Fermentation can be expected to enhance efficiency and sensory quality in modern winemaking. Conventional protocols can serve as a promising strategy to evaluate the potential of simultaneous inoculation. This study systematically investigated the effects of inoculation timing and specific strain combinations, including Saccharomyces cerevisiae, Oenococcus oeni, and Lactobacillus plantarum, on fermentation kinetics and the final aromatic profile in the grape juice medium. Alcoholic fermentation was initiated using the yeast strain S.cerevisiae ES 488. Two indigenous lactic acid bacteria (LAB) consortia were evaluated: one combining O.oeni ZX-1 and L.plantarum GF-26, and another pairing O.oeni GF-2 with L.plantarum GF-20. The experimental variable was selected as the timing of LAB inoculation: simultaneous inoculation introduced LAB at the start of alcoholic fermentation alongside yeast, whereas sequential inoculation was realized only after alcoholic fermentation. Biochemical parameters were dynamically monitored at regular intervals, including reducing sugar and L-malic acid degradation, as well as the microbial viability of all three strains. The real-time metabolic progression was captured after measurement. The volatile aroma compounds in the final wines were analyzed after stabilization using headspace solid-phase microextraction with gas chromatography-mass spectrometry (HS-SPME-GC-MS). The odor-active molecules were precisely identified and quantified. Results showed that the simultaneous inoculation significantly accelerated overall fermentation, reducing the total duration of alcoholic and malolactic fermentation by 7 to 19 days, compared with the sequential. 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 with the single-strain inoculations. The synergistic metabolic interactions enhanced the substrate utilization and stress tolerance. Ethanol levels after alcoholic fermentation were slightly lower in the simultaneous treatments, likely due to early competition for nutrients. The low-ethanol environment significantly promoted higher proliferation rates of the LAB. Otherwise, the inhibitory conditions were observed in the high-ethanol post-fermentation environment of sequential trials. The microbial activity facilitated higher malic acid consumption and greater metabolic diversity. The simultaneous treatment with the ZX-1 and GF-26 consortium was achieved in the rapid malic acid degradation to produce a sensorially superior wine. Chemical analysis revealed that there were 1.91 times higher concentrations of higher alcohols and 33.97% more total esters, compared with the sequential control. Consequently, this wine exhibited significantly high intensities of floral, fruity, and sweet aromatic notes, thereby contributing to a more balanced sensory perception. Statistical analysis confirmed that there was a strong positive correlation between these sensory attributes and elevated levels of key esters (ethyl octanoate, ethyl nonanoate, diethyl succinate, and ethyl caproate), which contributed to fruity aroma complexity. The microbial synergy was found in shaping wine aroma. In conclusion, the promising enological approach was obtained in the simultaneous inoculation with the tailored three-strain consortium of S.cerevisiae ES 488, O.oeni ZX-1, and L.plantarum GF-26. Dual benefits were integrated to substantially improve production efficiency by less fermentation time and operational costs. Tank turnover simultaneously enhanced the wine sensory quality using a more complex and optimal ester-rich aroma profile. The inoculation strategy was tailored for the practical application. The wines were produced with greater aromatic intensity, typicity, and consistency in a time-efficient and predictable manner. A reliable solution can offer to improve both economic viability and sensory appeal. The finding can also provide some insights and strategies into microbial co-cultivation dynamics of fermentation in the wine industry.

This study was conducted in order to explore the fermentation capacity and glycosidase activity of indigenous non-Saccharomyces isolated from naturally fermented grape juice from the eastern foothill of Helan Mountain using WL medium and esculin medium. The activity of β-D-glucosinase, β-D-xylosidase, α-L-rhamnosidase, and α-L-arabinosidase of the isolates were determined and compared using the p-nitrophenol method. The growth kinetics and glycosidase activity of six selected strains were monitored during the fermentation of synthetic grape must by each of them. The results showed that the six non-Saccharomyces strains with high glycosidase activity were identified as Hanseniaspora opuntiae, Metschnikowia pulcherrima, Hanseniaspora uvarum, and Torulaspora delbrueckii through sequence analysis of the D1/D2 domain of 26S rDNA, but their glycosidase activities were different. T. delbrueckii showed better fermentation capacity for synthetic grape must and higher cumulative glycosidase activity (94.10–127.70 mU/mL) than the other strains, which was 1.96–2.30 folds higher than the control strain. M. pulcherrima had high α-L-rhamnosidase and α-L-arabinosidase activity, while H. opuntiae and H. uvarum-3 showed high β-D-xylosidase and α-L-arabinosidase activity. In conclusion, these strains of non-Saccharomyces yeast have high glycosidase activity and possess good application potential for wine aroma enhancement.

