Introduction
The microbial flora present in grape juice and in wine have a striking impact on wine composition. The end products of microbial activity can directly impact wine aroma or flavor or can interact with grape components to enhance or mask varietal characters. Some volatile microbial components are detectable at quite low concentrations, well within the ranges found in wine. The more diverse the microbial flora, the more diverse the spectrum of end products of the wine will be. This may be positive or negative, depending upon the desired composition of the wine. Numerous microbes have been identified on grapes, on winery surfaces, in fermentations and during aging of wine. The biodiversity of grape and wine flora has been extensively examined using an array of technologies. Although there are some regional differences in the major genera found, a remarkably consistent progression of yeast species is seen.
A comprehensive understanding of the biodiversity of yeasts associated with grapes and the production of wine exists due to the large number of studies that have been conducted worldwide and the important role of biodiversity in the evolution of aroma and flavor compounds of the wine. The biodiversity within the genus Saccharomyces has only recently becomeappreciated, and this diversity is likewise important in creating the desired chemical constitution of the finished wine. The flora found on grapes shows a striking similarity from vineyards across the globe with differences in the dominant species being influenced by climate, altitude, vectors, grape variety, age of the grapes at harvest, disease pressure in the vineyard and intactness of the berries, and vineyard practices and seasonal conditions. The yeasts present initially during fermentation reflect the diversity of the species present on the grape surface at the time of harvest. The non-Saccharomyces yeasts can persist throughout fermentation or can be eliminated early on depending upon winemaking conditions, grape juice composition, winery practices, and type of inoculation used.
Yeast Classification
Yeast are classified taxonomically into genus and species. Taxonomic schemes can be based upon phenotypes or cellular properties such as morphology, or schemes can be based upon direct DNA sequence analysis and comparison. Taxonomic relationships among the yeast have changed over time as new analytical tools are developed and applied broadly to microbial taxonomy. The definition of a species is also variable. In general, strains of different species do not cross-breed while strains within a species may be quite different in genetic properties, but retain the ability to cross-breed. Different genera are characterized by a series of unique properties and genomic content, arrangement and similarity. Within a genus, strains are organized into species. In general, members of the same species have characteristic properties in common that distinguish them from other members of the genus. Finally, sub-species differences may also arise, leading to the appearance of genetic variants that retain the properties of the species but that are different in some recognizable way. Diversity may exist at any of these levels: genus, species or strain.
Factors Impacting Yeast Diversity
Several key factors have been shown to influence the types of yeasts present and their persistence during fermentation. Generally, the major species identified on the surface of grapes are the major species found at the onset of the alcoholic fermentation. These non-Saccharomyces yeasts impact the aroma and flavor composition of the finished wine, as well as determine its potential for microbial instability during aging and post-bottling. Significant diversity among wine isolates ofSaccharomyces has also been documented, and this diversity, likewise, impacts the composition of the finished wine. A thorough understanding of the flora present during the production of wine is important in determining the appropriate processing strategies to achieve the desired composition of the finished product.
Numerous yeast genera and species are found during the production of wine. The low pH of wine, high sugar content, rapidly generated anaerobic conditions, and presence of phenolic compounds creates the ideal environment to support the growth of yeasts and to enrich for these organisms over other microbes. The metabolic activities of yeast can have a profound impact on the composition of the wine and, therefore, of its aroma and flavor properties. Some wine styles in fact depend upon the metabolites of specific yeasts for their characteristic compositions. The yeasts that impact the composition of the wine are indigenous to the grapes and vineyard, are residents of the winery flora, and even can be spread by insect vectors such as fruit flies, bees and wasps. Any substance coming in contact with the grapes, must, or juice can be a source of inocula. The organisms found in wine can also derive from direct inoculation, using commercial yeast preparations.
Survey of yeast species found in wine production
Over twenty yeast genera have been identified from wines. In addition to this genus and species diversity, there is also significant biodiversity within a given species. Strains vary genetically from each other, leading to the expression of different biological properties. The extent and persistence of the diverse yeast populations is influenced by the winemaking conditions employed. For example, holding of must at low temperatures to increase extraction from the skins, termed a ‘cold soak’, results in a bloom of yeast species tolerant of low temperatures. The presence of these yeasts can then influence the metabolic behavior of the principle agent of the yeast fermentation, Saccharomyces, in addition to directly contributing aroma impact compounds to the wine.
