Physiological, Genetic, and Fermentative Traits of Oenococcus oeni Isolates from Spontaneous Malolactic Fermentation in Koshu Wine

Koshu wine, produced from the indigenous Japanese grape Vitis vinifera L. cv. Koshu exhibits a lower pH than other white wines, hindering malolactic fermentation (MLF) by lactic acid bacteria (LAB). Here, we aimed to isolate LAB strains capable of performing MLF under these challenging conditions to improve wine quality. Sixty-four Oenococcus oeni and one Lactobacillus hilgardii strain were isolated from Koshu grapes and wines that had undergone spontaneous MLF. MLF activity was assessed under varying pH, SO2, and ethanol conditions in modified basal medium (BM) and Koshu model wine media. Expression of stress-related genes was analyzed using real-time PCR. Carbon source utilization was evaluated via API 50CH assays. All isolates degraded malic acid and produced lactic acid at 15 °C and pH 3.2 in BM without reducing sugars. Seven strains, all identified as O. oeni, demonstrated MLF activity at pH 3.0 in modified BM lacking added reducing sugars or tomato juice. Six wine-derived strains tolerated up to 12% ethanol, whereas the grape-derived strain was inhibited at 10%. In a synthetic Koshu wine model (13% ethanol, pH 3.0), wine-derived isolates exhibited higher MLF activity than commercial starter strains. In high-performing strains, mleA was upregulated, and most isolates preferred fructose, arabinose, and ribose over glucose. These findings suggest that indigenous O. oeni strains from Koshu wine possess unique stress tolerance and metabolic traits, making them promising candidates for region-specific MLF starter cultures that could enhance Koshu wine quality and terroir expression.