Effects of lactic acid bacteria inoculation methods on fermentation characteristics and aroma compounds of simulated grape juice

This study systematically investigated the effects of inoculation timing and specific strain combinations involving Saccharomyces cerevisiae, Oenococcus oeni and Lactobacillus plantarum on fermentation kinetics and the final aromatic profile in a simulated grape juice medium. The primary objective was to evaluate the potential of simultaneous inoculation as a strategy to enhance both winemaking efficiency and sensory quality, thereby providing a scientific basis for optimizing traditional fermentation protocols. Alcoholic fermentation was initiated using the yeast strain S.cerevisiae ES 488. Two indigenous lactic acid bacteria (LAB) consortia were tested: one combining O.oeni ZX-1 and L.plantarum GF-26, and the other pairing O.oeni GF-2 with L.plantarum GF-20. The key experimental variable was the timing of LAB inoculation: simultaneous inoculation introduced LAB at the start of alcoholic fermentation alongside yeast, whereas sequential inoculation did so only after alcoholic fermentation was complete. Throughout the process, critical biochemical parameters including reducing sugar and L-malic acid degradation, as well as the microbial viability of all three strains were dynamically monitored at regular intervals to capture the real-time metabolic progression. After stabilization, volatile aroma compounds in the final wines were analyzed using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), enabling precise identification and quantification of key odor-active molecules. Results showed that simultaneous inoculation significantly accelerated overall fermentation, reducing the total duration of alcoholic and malolactic fermentation by 7 to 19 days compared to the sequential method. Notably, the consortium of ES 488, ZX-1, and GF-26 achieved full microbial stability within 14 days, indicating high process efficiency and potential for rapid turnaround in commercial settings. Moreover, the dual-species LAB inoculum shortened the malolactic fermentation phase by an additional 4 to 6 days compared to single-strain inoculations, suggesting synergistic metabolic interactions that enhance substrate utilization and stress tolerance. Although ethanol levels immediately following alcoholic fermentation were slightly lower in simultaneous treatments, likely due to early competition for nutrients, the resulting low-ethanol environment promoted significantly higher proliferation rates of the LAB, which otherwise face inhibitory conditions in the high-ethanol post-fermentation environment of sequential trials. This improved microbial activity facilitated faster malic acid consumption and greater metabolic diversity. The simultaneous treatment with the ZX-1 and GF-26 consortium stood out not only for its rapid malic acid degradation but also for producing a sensorially superior wine. Chemical analysis revealed 1.91 times higher concentrations of higher alcohols and 33.97% more total esters compared to the sequential control using the same strains. Consequently, this wine exhibited significantly enhanced intensities of floral, fruity, and sweet aromatic notes, contributing to a more balanced and appealing sensory perception. Statistical analysis confirmed a strong positive correlation between these sensory attributes and elevated levels of key esters (ethyl octanoate, ethyl nonanoate, diethyl succinate, and ethyl caproate), which are known to contribute to fruity aroma complexity. These findings underscore the role of microbial synergy in shaping wine aroma. In conclusion, simultaneous inoculation using the tailored three-strain consortium of S.cerevisiae ES 488, O.oeni ZX-1, and L.plantarum GF-26 represents a promising enological approach. The integrated strategy offers dual benefits: it substantially improves production efficiency by shortening fermentation time, increasing tank turnover, and reducing operational costs, while simultaneously enhancing wine sensory quality through a more complex and desirable ester-rich aroma profile. These outcomes support the tailored inoculation strategy’s practical application for producing wines with greater aromatic intensity, typicity, and consistency in a time-efficient and predictable manner, effectively addressing key challenges in modern winemaking and offering a reliable method to improve both economic viability and sensory appeal. The present study provides novel insights into microbial co-cultivation dynamics and offers actionable strategies for advancing fermentation technologies in the wine industry.