Methods Of Analysis Of Yeast Diversity
A critical factor in the analysis of yeast biodiversity concerns the methodology used to identify the microbes present. Yeast diversity can be assessed using direct plating methods or by direct molecular analysis of populations. In plating methods, yeasts are cultured on solid media to isolate colonies prior to identification by physiological or molecular analyses. The act of growing yeast colonies prior to identification may result in failure to detect some species that are present or skew the relative numbers of different yeasts, as minor populations might easily be missed, given their under-representation among the original cells in the population. Direct plating on non-selective, rich media favors the faster growing yeasts such asSaccharomyces, and may limit the growth of more slowly growing yeasts, so that they are not observed. Inclusion of conditions or inhibitors to prevent or limit the growth of fast growing yeasts often prevents or limits the growth of other yeast species and strains present.
Direct culturing methods of identification
One of the most frequently used methods to identify Saccharomyces versus non-Saccharomyces yeasts is plating on Lysine Agar. Saccharomyces cerevisiae will not grow on lysine as a sole nitrogen source, therefore only non-Saccharomyces yeasts will grow on these plates. In our experience, many “wild” Saccharomyces strains will grow slowly on lysine and some other non-Saccharomyces yeast may not grow well on lysine. Ambiguous results should always be independently verified before assuming a contaminant organism is present in the wine.
One can also use a direct selection to identify non-Saccharomyces yeasts, such as plating a wine on media containing cycloheximide, a standard selection for Brettanomyces yeast and other non-Saccharomyces yeast. The more restrictive the medium, the fewer the species that will be able to grow. Wine is often analyzed by plating simultaneously on rich, or non-selective media, and on selective media. Researchers attempt to select for a wide range of organisms by plating on non-selective media, such as Wallersteins Nutrient agar (WL), and then identifying yeast by colony morphology and dye uptake. However, this method may select against yeast that grow slowly on WL, but that otherwise are highly viable in the fermentation. A direct selection, such as plating a wine on media containing cycloheximide, a standard selection forBrettanomyces yeast, can also be used, but this is limited to determining only a few species, and not all representatives of that species. To get around these issues, people often plate on several different media that select for different types of yeast. Patterns of nutrient utilization and production of secondary metabolites, as well as sporulation and morphological characteristics, were traditionally used to identify organisms after isolation. However, given the extent of the natural diversity within species, spontaneously arising mutations can alter phenotypic properties, such that the yeast is eventually misidentified. As a consequence, these methods have been almost entirely supplanted in the last decade by molecular techniques.
Molecular methods of identification
Initially, molecular techniques were used to identify yeast isolates after isolation and growth in pure culture. Many different techniques have been used to for this purpose, including polymerase chain reaction (PCR) of the 26s ribosomal DNA and sequencing, and PCR and restriction enzyme digestion of internal transcribed spacers (ITS) from the 5.8s ribosomal DNA. These techniques still contain the bias inherent in the initial plating and isolation of the organism to be identified. To get around this type of bias, direct DNA sampling methods, coupled to molecular characterization of the consortium DNA and identification of different marker sequences are being used to determine the numbers and types of yeast in an environmental sample. Techniques such as PCR combined with denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (q-PCR) have been used with great success to study the ecological succession of microbes during fermentations and to identify spoilage organisms in wine. These methods allow the identification of organisms that do not grow on a given medium under given conditions. However, these methods also have their limitations. Analysis of DNA cannot distinguish between viable and nonviable cells, the methods often are limited to finding organisms only if they occur above a certain threshold frequency in the population, and, depending upon the technique used, are frequently limited to finding only those types of organisms that have been previously characterized molecularly. PCR based methods typically rely upon specific primers that select only organisms of a certain genus and/or species. If an organism that is not expected to occur in a specific environment being examined is present, it may not be detected using specific primers.
THE BIODIVERISTY OF GRAPE SURFACES
The diversity of yeast species on grapes has been investigated in vineyards worldwide. Using aggressive washing and analytical techniques, a concentration of 3 x 105 yeast cells/cm2 of the berry surface has been estimated. Other studies suggest a range of 104 to 106 cells/cm2.
