Cerevisiae Starter Cultures Research Articles

Differences in the Volatile Profile of Apple Cider Fermented with Schizosaccharomyces pombe and Schizosaccharomyces japonicus

In this study, two strains of Schizosaccharomyces pombe (NCAIM Y01474T and SBPS) and two strains of Schizosaccharomyces japonicus (DBVPG 6274T, M23B) were investigated for their capacity to ferment apple juice and influence the volatile compounds of cider compared to Saccharomyces cerevisiae EC1118. The ethanol tolerance and deacidification capacity of Schizosaccharomyces yeasts could make them potential substitutes for the commonly used S. cerevisiae starter cultures. Despite different time courses (10–30 d), all strains could complete the fermentation process, and Schizosaccharomyces strains reduced the concentration of malic acid in the apple juice. Results indicated that each yeast exerted a distinctive impact on the volatile profile of the apple cider, giving final products separated using a principal component analysis. The volatile composition of the cider exhibited significant differences in the concentration of alcohols, esters, and fatty acids. Particularly, the flocculant strain S. japonicus M23B increased the levels of ethyl acetate (315.44 ± 73.07 mg/L), isoamyl acetate (5.99 ± 0.13 mg/L), and isoamyl alcohol (24.77 ± 15.19 mg/L), while DBVPG 6274T incremented the levels of phenyl ethyl alcohol and methionol up to 6.19 ± 0.51 mg/L and 3.72 ± 0.71 mg/L, respectively. A large production of terpenes and ethyl esters (e.g., ethyl octanoate) was detected in the cider fermented by S. cerevisiae EC1118. This study demonstrates, for the first time, the possible application of S. japonicus in cider-making to provide products with distinctive aromatic notes”.

Influence of L. thermotolerans and S. cerevisiae Commercial Yeast Sequential Inoculation on Aroma Composition of Red Wines (Cv Trnjak, Babic, Blatina and Frankovka)

Even though Saccharomyces cerevisiae starter cultures are still largely used nowadays, the non-Saccharomyces contribution is re-evaluated, showing positive enological characteristics. Among them, Lachancea thermotolerans is one of the key yeast species that are desired for their contribution to wine sensory characteristics. The main goal of this work was to explore the impact of L. thermotolerans commercial yeast strain used in sequential inoculation with S. cerevisiae commercial yeast on the main enological parameters and volatile aroma profile of Trnjak, Babić, Blatina, and Frankovka red wines and compare it with wines produced by the use of S. cerevisiae commercial yeast strain. In all sequential fermented wines, lactic acid concentrations were significantly higher, ranging from 0.20 mg/L in Trnjak up to 0.92 mg/L in Frankovka wines, while reducing alcohol levels from 0.1% v/v in Trnjak up to 0.9% v/v in Frankovka wines. Among volatile compounds, a significant increase of ethyl lactate and isobutyl acetate, geraniol, and geranyl acetate was detected in all wines made by use of L. thermotolerans. In Babić wines, the strongest influence of sequential fermentation was connected with higher total terpenes and total ester concentrations, while Trnjak sequentially fermented wines stood up with higher total aldehyde, volatile phenol, and total lactone concentrations. Control wines, regardless of variety, stood up with higher concentrations of total higher alcohols, especially isoamyl alcohol. The present work contributed to a better understanding of the fermentation possibilities of selected non-Saccharomyces strains in the overall red wine quality modeling.

Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy

Saccharomyces cerevisiae starter cultures are largely used in winemaking to repress the wild microorganisms and achieve more predictable and desired outcomes. Notwithstanding, alternative microbial resources received increasing attention for their potential to produce wines with more distinctive and typical features. Our previous survey revealed a great inter- and intra-species diversity in an extensive collection of non-Saccharomyces wine yeasts from multiple regions of Italy. This study aimed to explore the detected biodiversity evaluating the quality of wines obtained by sequential inoculation of specific selected strains of the collection (Lachancea thermotolerans or Metschnikowia spp. or Starmerella bacillaris), and S. cerevisiae EC 1118. Fermentations of natural grape must at laboratory scale were followed by microbiological, chemical and sensorial analysis of the wines. The results indicated that each yeast species and strain exerted a distinctive impact on the wine, giving final products clearly separated with Principal Component Analysis. In particular, L. thermotolerans contributed producing relevant amounts of lactic acid and had the highest potential to reduce ethanol content; the presence of S. bacillaris increased the level of glycerol, and, remarkably, reduced acetaldehyde and total SO2; Metschnikowia spp. promoted the formation of higher alcohols and esters, and reduced volatile phenols. The sensory analysis based on the orthonasal aroma confirmed the separation between the wines obtained with the sequential fermentations and the control with single inoculation of EC 1118, although the three non-Saccharomyces species used could not be clearly distinguishable by the panelists. This study indicates that the use of selected native non-Saccharomyces strains in conjunction with S. cerevisiae positively modulates some relevant chemical parameters, and improves the aromatic intensity of wine, therefore justifying investments in non-conventional yeasts as co-starter cultures.

Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking.

The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.

Biocontrol activity of Starmerella bacillaris yeast against blue mold disease on apple fruit and its effect on cider fermentation.

The reduction of chemical fungicides in agriculture has led to the use of microorganisms as biocontrol agents. Starmerella bacillaris is a non-Saccharomyces yeast associated with overripe and botrytized grape berries microbiota. Its use has been proposed for wine fermentation because of yeast fructophilic character and high glycerol production. Recently, S. bacillaris has been demonstrated to possess antifungal activity against Botrytis cinerea on the grape. Penicillium expansum is the pathogen responsible for the blue mold rot, the most important postharvest disease of apples. These fruits are the raw material of the cider, an alcoholic beverage commonly produced using S. cerevisiae starter cultures. In this study 14 S. bacillaris strains have been studied to evaluate their postharvest antifungal activity against P. expansum on apples. Moreover, the fermentation performances in apple juice of these non-Saccharomyces strains were tested, both in single-strain fermentation and in sequential fermentation, together with S. cerevisiae. Four S. bacillaris strains, able to significantly decrease blue mold rot symptoms and to increase glycerol content during fermentation have been selected to improve apple and cider quality.

Geographical Distribution of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards in the Coastal Regions of the Western Cape in South Africa

Notwithstanding numerous studies on the yeast biota of grapes and grape must, the origin of the primary wine yeast Saccharomyces cerevisiae has been rather controversial. One school of thought claims that the primary source of S. cerevisiae is the vineyard, whereas another believes that ecological evidence points to a strict association with artificial, man-made environments such as wineries and fermentation plants. One of the main thrusts of these kinds of investigations is to understand the succession of yeasts during fermentation of wine and to determine the actual contribution of indigenous strains of S. cerevisiae and wild yeast species to the overall sensorial quality of the end product, even in guided fermentations using selected S. cerevisiae starter cultures. There is increasing interest within the wine community in the use of indigenous strains of S. cerevisiae and mixed starter cultures, tailored to reflect the characteristics of a given region. Against this background we have launched a comprehensive and long overdue biogeographical survey systematically cataloging yeasts in different climatic zones of the 350-year-old wine-producing regions of the Western Cape. The present paper represents the first phase of this programme aimed at preserving and exploiting the hidden oenological potential of the untapped yeast biodiversity in South Africa's primary grape-growing areas. Grapes were aseptically harvested from 13 sites in five areas in the coastal regions of the Western Cape. After fermentation, 30 yeast colonies per sample were isolated and examined for the presence of S. cerevisiae. Five sampling sites yielded no S. cerevisiae. CHEF-DNA analysis revealed the presence of 46 unique karyotypes in eight of the remaining sites. No dominant strain was identified and each site had its own unique collection of strains. The number of strains per site varied from two to 15. Only in four cases did one strain appear at two sites, while only one instance of a strain occurring at three sites was recorded. All sites contained killer and sensitive strains; however, killer strains did not always dominate. Commercial strains were recovered from three sites. Although commercial yeasts dominated the microflora at two sites, it appears that fears of commercial yeasts ultimately dominating the natural microflora seem to be exaggerated.

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production.

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.

Antimicrobial activity ofMetschnikowia pulcherrimaon wine yeasts

In the present study, it was investigated the antagonistic behaviour of Metschnikowia pulcherrima, as biocontrol agent, against the main wine yeast species involved in the winemaking process. Seven strains of M. pulcherrima were evaluated for the antimicrobial activity against 114 yeast strains belonging to Pichia, Candida, Hanseniaspora, Kluyveromyces, Saccharomycodes, Torulaspora, Brettanomyces and Saccharomyces genera. Results showed both different inter-generic and intra-generic responses to the antimicrobial action of M. pulcherrima strains. Interestingly, the antimicrobial activity of M. pulcherrima did not have any influence on the growth of Saccharomyces cerevisiae. Instead, M. pulcherrima displayed a broad and effective antimicrobial action on undesired wild spoilage yeasts, such as Brettanomyces/Dekkera, Hanseniaspora and Pichia genera. Fermentation trials carried out in synthetic grape must confirmed the antimicrobial activity of M. pulcherrima, determining the early death of the non-Saccharomyces co-inoculated cultures. The antimicrobial activity of M. pulcherrima does not seem due to proteinaceous compounds such as killer phenomenon, but to the pulcherriminic acid (the precursor of pulcherrimin pigment) that depletes iron present in the medium, making it not available to the other yeasts. These data agree with and further support the potential use of selected M. pulcherrima strains in controlled multistarter fermentations with S. cerevisiae starter cultures.

