Evaluation of Autochthonous Non-Saccharomyces Yeasts by Sequential Fermentation for Wine Differentiation in Galicia (NW Spain)

Non-Saccharomyces yeasts constitute a useful tool in winemaking because they secrete hydrolytic enzymes and produce metabolites that enhance wine quality; in addition, their ability to reduce alcohol content and/or to increase acidity can help to mitigate the effects of climatic change on wines. The purpose of this study was to evaluate the oenological traits of non-Saccharomyces yeast strains autochthonous from Galicia (NW Spain). To do that, we carried out sequential fermentation using 13 different species from the yeast collection of Estación de Viticultura e Enoloxía de Galicia (Evega) and Saccharomyces cerevisiae EC1118. The fermentation kinetics and yeast implantation were monitored using conventional methods and genetic techniques, respectively. The basic chemical parameters of wine were determined using the OIV official methodology, and the fermentative aroma compounds were determined by GC–FID. The results evidenced the limited fermentative power of these yeasts and the differences in their survival after the addition of S. cerevisiae to complete fermentation. Some strains reduced the alcohol and/or increased the total acidity of the wine. The positive effect on sensory wine properties as well as the production of desirable volatile compounds were confirmed for Metschnikowia spp. (Mf278 and Mp176), Lachancea thermotolerans Lt93, and Pichia kluyveri Pkl88. These strains could be used for wine diversification in Galicia.

Strain-Specific Responses by Saccharomyces cerevisiae to Competition by Non-Saccharomyces Yeasts

The use of non-Saccharomyces yeast species generally involves sequential or co-inoculation of a Saccharomyces cerevisiae strain to complete fermentation. While most studies have focused on characterising the impact that S. cerevisiae has on the growth and metabolic activity of these non-Saccharomyces species, microbial interactions work reciprocally. Antagonism or competition of non-Saccharomyces species against S. cerevisiae has been shown to impact subsequent fermentation performance. To date, it remains unclear whether these negative interactions are strain specific. Hence, characterisation of strain-specific responses to co-inoculation would enable the identification of specific S. cerevisiae strain/non-Saccharomyces combinations that minimise the negative impacts of sequential fermentation on fermentation performance. The competitive fitness response of 93 S. cerevisiae strains to several non-Saccharomyces species was simultaneously investigated using a barcoded library to address this knowledge gap. Strain-specific fitness differences were observed across non-Saccharomyces treatments. Results obtained from experiments using selected S. cerevisiae strains sequentially inoculated after Metschnikowia pulcherrima and Torulaspora delbrueckii were consistent with the competitive barcoded library observations. The results presented in this study indicate that strain selection will influence fermentation performance when using non-Saccharomyces species, therefore, appropriate strain/yeast combinations are required to optimise fermentation.

Use of a Sequential Fermentation Method for the Production of Aspergillus tamarii URM4634 Protease and a Kinetic/Thermodynamic Study of the Enzyme

Microbial proteases are commonly produced by submerged (SmF) or solid-state fermentation (SSF), whose combination results in an unconventional method, called sequential fermentation (SF), which has already been used only to produce cellulolytic enzymes. In this context, the aim of the present study was the development of a novel SF method for protease production using wheat bran as a substrate. Moreover, the kinetic and thermodynamic parameters of azocasein hydrolysis were estimated, thus providing a greater understanding of the catalytic reaction. In SF, an approximately 9-fold increase in protease activity was observed compared to the conventional SmF method. Optimization of glucose concentration and medium volume by statistical means allowed us to achieve a maximum protease activity of 180.17 U mL−1. The obtained enzyme had an optimum pH and temperature of 7.0 and 50 °C, respectively. Kinetic and thermodynamic parameters highlighted that such a neutral protease is satisfactorily thermostable at 50 °C, a temperature commonly used in many applications in the food industry. The results obtained suggested not only that SF could be a promising alternative to produce proteases, but also that it could be adapted to produce several other enzymes.

Starmerella bombicola and Saccharomyces cerevisiae in Wine Sequential Fermentation in Aeration Condition: Evaluation of Ethanol Reduction and Analytical Profile

In the last few decades, the increase of ethanol in wine, due to global climate change and consumers’ choice is one of the main concerns in winemaking. One of the most promising approaches in reducing the ethanol content in wine is the use of non-Saccharomyces yeasts in co-fermentation or sequential fermentation with Saccharomyces cerevisiae. In this work, we evaluate the use of Starmerella bombicola and S. cerevisiae in sequential fermentation under aeration condition with the aim of reducing the ethanol content with valuable analytical profile. After a preliminary screening in synthetic grape juice, bench-top fermentation trials were conducted in natural grape juice by evaluating the aeration condition (20 mL/L/min during the first 72 h) on ethanol reduction and on the analytical profile of wines. The results showed that S. bombicola/S. cerevisiae sequential fermentation under aeration condition determined an ethanol reduction of 1.46% (v/v) compared with S. cerevisiae pure fermentation. Aeration condition did not negatively affect the analytical profile of sequential fermentation S. bombicola/S. cerevisiae particularly an overproduction of volatile acidity and ethyl acetate. On the other hand, these conditions strongly improved the production of glycerol and succinic acid that positively affect the structure and body of wine.