The factors impacting which genera and species are found have also been evaluated. The methodologies have differed, but there is a striking similarity of the main genera and species found. There are three principal genera found on grapes:Hanseniaspora uvarum (anamorph: Kloeckera apiculata) and Metschnikowia pulcherrima (anamorph: Candida pulcherrima) and Candida stellata. In some reports, Hanseniaspora is the dominant species and in others it is Candida. Candida has been shown to complete the alcoholic fermentation in some cases. Several of the Candida stellata isolates from wine are actually Candida zemplinina. In one study of grapes from cooler climates, the basidiomycetes, Cryptococcus andRhodotorula, dominated in number over the ascomycete yeasts. In another, the dimorphic fungus, Aureobasidium, was found as the dominant yeast on grape surfaces in addition to Cryptococcus, followed by Rhodotorula and Rhodosporiduim,depending upon the grape variety. A key factor determining the species present on the surface of grape appears to be the amount of damage to the fruit. The leakage of sugar substrates, either through physical damage mediated by insects, birds or invasive fungal species, or as a consequence of berry aging and shrivel on the vine due to dehydration, enriches for the ascomycetes. The amount of natural seepage varies with different grape varieties and the tightness of the clusters, so it is not surprising that some studies have seen a strong correlation of the variety with the biodiversity of the fruit surface. The first of the ascomycetous yeasts to appear are Hanseniaspora, Candida and Metschnikowia. These yeasts dominate the grape surface flora as the grapes ripen. Thus, some of the variation in species identified in comparing different published reports is a function of the physiological ripeness and integrity of the grapes when harvested for the analysis.
Other yeasts can be commonly found, although they are not as universal. Saccharomyces can be detected, but is present on grape surfaces at very low levels, and in some studies has been undetectable. In a comprehensive study using direct DNA profiling of grape surface microbes, 52 species of yeast were identified from the following 22 genera: Auerobasidium, Auriculibuller, Brettanomyces, Bulleromyces, Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Issatchenka, Kluyveromyces, Lipomyces, Metschnikowia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporidiobolus, Sporobolomyces, Torulaspora, Yarrowia, Zygoascus, and Zygosaccharomyces. Other researchers have also foundHansenula. Saccharomyces is more commonly isolated from heavily damaged grapes, or in cases of rot.
The change in species on the surface of grapes that occurs during ripening follows a pattern of early dominance by the basidiomycetous yeasts, Aureobassidium, Cryptococcus, Rhodosporidium and Rhodotorula pre-veraison, and during early ripening. These give way to the ascomycetous yeast, particularly Hanseniaspora, Metschnikowia and Candida, as the fruit ripens. Berry damage that occurs later in ripening due to physical or biological factors enriches for these yeasts, as well as fermenting yeasts, such as Saccharomyces. The presence of other yeast genera depends upon regional and climactic influences, the grape variety, disease pressure and level of damage of the grapes, and vineyard practices.
A direct comparison of plating to obtain viable isolates to total DNA extraction analysis of species present on the surface of grapes indicated that different organisms were obtained by the two methods, most likely due to differences in relative sensitivities and abilities to grow on the selective medium. The major species identified using either methodology were the same, but a greater number and diversity of yeasts were detected in the direct DNA isolation studies.
Many factors in addition to stage of ripening have been identified that impact the presence and numbers of yeasts on the surface of grapes. In general, the number of yeasts present on grapes increases with ripening and the numbers are higher by one or two orders of magnitude nearer the peduncle. Seasonal variation has also been observed with warmer and dryer years yielding increased yeast populations. Infection with molds such as Botryis that can penetrate the berry surface releasing nutrients can impact the microbial flora of the surface of the grape. Infection with Botrytis was found to increase the numbers of yeasts by three orders of magnitude. Another study of Botryis infected grapes demonstrated the presence ofMetschnikowia strains that were then inhibitory to other yeasts, fungi and bacteria. The mechanism of inhibition was thought to be the sequestration of iron.
The insect pressure in a vineyard is also an important factor. Bees, wasps and the fruit fly Drosophila have all been shown to be vectors of yeast species in vineyards. Microorganisms can adhere to the surfaces of the insects and be deposited on other fruit surfaces as the insect travels about the vineyard. Since the insects are attracted to damaged fruit, they can spread the yeasts from the surface of the damaged fruit to other sectors of the vineyard. The application of fungicides, such as elemental sulfur, in the vineyard may also impact the yeast species present. The regional climate and altitude of the vineyard can affect the yeast found. The type of grape variety may also impact the yeast species found on the grape surface.
It was thought that the higher levels of Saccharomyces seen in some vineyards may be due to the practice of placing yeast lees from the fermentation in the vineyard as a source of vine fertilization. To test this hypothesis, the effect of deliberate inoculation of vineyards with Saccharomyces on the presence of Saccharomyces at the time of harvest has been investigated. The winery residents and vineyard inocula did not become established in the berry flora in spite of high inoculation levels. Puncturing the grapes to induce berry seepage and damage did not improve the chances of colonization by theSaccharomyces inoculum.