In order to study the kinetic changes in microbial growth, substrate consumption and product generation during the co-fermentation of Saccharomyces cerevisiae and Oenococcus oeni, indigenous O. oeni ZX-1 and MG-1 were co-inoculated with S. cerevisiae VW and AW into a model grape system, separately, and the viable count of S. cerevisiae, reducing sugar content, alcohol content, O. oeni viable count and L-malic acid content in the fermentation system were measured every day during the fermentation period. The classical Logistic, Boltzmann, SGompertz and DoseResp models were adopted to nonlinearly fit the measured data. The results showed that the Boltzmann model could better describe the growth of S. cerevisiae and the change of alcohol content; the logistic model had the best fitting effect on the change of reducing sugar content. For each of the four co-culture groups, the fitting coefficients R² of the Boltzmann and DoseResp models for O. oeni growth and L-malic acid content change were consistent, indicating both models could well predict the growth of O. oeni and the trend of L-malic acid content during the co-fermentation process. Taken together, we concluded the tested strains O. oeni have good biocompatibility with S. cerevisiae VW and AW, and the established kinetic models can provide data support and a theoretical basis for the control of mixed culture fermentation for industrial wine production.

Non-Saccharomyces yeast (NS) strains can be adapted to generate diverse secondary metabolites, in order to alter the flavor and aroma characteristics of wines. Particularly, the NS strains are co-inoculated with Saccharomyces cerevisiae in the terroir of the production area. Therefore, it is of great significance to identify the differences in the oenological characteristics of autochthonous NS strains, in order to regulate the wine flavor. In this experiment, the test strains were selected as Hanseniaspora uvarum (HX-1, HX-3), Metschnikowia pulcherrima (HX-2, HX-4), and Torulaspora delbrueckii (HX-6, HX-7) isolated from spontaneously grape juice in Hexi Corridor wine region. A systematic measurement was performed on the growth curves of these 6 non-Saccharomyces yeast strains, together with their tolerance to vinification factors. The results showed that the NS strains belonging to the same genus showed similar growth curves, and all strains were well tolerant to the high concentrations of glucose and SO₂, among which HX-2, HX-6, and HX-7 still grew normally at a glucose concentration of 300 g/L. Except for the HX-6 strain, the growth of the rest strains was only slightly inhibited at an SO₂ concentration of 300 mg/L. Notably, there were significant differences among the tested strains under different culture temperatures, pH, and ethanol levels. Among them, HX-1, HX-3, HX-6, and HX-7 strains showed excellent low-temperature tolerance, where their OD₆₀₀ values reached more than 0.6 under the fermentation temperature of 15℃. In addition, HX-1, HX-2, HX-3, and HX-4 strains grew normally under low pH conditions. HX-6 and HX-7 strains showed the strongest tolerance to ethanol. In particular, the growth of HX-6 was slightly inhibited, where the absorbance value reached 0.569 when the ethanol level reached 9%. All the test strains were inoculated into Merlot grape juice for pure fermentation, in order to investigate the vinification performance of these NS strains. The oenological parameters of the wine samples were determined to detect the volatile aroma compounds using HS-SPME-GC-MS. Pure culture fermentation experiments showed that the HX-6 and HX-7 strains exhibited strong fermentation ability and high production of ethanol and glycerol during Merlot wine making. Torulaspora delbrueckii strain HX-7 was closely related to the formation of volatile compounds, such as 1-heptanol, isoamyl alcohol, isoamyl acetate, isoamyl acetate, and ethyl acetate, while the HX-6 strain was effectively improved the content of ester compounds, such as ethyl caprate and ethyl caprylate in wine samples. Hanseniaspora uvarum strains HX-1 and HX-3 significantly enhanced the content of total acidity and malic acid, with great contribution to the formation of C13-norisopentadiene in the wines. The limited reducing sugar by the HX-1 strain resulted in the alcohol fermentation stagnation on the 8th day after inoculation. The HX-4 strain shared a high yield of volatile acidity. The HX-2 strain significantly increased the content of varietal aroma compounds, such as citronellol and linalool in Merlot wine. Hierarchical clustering of the oenological parameters combined with the aroma compounds revealed that there were significant intraspecific or interspecific differences in the fermentation characteristics of the tested NS strains. Comprehensive analysis showed that the oenological parameters and aroma profile of the autochthonous Torulaspora delbrueckii and Hanseniaspora uvarum strains during pure culture fermentation showed intraspecies similarity, while the fermentation characteristics of Metschnikowia pulcherrima showed strain dependence. The finding can provide a scientific reference to regulate the flavor quality of wines using mixed fermentation.