Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Inventory and monitoring of wine microbial consortia

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.     

Three‐phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of ‘soidon’ (indigenous fermented food) in North‐east India were studied using cultivation‐dependent and cultivation‐independent molecular approaches. Cultivation‐dependent analyses (PCR‐amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation‐independent analyses (PCR‐DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three‐phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1–2 days) which was joined by Leuconostoc citreum during the mid‐phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5–7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation‐dependent studies complemented with cultivation‐independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.     

A metabolomics based approach for understanding the influence of terroir in Vitis Vinifera L.

This study aims to understand the contribution of ‘terroir’ during the cultivation of Vitis vinifera L. The concept of terroir stems from the French ideal that a region’s soil and local vineyard topography together with a region’s macroclimate, including the mesoclimate and vine microclimate, together define the unique characteristics of a wine. In this current study we have utilized high performance liquid chromatography combined with a Q Exactive quadrupole Orbitrap™ mass analyzer for the direct injection analysis of Vitis vinifera juice samples sourced from two different vineyards from the Santa Ynez AVA of Santa Barbara county. Analysis of the mass spectral data was facilitated by a differential analysis software program—SIEVE 2.0™. Distinct metabolomic signatures in freshly crushed juice samples were elucidated. Interestingly, important and distinct information was revealed from the analysis of both the positive and negative ion data. Hierarchical clustering indicated the negative ion data displayed similarity based on varietal character while results obtained in the positive ion mode clustered primarily on terroir. This may indicate that more acidic compounds are influenced by varietal character while more basic compounds are influenced by terroir. Using a feature of SIEVE 2.0 a flavonoid database was utilized to search the raw data for flavonoids present in the juice samples. This targeted analysis indicated the flavonoid profile of juice samples appears to be a good indicator of varietal character independent of terroir. The analysis presented in this study suggests distinct Vitis vinifera grape juice chemical signatures are present prior to fermentation. Further analysis will aim to attribute which of these compounds is influenced by varietal character and/or terroir.

     

Genomic variations of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strains and the potential to impact on malolactic fermentation and aroma compounds in wine

Malolactic fermentation (MLF) is the bacterially driven decarboxylation of l-malic acid to l-lactic acid and carbon dioxide, and brings about deacidification, flavour modification and microbial stability of wine. The main objective of MLF is to decrease wine sourness by a small increase in wine pH via the metabolism of l-malic acid. Oenococcus oeni is the main lactic acid bacterium to conduct MLF in virtually all red wine and an increasing number of white and sparkling wine bases. Over the last decade, it is becoming increasingly recognized that O. oeni exhibits a diverse array of secondary metabolic activities during MLF which can modify the sensory properties of wine. These secondary activities include the metabolism of organic acids, carbohydrates, polysaccharides and amino acids, and numerous enzymes such as glycosidases, esterases and proteases, which generate volatile compounds well above their odour detection threshold. Phenotypic variation between O. oeni strains is central for producing different wine styles. Recent studies using array-based comparative genome hybridization and genome sequencing of three O. oeni strains have revealed the large genomic diversity within this species. This review will explore the links between O. oeni metabolism, genomic diversity and wine sensory attributes.     

Evolutionary trends on extant cat skull morphology (Carnivora: Felidae): a three‐dimensional geometrical approach

The skulls of 33 extant cat species were characterized through three‐dimensional geometric morphometrics using 20 landmarks. A principal component analysis (PCA) was performed with Procrustes fitted coordinates, and the PC‐scores were phylogenetically corrected by independent contrasts method. Three PCs allowed for the definition of five cat skull patterns. PC1: ‘snouted/massive‐headed cats’ (genus Panthera) opposing the ‘round‐headed small cats’ (genus Oncifelis, Prionailurus rubiginosus, Prionailurus bengalensis, among other small cats); PC2: ‘tapering‐headed cats’ (Neofelis nebulosa, Herpailurus yagouaroundi, Prionailurus planiceps) opposing the ‘stout‐headed cats’ (Acinonyx jubatus, Uncia uncia, Otocolobus manul, Felis margarita, and Felis nigripes); and PC3: ‘low profiled‐headed cats’ (mostly, Pr. planiceps). A sixth pattern, the ‘generalized skull’, observed in the Caracal lineage, genus Lynx, Leopardus pardalis, and Catopuma temminckii, indicates a morphological convergence among midsized‐cats. The morphological trends ‘snouted/massive’ and ‘round’ clearly denote a co‐evolution between size and shape. The other skull patterns evolved unrelatedly to the size (i.e. their allometric variations are not a size function). Nevertheless, each species comprises an amalgam of these patterns, so the influence of the size permeates, in some extent, the skull morphology along all cat lineages. The felid ecomorphological fit to hypercarnivory is obvious; however, different skull shapes in same‐sized species with similar habits, indicate that the variation in the skull morphology may result from phenotypic fluctuations, whose adaptive value (if indeed there is any) is still obscure. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 176–190.     

Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of “fino” sherry wine making. The four races of “flor” Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Genetic variation at the alcohol dehydrogenase locus in Drosophila melanogaster in relation to environmental variation: Ethanol levels in breeding sites and allozyme frequencies

Summary Ethanol levels in Drosophila breeding sites in seepages of unfortified wine inside wineries have been found to be similar to those in many decaying fruits and vegetables. Fortified wine seepages inside wineries have ethanol levels on average three times as high as other breeding sites. However there was no evidence that this variation in ethanol levels was associated with differences in Adh ^F frequencies in D. melanogaster at sites either within wineries or outside wineries. D. simulans was at lower frequencies at sites inside wineries compared to sites outside although this difference may not be related to ethanol levels. It is concluded that adaptation to natural levels of environmental ethanol by D. melanogaster does not necessarily modify Adh frequencies.     

Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Production of higher alcohols,ethyl acetate,acetaldehyde and other compounds by 14Saccharomyces cerevisiae wine strains isolated from the same region (Salnés,N.W. Spain)

Fourteen strains of the yeastSaccharomyces cerevisiae were isolated from three wineries in the Salnés wine region (N.W. Spain) at the three different periods of the natural fermentation. Each wild yeast was screened for production of acetaldehyde, ethyl acetate, isobutanol,n-propanol, amylic alcohol and other important enological compounds during laboratory scale fermentations of grape juice. After 25 days at 20°C, the analytical results evidenced variations in the production of acetaldehyde (from 13.1 to 24.3 mg/l), isobutanol (from 27.7 to 51.1 mg/l), amyl alcohols (from 111 to 183 mg/l) and ethyl acetate (from 19.3 to 43.7 mg/l). Although isolated from the same wine region, differences in the wine composition were observed depending on the particular yeast strain used for the vinification experiments.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Bacteriocinogenic Potential of <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated from Wine

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.     

Evolution of ecological dominance of yeast species in high‐sugar environments

In budding yeasts, fermentation in the presence of oxygen evolved around the time of a whole genome duplication (WGD) and is thought to confer dominance in high‐sugar environments because ethanol is toxic to many species. Although there are many fermentative yeast species, only Saccharomyces cerevisiae consistently dominates wine fermentations. In this study, we use coculture experiments and intrinsic growth rate assays to examine the relative fitness of non‐WGD and WGD yeast species across environments to assess when S. cerevisiae’s ability to dominate high‐sugar environments arose. We show that S. cerevisiae dominates nearly all other non‐WGD and WGD species except for its sibling species S. paradoxus in both grape juice and a high‐sugar rich medium. Of the species we tested, S. cerevisiae and S. paradoxus have evolved the highest ethanol tolerance and intrinsic growth rate in grape juice. However, the ability of S. cerevisiae and S. paradoxus to dominate certain species depends on the temperature and the type of high‐sugar environment. Our results indicate that dominance of high‐sugar environments evolved much more recently than the WGD, most likely just prior to or during the differentiation of Saccharomyces species, and that evolution of multiple traits contributes to S. cerevisiae's ability to dominate wine fermentations.     

Early post‐fire succession in northwestern Patagonia grasslands

Abstract. Large‐scale disturbances, notably fire and grazing, structure grass and shrubland dynamics in semi‐arid environments. We studied early post‐fire succession in two burned grasslands, one unburned grassland, and one shrubland near the burned area. We observed three processes: (1) establishment of a ‘phantom’ community comprised of fugitive species. Although transient, these species increase diversity and recharge the seed bank before the next disturbance; (2) regeneration of the original community by persistence of resprouter species and by auto‐replacement; (3) early stages of invasion by seedlings of the shrub Fabiana imbricata, which germinate next to shrubland and create new F. imbricata patches. Weed invasion was principally due to the ruderal exotic species Verbascum thapsus from the nearby road verge and by rapid increase of Rumex acetosella cover, another exotic species present before the fire. Although post‐fire climatic conditions are particularly important in semi‐arid environments, succession depends greatly on the regeneration strategies and dispersal abilities of the species present in the burned area. The phantom community occurs only at the first stage of succession when there is little competition for resources. We could call this process ‘the race for occupation of the area’. The second stage, when competition for resources becomes progressively more important, could be called ‘the effort to maintain space’.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Wine,geology mapping and the value of place in McLaren Vale

In the wine‐producing region of McLaren Vale, South Australia, the development of a detailed geology map has worked to increase understandings about the physical diversity of vineyard land. I argue that the production and deployment of social/scientific knowledge through the map is instrumental in strategic imaginings of the viticultural space of McLaren Vale. Ethnographic examination of ‘District Tastings’ of single‐vineyard Shiraz wines shows how the geology map provides a framework for understanding and promoting relationships between vineyard geology and wine flavours. This process feeds into debates over possible delineation of official winemaking ‘subregions’, which may provide a mechanism for the generation of economic value based on discourses of quality and distinctiveness. The map's depiction of geological diversity is embraced by wine producers, who seek to add value to their product through appeals to a purported uniqueness, scarcity or specialness based on geological terroir.     

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.     

Phytoplasma Transmission by Heterologous Grafting Influences Viability of the Scion and Results in Early Symptom Development in Periwinkle Rootstock

The stolbur phytoplasma ‘Candidatus Phytoplasma solani’ is responsible for the grapevine disease ‘bois noir’ affecting a number of wine‐growing areas in Europe. Transmission of stolbur phytoplasma to different laboratory hosts can be difficult due to the requirement of transmitting insect vectors or parasite plants. Here, heterologous grafting was used as an alternative technique for transmission of common and strongly symptomatic stolbur genotypes CPsM4_At1 and CPsM4_At6 of ‘Ca. P. solani’ to experimental host plants such as Catharanthus roseus and tomato making phytoplasma strains more accessible for molecular and experimental investigations in different plant species. Transmission was confirmed by quantitative PCR, microscopy and nested PCR followed by marker gene sequencing. In our study, the transmission of different genotypes of ‘Ca. P. solani’ resulted in distinguishable symptom development in the laboratory host C. roseus. Symptom development in grafted rootstock was observed three to 7 weeks after heterologous grafting. Survival of the graft unit was influenced by the presence of ‘Ca. P. solani’ in the scions and was clearly reduced in phytoplasma free scion – rootstock combinations.