THE BIODIVERSITY OF WINERIES
Significantly fewer studies have been conducted of the yeast flora found on winery surfaces and equipment. It has been demonstrated that the winery flora represent a significant source of inoculation for the juice, must and wine. Following grape processing the numbers of Saccharomyces found per unit volume can increase by three orders of magnitude or more. Biofilms readily form on winery surfaces. Stainless steel is commonly used for fermentation, but juices are also fermented in more porous containers such as wooden barrels and vats. These are notoriously difficulty to clean let alone sanitize and cannot be sterilized without loss of integrity. Microbial flora often also coat walls, outer barrel surfaces, hoses and drains, particularly during barrel ageing as this is typically done under conditions of humidity to prevent evaporative loss of wine volume. Sanitation practices vary widely as does the practice of supplementation with nutrients. All of these factors impact winery flora.
Only a few studies of the flora found on winery surfaces have been conducted. Analysis of the surfaces of barrels indicated high numbers of Saccharomyces, with Candida, Cryptococcus and Brettanomyces also commonly present, although in lower concentrations. Bacteria and molds can be more commonly found on winery surfaces except during active fermentation when the populations of yeasts can be high. There is considerable diversity of mold species present in wineries.
A current controversy concerns the origin of the Saccharomyces species that arise during a spontaneous or uninoculated fermentation. A direct analysis of the presence of Saccharomyces isolates on grape surfaces was undertaken using aseptically harvested grapes immediately processed under sterile fermentation conditions without benefit of possible inoculation by contaminated winery surfaces. In this study, 68% of the vineyard samples were able to initiate fermentation. However only 42% of the completed fermentations, or 28% of the total aseptic samples taken from the vineyard, were dominated by Saccharomyces. In another study that also used aseptic grape handling techniques, the major species found during the alcoholic fermentation was Candida stellata with Saccharomyces only rarely found and often not in high numbers. These studies demonstrate that Saccharomyces can indeed be found in vineyards and that in some cases the level ofSaccharomyces yeasts coming in with the grapes is sufficient to initiate fermentation. However this is not always the case and it is also true that the yeast conducting the fermentation may derive from the winery flora. Since there can be a significantSaccharomyces bioflora on winery surface, if the number of Saccharomyces yeasts derived from the winery surfaces dominates the number of those coming from the vineyard, the winery yeasts will be the major species present during fermentation. This is also true if an inoculum is used. Thus whether the grapes or the winery flora are the major source of the fermentation flora depends upon the relative numbers of Saccharomyces coming from the surface of the grapes versus the surfaces of the winery and winery equipment.
THE BIODIVERISTY OF WINE FERMENTATIONS
Many analyses of the yeast flora found during wine fermentation have been conducted. Wine fermentations can be divided into two types: directly inoculated and uninoculated. Uninoculated fermentations are also called native flora, spontaneous or natural fermentations and rely on the indigenous flora of the grapes and winery for fermentation. In both cases following crushing of the grapes the must (grape solids and accompanying juice) generally displays high concentrations of the yeasts present on the grape berry. These yeasts initiate the bioconversion of grape juice into wine. How long the non-Saccharomycesyeast persist depends upon the winemaking conditions and relative levels of the major species present. The factors affecting the yeasts found in fermentations are similar to those affecting the flora on the berry such as the maturity of the fruit, age of the vineyard, variety, use of antifungal agents, climate and vineyard location. The use of antifungal agents in the vineyard results in increased populations of Metschnikowia and decreased populations of Saccharomyces. In addition, harvesting techniques can also impact the yeasts present in the fermentation, particularly if the berries are damaged during harvest and microbial growth occurs during shipping to the winery.
Numerous studies have categorized the changes and persistence of non-Saccharomyces flora during uninoculated fermentations. These studies all demonstrate a similar pattern of species evolution during fermentation. In the beginning the species present on the surface of the grape appear to dominate the species found in the fermentation, including the basidiomycetous yeasts and the oxidative ascomycetes. As fermentation progresses, the levels of these yeasts decrease while that of Saccharomyces increases. By the end of fermentation Saccharomyces is the majority of the yeast found and often the only yeast isolated.
Several additional factors have been found to affect the persistence of the non-Saccharomyces yeasts during fermentation. Sanitation practices can have a dramatic effect on the organisms present during fermentation. In one study wineries with poorer sanitation practices had higher levels of the fermentative yeasts presumably because these yeasts had colonized winery equipment. Surprisingly, sulfur dioxide, used as an antimicrobial agent typically added to juice upon crushing of the fruit does not show a significant effect on the wild fermentative yeast species. Other studies have seen a slight effect in a decrease in yeast cell numbers with use of sulfite, but have not seen an effect on the aroma profile of the resulting wines. In contrast, the basidiomycetous yeasts seem to show a greater sensitivity to sulfite, with one study reporting decreases of these yeasts up to 90%.
