidates.

Malolactic fermentation (MLF) by lactic acid bacteria (LAB) is essential for most red wine production, yet commercial starter cultures are largely limited to Oenococcus oeni, a species constrained by slow growth, fastidious nutritional requirements, and poor co-inoculation competitiveness. To identify novel MLF candidates from diverse ecological niches, we assembled 714 wild LAB strains from seven Australian geographic regions and eight ecological sources, including insects, flowers, honey, and fermented foods, spanning 21 genera. Four hundred strains were screened in a microplate assay quantifying nine metabolites in Shiraz grape juice and classified into six functional categories based on thresholds for malic acid reduction, growth, and spoilage compound production. Fourteen strains (3.5%) achieved High Performance, dominated by Lactiplantibacillus (9/14). Two Apilactobacillus strains, a genus known primarily for wine spoilage, also completed MLF without spoilage compound production. Twenty-three strains were validated in co-fermentation with Saccharomyces cerevisiae using an automated robotic platform. An insect-derived Lactiplantibacillus plantarum from Kangaroo Island completed MLF within 48 h, outperforming the commercial Lp. plantarum control, while all 14 Fructilactobacillus strains failed in co-fermentation despite demonstrating MLF activity in juice, indicating that wine conditions rather than metabolic capacity determined MLF outcome. Volatile profiling of five selected candidates confirmed wild strains preserved yeast-derived aroma profiles. Geographic origin significantly predicted functional category (P < 0.001), with grape-associated environments enriched for MLF-competent strains. This study validates environmental bioprospecting as a strategy for wine starter culture discovery and provides a curated collection of 714 LAB for fermentation applications.