Adriana C. Caballero

Patagonian wines: the selection of an indigenous yeast starter.

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.

Killer behaviour in wild wine yeasts associated with Merlot and Malbec type musts spontaneously fermented from Northwestern Patagonia (Argentina)

The occurrence of killer wine yeasts in Comahue Region (Patagonia, Argentina) was studied. Wild wine yeasts were isolated from spontaneously fermenting Merlot and Malbec type musts. Out of 135 isolates analyzed 37% were sensitive to some well characterized killer toxins as K1–K10 and did not show killer activity (sensitive phenotype, S), 21% showed neutral phenotype (N) and 42% demonstrated killer activity (killer phenotype, K). All but two killer strains, identified as Candida pulcherrima and Kluyveromyces marxianus, were Saccharomyces cerevisiae. Additionally, all killer strains were sensitive to some killer reference strains, showing a killer‐sensitive phenotype (KS); neither Saccharomyces or non‐Saccharomyces wild yeasts were phenotype killer‐resistant (KR).

Combined use of killer biotype and mtDNA-RFLP patterns in a Patagonian wine Saccharomyces cerevisiae diversity study

The aim of this work was to characterize the indigenous wine Saccharomyces cerevisiae diversity within the Argentinean Patagonia. Two cellars with particular enological practices located in different winegrowing areas were selected and 112 indigenous S. cerevisiae isolates were obtained from spontaneous red wine fermentations carried out in them. Thirty-five and 19 patterns were distinguished among the total indigenous isolates using mtDNA-RFLP and killer biotype analysis, respectively. The combination of both typing techniques rendered a higher variability with 42 different patterns, i.e. 42 strains, evidencing a great diversity in S. cerevisiae populations associated with spontaneous red wine fermentations in Northwestern Patagonia. The analysis of the relatedness among strains using Principal Coordinates Analysis from combined data allowed the clustering of the strains into two populations significantly related to their origin fermentations. The combined use of the mtDNA-RFLP analysis together with the killer biotype method proved to be a powerful tool in the fingerprinting of the enological S. cerevisiae strains.

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.

OENOCOCCUS OENI FROM PATAGONIAN RED WINES: ISOLATION, CHARACTERIZATION AND TECHNOLOGICAL PROPERTIES

Pinot noir vinifications carried out at industrial scale during 2008 vintage were monitored in Patagonian region, Argentina, and several lactic acid bacteria were obtained. By analysis of a fragment of rpo B gene, species- specific PCR of malolactic enzyme and sequencing of 16S rRNA gene, three isolates were identified as Oenococcus oeni. Polyphasic typing made by carbohydrate fermentation behaviour and RAPD-PCR grouped the isolates and O. oeni reference strain in three different clusters. Additionally, the effect of ethanol on bacterial growth and malolactic activity was evaluated. In spite of some inhibitory effects were found, the three isolates were able to growth in all ethanol concentration tested. A clear correlation was found between the different clusters obtained and their ethanol tolerance. All indigenous O. oeni showed a strong malolactic activity at all ethanol concentrations assayed, indicating that this alcohol just affect the growing parameters of bacteria, but not the malolactic activity. These promissory results suggest a potential enological application of the O. oeni indigenous isolates as malolactic fermentation starters in winemaking. Keywords- Pinot noir wine, Argentinean North Patagonia, malolactic fermentation, Oenococcus oeni

Advantages of Using Blend Cultures of Native L. plantarum and O. oeni Strains to Induce Malolactic Fermentation of Patagonian Malbec Wine

The malolactic fermentation (MLF) of Patagonian Malbec wine inoculated with blend cultures of selected native strains of Lactobacillus plantarum and Oenococcus oeni was monitored during 14 days, analyzing the strains ability to modify the content of some organic acids and to change the volatile compounds profile. The performance of the LAB strains was tested as single and blends cultures of both species. An implantation control by RAPD PCR was also carried out to differentiate among indigenous and inoculated strains. The L. plantarum strains UNQLp11 and UNQLp155 and the O. oeni strain UNQOe73.2 were able to remain viable during the monitoring time of MLF, whereas the O. oeni strain UNQOe31b showed a decrease of five log CFU at day 14. The four strains assayed showed a similar behavior in wine whether they were inoculated individually or as blend cultures. All strains were able to consume L-malic acid, particularly the L. plantarum strains, which showed the highest consumption values at day 14, both as single or blend cultures. The changes in the volatile compounds profile of Malbec wine samples, before and after MLF, were determined by HS-SPME and GC-MS technique. Wines inoculated with blend cultures containing strain UNQLp155 showed a decrease in the total alcohols content and an increase in the total esters content. On the other hand, wines inoculated with single cultures of strains UNQLp155, UNQOe31b or UNQOe73.2 showed no significant decrease in the total alcohols concentration but a significant increase in the total esters content. When strain UNQLp11 was inoculated as single or as blend culture with strain UNQOe31b, wines exhibited an increase in the total alcohols content, and a decrease in the total esters content. The content of diethyl succinate showed the greatest increase at final of MLF, and a particular synergistic effect in its synthesis was observed with a blend culture of strains UNQLp155 and UNQOe73.2. These results suggest that the use of blend cultures formulated with strains belonging to L. plantarum and O. oeni species could offer an interesting advantage to induce MLF in Malbec wines, contributing to diversify their aromatic profiles.

Wild Yeasts Selection for High-Quality Patagonian Wines

The use of yeast starters indigenous to each winegrowing area is today a worldwide oenological practice aiming to imprint a differential character to the wine. This chapter summarizes the research carried out towards the design and development of indigenous wine yeast starters from Argentinean Patagonia that are currently nonexistent in the market. The results shown include diversity and criteria for Saccharomyces and non-Saccharomyces wild yeast strain selection as well as their contribution to the physicochemical and sensorial qualities of wines when they are used either singly or in combined forms. The advantages of these indigenous starters over the foreign commercial ones to imprint a differential character on regional wines are also discussed.

The Use of Indigenous Yeast to Develop High-Quality Patagonian Wines

In young wines, the compounds responsible for wine flavor come from two possible origins: grapes and microorganisms involved in winemaking. Yeasts play the most important role in flavor influence because of their role in conducting the alcoholic fermentation (FA), the key process of winemaking. Ecological studies show that yeast diversity is significantly influenced by geographical and technological features of each particular winegrowing region. Wines from Argentine have achieved high-quality certifications, and particularly, in the Comahue region, wine production is mainly oriented to young red wines varieties, some of which found in this region optimal ecological condition to express all their enological potential. Despite this, the need to satisfy the demands of an increasingly competitive and globalized international market and the consumer demand for new wine styles with the best quality/price ratio imposes the regional productive sector new challenges that require technological innovation. The use of starter cultures developed from indigenous yeast isolated from our region, specially selected for its enological properties, appears as a valuable tool for differentiation, diversification, and quality improvement of wines. In this context, conventional and non-conventional yeasts were isolated and selected over the years and used for vinifications in red grape varieties (Pinot noir and Malbec). Assays were carried out at laboratory and pilot scale, in the 2010–2015 vintages. The experiences developed along the years contribute to a better understanding of the processes involved in the production of improved wines by autochthonous strains, an important practice to develop a more competitive regional wine industry.