Effect of co-fermentation with Saccharomyces cerevisiae and Torulaspora delbrueckii or Metschnikowia pulcherrima on the aroma and sensory properties of mango wine

The effect of pure and mixed culture fermentations by Saccharomyces cerevisiae and Metschnikowia pulcherrima or Torulaspora delbrueckii on physicochemical and sensory qualities of the mango wines were investigated under laboratory conditions. S. cerevisiae produced alcohol at 11.9% from sugar, while one M. pulcherrima and two T. delbrueckii strains (NCIM and IIHR) produced alcohol at 3.8, 7.2 and 6.9% (v/v) in their mono-cultures, respectively. However, in their co-fermentation, they produced similar alcohol content to that of S. cerevisiae mono-culture: 11.04, 11.53, 11.35% (v/v) for S. cerevisiae + M. pulcherrima and S. cerevisiae + T. delbrueckii strains (NCIM and IIHR), respectively. The formation of major volatile compounds in mango wine was assessed by gas chromatography and the analysis showed that the wines from mixed cultures presented differences in the concentration of volatiles. Further, the wines produced by co-fermentation indicated that these non-Saccharomyces strains could be used with S. cerevisiae starter cultures to increase glycerol ranging from 5.4 to 7.6 and to reduce volatile acidity from 1.28 to 0.18 as well as the total acidity from 5.5 to 3.8 (g/l) of the final wines. These characteristics positively influenced the sensory qualities of the wines produced with mixed cultures, which was reflected in the preferences of these wines by panelists. The results emphasized the potential of employing indigenous non-Saccharomyces yeast strains for the production of mango wines with improved flavor.

Selected non- Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non- Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non- Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non- Saccharomyces yeasts of the genera Candida, Lachancea ( Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non- Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non- Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non- Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non- Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non- Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).

Non-Saccharomyces wine yeasts have a promising role in biotechnological approaches to winemaking

Biotechnology as applied to winemaking includes several aspects of the fermentation industry, such as monitoring of microbial populations, use of selected starter cultures, and control of undesired yeasts. Over the last few decades, the control of microorganisms using biotechnological approaches has become of increasing importance in the winemaking field. The profusion of selected starter strains has allowed more extensive use of inoculated fermentations, with a consequent improvement in the control of fermentation combined with the use of new biotechnological processes in winemaking. As a consequence of this re-evaluation of the role of non-Saccharomyces yeasts in winemaking over the last few years, several studies have evaluated the use of controlled mixed fermentations using Saccharomyces and different, non-Saccharomyces, yeast species that are a part of the winemaking environment. In this context, mixed fermentations using controlled inoculations of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a practical way towards improving wine complexity and enhancing specific characteristics of a wine. Another trait in the use of non-Saccharomyces yeasts in winemaking relates to the control of spoilage microorganisms. Indeed, more strict control of undesirable yeasts is required during the various phases of wine fermentation. Moreover, there is now increasing interest in the use of natural antimicrobial agents in foods and, in this context, killer yeasts might have important roles in the control of spontaneous and/or spoilage microflora. Thus, killer toxins appear to represent an attractive solution for use as antimicrobial agents, to partially, or even completely, substitution for chemical agent use even if application costs could limit their use in winemaking.

Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking

Mixed fermentations using controlled inoculation of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a feasible way towards improving the complexity and enhancing the particular and specific characteristics of wines. The profusion of selected starter cultures has allowed the more widespread use of inoculated fermentations, with consequent improvements to the control of the fermentation process, and the use of new biotechnological processes in winemaking. Over the last few years, as a consequence of the re-evaluation of the role of non-Saccharomyces yeasts in winemaking, there have been several studies that have evaluated the use of controlled mixed fermentations using Saccharomyces and different non-Saccharomyces yeast species from the wine environment. The combined use of different species often results in unpredictable compounds and/or different levels of fermentation products being produced, which can affect both the chemical and the aromatic composition of wines. Moreover, possible synergistic interactions between different yeasts might provide a tool for the implementation of new fermentation technologies. Thus, knowledge of the Saccharomyces and non-Saccharomyces wine yeast interactions during wine fermentation needs to be improved. To reach this goal, further investigations into the genetic and physiological background of such non-Saccharomyces wine yeasts are needed, so as to apply '-omics' approaches to mixed culture fermentations.

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.