Factors such as pH and temperature of fermentation can impact the persistence of the yeast species present. Incubation of the juice at low temperatures to settle solids has been shown to impact yeast populations. In one study, the generaHansenula, Issatchenkia and Saccharomyces decreased dramatically while Hanseniaspora and Candida species increased. In a similar study using a cold soak of must from a red grape variety, again Hanseniaspora and Candida species persisted during this incubation at low temperature, however, these species showed a greater dominance during the alcoholic fermentation. Interestingly this study also showed that during the fermentation Pichia emerged along with Saccharomyces. Thus the changes in flora accompanying the cold settling altered the microbial dynamics much later during the fermentation. The variation in persistence of yeast species during fermentation is also dependent upon the variety. One factor that does impact the persistence of non-Saccharomyces flora is the inoculation with commercial strains of Saccharomyces. Inoculation with Saccharomyces leads to a faster domination of the fermentation and more rapid inhibition of the other yeasts present.
THE BIODIVERSITY OF SACCHAROMYCES STRAINS
Two principal species of Saccharomyces are found during the alcoholic fermentation: Saccharomyces cerevisiae andSaccharomyces bayanus (formerly S. uvarum). Occasionally S. pastorianus can be found. The S. bayanus group includes cryptophilic strains able to ferment melibiose. S. cerevisiae has recently been divided taxonomically into six groups: cerevisiae, cheresanus, diastatcus, ellipsoideus, logos and oviformis. Strains previously designated as S. cerevisiae varbayanus are now classified in the oviformis group. Although yeasts from all six groups have been found in wine, the major wine yeasts are from the ellipsoideus and oviformis groups.
In addition to the diversity of non-Saccharomyces yeasts, genetic diversity within Saccharomyces cerevisiae has been well documented. In one comprehensive study, over 1,600 isolates from 54 spontaneous fermentations were examined and found to comprise 297 unique strains. An even higher ratio of unique genotypes (91) to total isolates (104) was found in a similar analysis. In one study that examined yeast biodiversity over two vintages 60 and 65 different yeast strains as determined by analysis of mitochondrial DNA were found with only 21 of these in common for the two vintages. A study in Argentina found similar results that 9 out of 29 genotypes were dominant during fermentation and of these only 5 were common across vintages. Other studies have found less, but still significant yeast diversity. Most of these studies find the greatest number of genotypes are represented by a single isolate, indicating that the true extent of the diversity present is still being underestimated. Some studies have found that one or a few strains dominate throughout fermentation while others have seen a different strains dominate at different stages of the fermentation. Other studies have seen no clear dominance of one strain during fermentation and several strains of Saccharomyces appear to be simultaneously present in equivalently high numbers. In cases where a single strain dominates it has been shown to carry the killer phenotype. Significant diversity among strains of S. bayanus has also been found.
The diversity of wine yeasts has been documented using genomic sequence comparisons and functional genomic analysis of transcript profiles. Even strains that are similar in genetic composition may show changes in important enological phenotypes if the genetic differences are targeted to high impact genes (such as transcription factors) or genes involved in flavor modification or production. The biodiversity of wine strains of Saccharomyces is likely a consequence of both natural selection and random mutagenesis and accumulation of mutations. Wild yeasts show elevated rates of spontaneous mutagenesis which, if followed by sporulation and diploidization can lead to the creation of significant diversity rapidly across a population. The return to a homozygous state has been termed ‘genome renewal’, and is likely a key feature of life in the wild for Saccharomyces.
THE BIODIVERSITY OF YEASTS DURING WINE AGING
Yeasts are also present during the aging of wines and can play an important role in the evolution of wine composition throughout the aging process. The type of flora present during aging depends upon the type of vessel used and winery sanitation practices. Both stainless steel and barrel surfaces can support yeast biofilm formation. Stainless steel is easier to sanitize than porous wooden surfaces which tend to build up significant numbers of yeast over the years of use. TheSaccharomyces and non-Saccharomyces yeasts found during the fermentation can persist through aging, although these yeasts are usually not biologically active. Species of Candida, Pichia and particularly Brettanomyces can be found in wines in barrel and can lead to cosmetic (film) or organoleptic defects in the wine. Significant diversity is found among isolates ofBrettanomyces as well. Zygosaccharomyces, due to its tolerance of both sulfur dioxide and sorbate, can also be found as a contaminant of wine.
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