Effects of inoculation timing and mixed fermentation with Pichia fermentans on Oenococcus oeni viability, fermentation duration and aroma production during wine malolactic fermentation

There has been a growing interest in developing co-inoculum of Oenococcus oeni and Saccharomyces cerevisiae/non-Saccharomyces for simultaneous malolactic fermentation (MLF) and alcoholic fermentation (AF) of wines. This study sought to elucidate the effects of two crucial factors (inoculation timing and paired yeast) on the fermentation performance and aroma production of Merlot wine. O. oeni used for MLF was concurrently or sequentially inoculated with two yeast cultures (i.e., single S. cerevisiae and mixed S. cerevisiae /Pichia fermentans H5Y-28) used for AF. Inoculation timing determined the overall vinification duration, and conditioned the production of principle higher alcohols, terpene and O. oeni-mediated volatiles. In contrast, paired yeast improved O. oeni viability, and showed significant effect on aromatic esters and volatile fatty acids. Possibly due to lower ethanol stress, co-inoculum allowed O. oeni to initiate MLF during AF, resulting in 45% reduction of total fermentation time. Meanwhile, O. oeni growth was stimulated by P. fermentans, with 1.7-fold of the maximum population higher than that in co-fermentation without P. fermentans. Such stimulation of O. oeni growth also occurred in sequential fermentation where P. fermentans had been replaced by S. cerevisiae. Only in sequential inoculum, P. fermentans induced high levels of 3-methylbutyl acetate, ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, which may result in enhanced fresh fruity trait of wines. These findings suggested a positive effect of P. fermentans H5Y-28 on O. oeni and MLF. This work provides an alternative approach to improve wine MLF and aroma outcomes using friendly non-Saccharomyces yeast with appropriate inoculation strategy.