Potential application of indigenous Pichia kluyveri for enhanced wine aroma quality

Aims: In previous work, five indigenous Pichia kluyveri strains, GS1-1, FS-2-7, HS-2-1, C730 and C732, were isolated and selected from spontaneous fermented wines from Ningxia and Gansu. The aims of this study were to 1) evaluate resistance of these strains to environmental stressors that may restrict their growth and the progress of alcoholic fermentation; 2) Investigate their fermentation dynamics; 3) Characterise aroma profiles of Cabernet Sauvignon wines made from mixed cultures of P. kluyveri and Saccharomyces cerevisiae. 

Methods and Results: Tolerance assays were conducted in YEPD medium to test resistance of each Pichia kluyveri strain to sugar, pH, ethanol, temperature and free SO₂. All strains except FS-2-7 were able to tolerate 60% w/v glucose, low pH of 2.0, 16% v/v ethanol, extreme fermentation temperatures (11˚C and 44˚C), and 500 mg/L total SO₂. Following this, these strains were inoculated into a synthetic grape juice medium to test their fermentation performance and evaluate basic parameters of the final synthetic wine. Strain HS-2-1 was the first to initiate fermentation, and produced significantly higher amounts of total organic acids and less volatile acids compared to other strains. Thus, strain HS-2-1 was chosen for further characterisation in Cabernet Sauvignon fermentation trials co-fermented with S. cerevisiae NX11424 at different ratios. Viable yeast cell numbers were determined by plate counting. Yeast-derived volatile compounds of the final wine were analysed using head space-solid phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC/MS). Mixed fermentation did not inhibit HS-2-1 growth, and also produced less volatile acid, and significantly more esters and higher alcohols compared to single fermentation by S. cerevisiae. Notably, concentrations of isopentanol, ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl 9-decenoate and ethyl lactate increased in line with the increased proportion of HS-2-1 in the inoculum.

Conclusions: 

This study shows indigenous P. kluyveri HS-2-1 has good resistance to alcoholic fermentation associated common stressors, better fermentation performance, and excellent oenological characteristics when co-fermenting with S. cerevisiae. 

Significance and Impact of the Study: Chinese wine regions such as Ningxia and Gansu have developed dramatically in recent years. These wine regions are in great need to produce wines with typical regional characteristics. To promote regional typicity, using selected indigenous yeasts could introduce a unique local character or “terroir” during winemaking. Pichia kluyveri widely occurs at earlier stages of spontaneous fermentation, however limited research has been done on its oenological characteristics. This study comprehensively investigated the features of indigenous P. kluyveri strain(s), and highlighted the potential application of strain HS-2-1 in winemaking by co-fermenting with S. cerevisiae for improving the fruity and floral aroma profile of these Chinese wines.