Stimulation of malo-lactic fermentation in eastern grape musts

Induced malo-lactic fermentation was stimulated in Eastern grape musts by the addition of a new fermentation enhancer product.     

Biodiversity of yeasts isolated during spontaneous fermentation of cool climate grape musts

Archives of Microbiology - Biodiversity of native yeasts, especially in winemaking, has hidden potential. In order to use the value of non-Saccharomyces strains in wine production and to minimise...     

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Functional yeast starter cultures for cocoa fermentation

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Two-step fermentation of acidic grape musts: Interactions between Schizosaccharomyces pombe and Saccharomyces spp.

The interactions between Schizosaccharomyces pombe and Saccharomyces spp. (S. cerevisiae, S. cerevisiae sake, S. bayanus, S. uvarum) were investigated by growing the yeasts in sterile, partially fermented glucose asparagine medium in flasks, and also in the Ecologen containing either synthetic medium or grape must be separating the adjacent chambers with membranes which allow free movement of medium but not of cells. The growth of Sch. pombe was inhibited by Saccharomyces spp. to a varied extent, but the reverse was not observed. Saccharomyces uvarum, and S. cerevisiae more strongly inhibited Sch. pombe than the other species tested. All three strains of Sch. pombe (ICV-M, BG, ATCC-16979) were inhibited by S. cerevisiae although ICV-M and ATCC strains were more sensitive than BG. The higher growth rate of S. cerevisiae resulted in the exhaustion of nutrients, and its metabolic products could possibly be responsible for the growth inhibition of Sch. pombe. In the light of the present experimental results, the suitability of a two-step fermentation process for making better quality wines from acidic grape musts is discussed.     

Influence of skin maceration and oxygen on anaerobic fermentation of grape musts with high sugar content

Saccharomyces cerevisiae race cerevisiae was used to ferment grape musts in strictly anaerobic conditions, subjected to a prefermentative treatment of skin maceration and following a short aeration after 48 h of fermentation. Skin maceration caused an increase in the cellular phospholipid content which affected neither viability nor the fermentative capacity of the yeasts. The short aeration had no significant effect on the unsaturation index of the cellular fatty acids, although it did increase the ergosterol/phospholipid ratio. This was reflected by an increase in the growth rate, viability and fermentative capacity of the yeasts. Maceration did not increase the effect of aeration.     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

Analysis of non‐Saccharomyces yeast populations isolated from grape musts from Sicily (Italy)

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.     

Ethyl esterification of long-chain unsaturated fatty acids derived from grape must by yeast during alcoholic fermentation

The composition of total fatty acid ethyl ester (FAEE) in yeast cells and the liquid phase separated from grape must during alcoholic fermentation at different temperatures was investigated by using the solid-phase extraction method. Thirteen FAEE from butyric to linolenic acids were detected during fermentation. Significant amounts of long-chain unsaturated FAEE, including linoleic and linolenic acids derived from grape material, had already accumulated in the yeast cells by day 3 during fermentation.     

Membrane-integrated fermentation system for improving the optical purity of D-lactic acid produced during continuous fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.     

Biodiversity of Saccharomyces yeast strains from grape berries of wine-producing areas using starter commercial yeasts

The use of commercial wine yeast strains as starters has grown extensively over the past two decades. In this study, a large-scale sampling plan was devised over a period of 3 years in three different vineyards in the south of France, to evaluate autochthonous wine yeast biodiversity in vineyards around wineries where active dry yeasts have been used as fermentation starters for more than 5 years. Seventy-two spontaneous fermentations were completed from a total of 106 grape samples, and 2160 colonies were isolated. Among these, 608 Saccharomyces strains were identified and 104 different chromosomal patterns found. The large majority of these (91) were found as unique patterns, indicating great biodiversity. There were differences in biodiversity according to the vineyard and year, showing that the biodiversity of Saccharomyces strains is influenced by climatic conditions and specific factors associated with the vineyards, such as age and size. Strains that were terroir yeast candidates were not found. The biodiversity of S. cerevisiae strains after harvest was similar to that in the early campaign; moreover, a temporal succession of S. cerevisiae strains is shown. This fact, together with the differences in biodiversity levels verifies that other factors were more important than commercial yeast utilization in the biodiversity of the vineyard.     

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5′-phosphate decarboxylase (URA3) or orotidine-5′-pyrophosphatase (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A␣transformation frequency of approximately 10⁴/μg DNA was obtained, which is tenfold higher than results described in earlier reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid. Received: 19 August 1996 / Received last revision: 30 April 1997 / Accepted: 4 May 1997     

The effects of temperature and pH on the growth of yeast species during the fermentation of grape juice

The effects of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Candida krusei, Candida pulcherrima and Hansenula anomala were examined during mixed culture in grape juice. At 25°C, pH 3.0 and pH 3.5, S. cerevisiae dominated the fermentation and the other species died off before fermentation was completed. Saccharomyces cerevisiae also dominated the fermentation at 20°C but there was increased growth and survival of the other species. At 10°C the fermentation was dominated by the growth of both S. cerevisiae and K. apiculata and there was extended growth and survival of C. stellata and C. krusei. Juices fermented at 10°C exhibited ethanol concentrations between 7.4 and 13.4% and populations of K. apiculata, C. stellata and C. krusei in the range 10⁶-10⁸ cells/ml. However, these species produced maximum ethanol concentrations in the range 2.7–6.6% when grown as single cultures in grape juice.     

Diversity and evolution of non-Saccharomyces yeast populations during wine fermentation: effect of grape ripeness and cold maceration

We have evaluated the effect of grape maturity and cold maceration prior to fermentation on the yeast ecology during wine fermentation. Non-Saccharomyces strains were selectively isolated and identified using two rapid PCR techniques, namely enterobacterial repetitve intergenic consensus-PCR and PCR-intron splice sites, in various wine fermentation conditions. These identifications were further complemented and confirmed by restriction fragment length poymorphism and sequencing analysis of the 5.8S-ITS and D1/D2 ribosomal regions, respectively. Eleven species belonging to five genera were identified. Candida stellata, Hanseniaspora uvarum and Hanseniaspora osmophila were the dominant species, representing almost 90% of the isolates. Minor strains presented different species of the genera Candida, Issatchenkia, Zygoascus and Zygosaccharomyces. Selective isolation made it possible to isolate some species that were hardly related to the wine-making process, such as Issatchenkia hanoiensis, a new species that has only been described recently.     

Transformation of lignins from grape solids during alcoholic fermentation

Conversion of lignins contained in solid parts of Rkatsiteli grapes (crests, seeds, and skin) during alcoholic fermentation by wine yeast in Reader's medium was studied. Various species of wine yeast were used: Saccharomyces oviformis, S. vini Kakhuri 42, S. chodati Teliani 79, and S. uvarum Tsinandali 77. We found that lignins from solid parts of grapes are partially decomposed during alcoholic fermentation, which releases low-molecular-weight aromatic compounds into the medium. A peculiar feature of lignin decomposition during alcoholic fermentation is the formation of reduction products.     

Intracellular pH of yeast during brewery fermentation

The intracellular pH value of Saccharomyces cerevisiae NCYC 1681 was measured using radiolabelled [¹⁴C]-propionic acid. Errors, due to the binding of radioactive material to trub, were eliminated using silicone oil centrifugation. Replication of analyses reduced the variations associated with low cell counts during fermentation. Whilst fermenting brewer's wort, yeast intracellular pH values were maintained within a narrow range (5.9–6.4). Cellular ATP concentrations were highly conserved in spite of the fact that the cells were exposed to an increasing concentration of ethanol as the fermentation progressed.