Diversity And Dynamic Change Of Yeasts During Icewine Fermentation

Icewine is honoured as the liquid gold for its unique, full-bodied aroma, high value andlong brewing process. The high sugar and acid of icewine grapes and low temperature duringfermentation process gives strict requirement to the yeasts.The evolution of yeast species and Saccharomyces cerevisie genotypes during icewinefermentations of two main grape varieties (Merlot, Italian Riesling) in Gansu region of Chinawere followed in this study. Using a combination of colony morphology on WallersteinsLaboratory Nutrient (WLN) medium, sequence analysis of the26S rDNA D1/D2domain andinterdelta sequence fingerprinting analysis, a total of1214isolates were identified at thespecies level. The aim of this study were to discover the wine-related yeasts resources anddiversity in Gansu icewine fermentation, to provide the basis for microbial control, flavor andquality improvement of icewine and to screen out more superior indigenous S. cerevisie foricewine in the future. The main research results are as follows:1: The524colonies isolated from spontaneous and inoculated fermentations of Merloticewine represented12types on WLN medium belonging to6species of6genera, namely, S.cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans (Kluyveromycesthermotolerans), Hanseniaspora uvarum, Metschnikowia aff. Fructicola, Pichiasubpelliculosa, with the proportion of65.5%、2.7%、19.1%、9.9%、0.2%、2.7%,respectively. In addition, S. cerevisiae (A) and H. uvarum (D) had two different WLN types;T.delbrueckii (B) and L.thermotolerans (C) had three different WLN types, respectively.The690colonies isolated from spontaneous and inoculated fermentations of ItalianRiesling icewine represented14types on WLN medium belonging to8species of6genera,namely, S. cerevisiae, Saccharomyces uvarum, T. delbrueckii, H. uvarum, L. thermotolerans(K. thermotolerans), M. aff. Fructicola, Hanseniaspora osmophila, Schizosaccharomycespombe, with the proportion of90.58%、2.46%、1.01%、1.45%、2.90%、0.29%、0.86%、0.43%, respectively. In addition, T.delbrueckii (Cc) and H. osmophila (Gg) had two differentWLN types; S. cerevisiae (Aa) and L. thermotolerans (Ee) had three different WLN types,respectively.2: The species, strain quantity and proportion of yeast showed some regularity duringdifferent stages of Merlot icewine fermentation. The non-saccharomyces here displayed strong competitiveness and started Merlot icewine fermentation. After crushing we could finda mass of non-saccharomyces (49.3%) mainly consisting of T. delbrueckii (17.6%) and L.thermotolerans (30.4%). The early stage of the spontaneous fermentation was dominated bythe non-saccharomyces(68.4%) which were the main strains starting Merlot icewinefermentation and the proportion of the S. cerevisie is not high which occupied31.6%. Aboveall, L. thermotolerans and H. uvarum (35.6%and26.9%, respectively) have become thepreponderant strains to start the fermentation. Some non-saccharomyces were sensitive toalcohol, died out gradually during middle stage of the fermentation. While S. cerevisiae grewwell and dominated the fermentation (92.3%) during this stage since it has good tolerance ofalcohol. At the end stage, non-saccharomyces died out and S. cerevisiae gained priority(100%) and finished the fermentation to the end. Compared with spontaneous fermentation,the inoculated fermentation showed more activity at the middle stage of fermentation. At theearly stage of inoculated fermentation there were more non-saccharomyces (94.7%) than S.cerevisiae (5.3%). L. thermotolerans and H. uvarum (48.9%and27.5%) displayed moreactivity in inoculated fermentation and became the preponderant starting strains. At themiddle stage, S. cerevisiae adapted gradually to the environment, grew well and replaced thenon-saccharomyces to be the dominant strains, while non-saccharomyces died out graduallyand dropped to13.4%but maintaining some vitality. At the end stage, non-saccharomycesdied out and S. cerevisiae became predominant (100%) and finished the fermentation to theend.Italian Riesling icewine fermentation also showed some regularity: Thenon-saccharomyces here showed less competitiveness and died out gradually from the earlystage. After crushing we could found plentiful non-saccharomyces including S. uvarum, T.delbrueckii, L. thermotolerans, M. fructicol, H. osmophila, S. pombe mainly consisting of S.uvarum (32.6%) and S. pombe (5.4%) which could found only in the grape raw material. Theearly stage of the spontaneous fermentation is dominated by the S. cerevisiae (96.0%) whichwere also the main starting fermentation strains. From now on S. cerevisiae dominated andfinished the fermentation to the end. Compared with spontaneous fermentation, S. cerevisiaeshowed more activity in the inoculated fermentation with the proportion of93.2%、91.0%、88.4%、99.0%in the four stages (T2、T3、T4、T5) of Italian Riesling inoculated fermentation.3: Twelve genotypes (G1~G12) of S. cerevisiae were distinguished by interdeltasequence analysis among the total of randomly selected125S. cerevisiae isolates from Merloticewine spontaneous and inoculated fermentation, with the proportion of7.1%、54.8%、2.4%、12.7%、12.7%、4.0%、0.8%、0.8%、0.8%、0.8%、1.6%、0.8%, respectively. Eighteen genotypes (g1~g18) of S. cerevisiae were distinguished by interdelta sequenceanalysis among the total of randomly selected211S. cerevisiae isolates from Italian Rieslingicewine spontaneous and inoculated fermentation, with the proportion of12.3%、4.3%、6.2%、8.1%、32.2%、14.2%、2.8%、1.4%、4.7%、5.2%、4.7%、0.9%、0.5%、0.5%、0.5%、0.5%、0.5%、0.5%, respectively.Compared with the Merlot and Italian Riesling icewine fermentation, we found that G8、G2、G4have the same genetype to g1、g3、g17, respectively, and got9preponderantgenotypes(G1、G2、G4、G5、G11、g1、g4、g5、g6) for comparing their proportion, competitivecapacity, sustainable growth and the contribution of completing the fermentation. Focusing onresearching the vinification characteristics of those strains in the future could foundpreponderant yeasts for wine fermentation even icewine fermentation.Unexpectly, the inoculated wine yeast (Levuline SEWA) was detected neither in Merlotinoculated fermentation nor in Italian Riesling inoculated fermentation. Therefore, it isconcluded that the inoculated wine yeast did not conduct and dominate the two icewinefermentations and it was that the indigenous S. cerevisiae fulfilled the fermentations, Inaddition, the winery commercial yeast was not detected, which indicated that icewine grapeswere not affected by the winery environment.