non-Saccharomyces Populations Research Articles

Influence of Cryoextraction and Cold Pre-Fermentative Maceration on the Yeast Microbiota and the Volatile Compounds Profile of Sangiovese Wine

Low-temperature treatments can be applied to grapes or must before alcoholic fermentation to enhance the wine’s sensory characteristics. Several studies have shown that such practices have a positive effect on the polyphenol profile of the wine, but only a few surveys have examined the effect of these treatments on the yeast microbiota of grapes and wine. Therefore, this study aimed to evaluate how cryoextraction (freezing the grape with liquid nitrogen) and cold pre-fermentative maceration (at 5 °C for 48 h) affect the Saccharomyces and non-Saccharomyces populations during the winemaking process of red grapes, cv Sangiovese, conducted at two temperatures (20 and 30 °C). This research analyzed the concentration of various yeast species, their fermentation abilities, and the resulting wine’s aromatic profile. The Principal Component Analysis performed on yeast concentrations during the fermentations of various wines did not group the experimental wines based on treatment. However, the same groupings were highlighted when the concentrations of the volatile compounds, quantified in the experimental wines, were processed using the same statistical approach. Therefore, cryoextraction and cold pre-fermentative maceration seem to contribute less to the aromatic profile than the yeasts involved in the fermentation process.

Optimization of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native yeasts

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 106 cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 106 cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels.

Influence of Cold Maceration Time on Chromatic and Microbiological Characteristics of Cabernet Sauvignon Wines

The pre-fermentative cold soak (CS) is proposed so as to extract and stabilise polyphenolic compounds in wines. CS is applied empirically to Cabernet Sauvignon in Cuyo, Argentina; however, there is poor scientific background on this technique. The aim was analyse the effect of cold soak time on colour parameters such as total polyphenols, total anthocyanins, tannins, colour and polymerisation indexes. Moreover, Saccharomyces and non-Saccharomyces populations were studied to evaluate their impact on colour. Cabernet Sauvignon must (Pedernal, San Juan) was distributed into four vessels: CONTROL (CT): simultaneous maceration and alcoholic fermentation (AF) with commercial yeast D254; T1: CS for two days; T2: CS for five days; and T3: CS for seven days. T1, T2 and T3 were maintained at 4 ± 1°C during CS. They were later inoculated with D254 (AF: 22 ± 1°C). The total polyphenol index (TPI), total anthocyanins (TA) and tannins were quantified during CS and AF. The colour index (CI) and polymerisation index (PI) were determined at the end of AF. The total yeast populations of Saccharomyces and non-Saccharomyces were quantified during CS and AF. The highest values of TA, tannins, TPI and PI were obtained by CT. This treatment also registered the highest total yeast population and the highest total yeast death. The highest CI was observed in T3. When Saccharomyces was found at the end of CS (T1 and T2), it dominated the alcoholic fermentation in the early stages (not observed in CT and T3). The use of the pre-fermentative CS technique in Cabernet Sauvignon wines is promising for young wines due to the increased colour obtained.

Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation

During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation.

Yeast population dynamics during prefermentative cold soak of Cabernet Sauvignon and Malbec wines

Prefermentative cold soak is a widely used technique in red wine production, but the impact on the development of native yeast species is hardly described. The aim of this work was to analyse the dynamics and diversity of yeast populations during prefermentative cold soak in red wines. Three different temperatures (14±1°C; 8±1°C and 2.5±1°C) were used for prefermentative cold soak in Cabernet Sauvignon and Malbec grape musts. Saccharomyces and non-Saccharomyces populations during cold soak and alcoholic fermentation were analysed. In addition, the impact on chemical and sensory properties of the wines was examined. Yeast dynamics during prefermentative cold soak were temperature dependent. At 14±1°C, the total yeast population progressively increased throughout the cold soak period. Conversely, at 2.5±1°C, the yeast populations maintained stable during the same period. Prefermentative cold soak conducted at 14±1°C favoured development of Hanseniospora uvarum and Candida zemplinina, whereas cold soak conducted at 8±1°C favoured growth of Saccharomyces cerevisiae. At 2.5±1°C, no changes in yeast species were recorded. Acidity and bitterness, two sensory descriptors, appear to be related to wines produced with prefermentative cold soak carried out at 14±1°C. This fact could be associated with the increase in non-Saccharomyces during the prefermentation stage. Our results emphasise the importance of the temperature as a determinant factor to allow an increase in non-Saccharomyces population during prefermentative cold soak and consequently to modify sensorial attributes of wines as well as their sensorial impact.

Influence of organic viticulture on non-Saccharomyces wine yeast populations

This study evaluated the population dynamics of non-Saccharomyces biota during spontaneous fermentation of organic musts. Non-Saccharomyces yeasts were found to be present at high levels during all fermentations. A total of 543 yeast colonies were isolated, 190 from Lysine-Medium (LM) agar, 254 from Wallerstein Laboratory Nutrient (WLN) agar and 99 from YPD agar. To estimate yeast population dynamics during spontaneous fermentation a genotypic approach was applied. PCR-RFLP of the ITS1-5.8S rDNA-ITS2 region and sequence determination of the D1/D2 region of the 26S rRNA gene enabled identification of the yeast isolates at the species level. Hanseniaspora uvarum, Metschnikowia fructicola and Candida zemplinina predominated, while Issatchenkia terricola, Issatchenkia orientalis and Pichia sp. were identified with a lower frequency. Hanseniaspora uvarum, M. fructicola and C. zemplinina represented 43%, 31% and 11% of the total non-Saccharomyces population isolated, respectively. Some yeast isolates were shown to be closely related to Hanseniaspora spp. and Candida spp. on the basis of the D1/D2 sequences. Based on those results, the coexistence of different Hanseniaspora and Pichia species in grape musts was supposed, and their complete identification was achieved using additional molecular markers. Moreover, strain typing and differentiation was carried out by RAPD-PCR. High strain polymorphisms were observed in the different species. For some strains, appreciable properties were demonstrated both in vitro by the API-ZYM test and in must. In must microvinification some strains showed good fermentation performances, low production of acetic acid and a partial capability to degrade malic acid.

Yeast Flora During the Fermentation of Wines made from Vitis vinifera L. cv. Emir and Kalecik Karasi grown in Anatolia

A study was conducted of the growth of Saccharomyces (S.) and non-Saccharomyces populations during alcoholic fermentation of Emir and Kalecik karasi grape varieties in the 1998 and 1999 vintages. Kloeckera (Kl.) apiculata, Kluyveromyces (K.) thermotolerans, S. cerevisiae and Candida (C.) pulcherrima were the dominant yeasts in fermentation of the 1998 vintage in Emir must. Kl. apiculata and K. thermotolerans proliferated at the beginning of the fermentation. The number of these yeasts eventually decreased when S. cerevisiae appeared as the dominant yeast on day four. But they remained until the end of the fermentation. Kl. apiculata, C. dattilla, C. pulcherrima, C. krusei and S. cerevisiae were found during the fermentation of the 1999 vintage Emir must. The count of S. cerevisiae was very high in the juice, but after skin fermentation and cold treatment, it disappeared and could not be isolated until day six of the fermentation. Kl. apiculata and C. dattilla were the dominant yeasts until S. cerevisiae started proliferation after the middle of the fermentation. Kl. apiculata, Metschnikowia (M.) pulcherrima, S. cerevisiae, C. holmii, C. valida, C. guillermondii and Candida sp. were isolated during the fermentation of Kalecik karasi must in 1998. Kl. apiculata, C. pulcherrima, S. cerevisiae, C. holmii and C. valida were identified in fermentation of must in the 1999 vintage.

Screening and typing of Patagonian wine yeasts for glycosidase activities.

The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines. The study was carried out in oenological autochthonous yeasts from Comahue region (Argentinean North Patagonia). A set of screenable yeast phenotypic characteristics indicative of their potential usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-Saccharomyces populations. Twelve isolates showing six different glycosidase phenotypes were selected and they were characterized at species and strain levels using molecular methods. A close correlation between molecular and phenotypic characteristics was observed. Five strains belonging to Candida guilliermondii, C. pulcherrima and Kloeckera apiculata with highest constitutive beta-glucosidase activity levels without anthocyanase activity were discriminated. Some of them also showed constitutive beta-xylosidase and inductive alpha-rhamnosidase activities. The extension of the selection of oenological yeast to non-Saccharomyces species provided strains possessing novel and interesting oenological characteristics which could have significant implications in the production of more aromatic young red wine. As these non-Saccharomyces are indigenous to wine, they can be used in mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine nuance that is typical of this specific area.