Influence of different floor management strategies of the vineyard on the natural yeast population associated with grape berries

Some oenological practices, such as the massive utilisation of commercial yeast and the consequent colonisation of wineries, can contribute to reducing the native yeast biodiversity. In this context, the vineyard could be a reservoir of autochthonous yeasts of oenological interest. Thus, the evaluation of the influence of different agricultural parameters on the biodiversity of yeast population in the vineyard is necessary. This work shows the results of the influence of some floor management strategies of the vineyard in the natural yeast population associated with the grape-berries. With this objective, a three year sampling plan was designed in the Shiraz vineyards of the Madrid region using three floor management strategies: bare soil by tillage, bare soil maintained with herbicides and soil maintained with cover crop. The results of this study have shown that bare soil by tillage could be a sustainable alternative for managing the soil, due to the reduced use of agrochemicals and the resulting high yeasts biodiversity. Nevertheless, the presence of herbicides in the vineyard has a minor impact on the diversity of grape associated yeast communities, and this could have increased the yeast populations. Hence, from the fermentative yeasts' (like Saccharomyces) point of view, in hot and arid environments where soils may be affected by the tillage management, the best option could be the maintenance of the bare soil with the use of herbicides.     

Biodiversity of Saccharomyces cerevisiae populations in Malbec vineyards from the "Zona Alta del Río Mendoza" region in Argentina

The “Zona Alta del Río Mendoza” (ZARM) is the major Malbec grape viticulture region of Argentina. The aim of the present study was to explore Saccharomyces cerevisiae biodiversity in ZARM vineyards. Interdelta PCR and RFLP mtDNA molecular markers were applied to differentiate S. cerevisiae strains. The presence of commercial strains on ZARM vineyards was also assessed. Our results reveal a highly diverse, but genetically closely related, S. cerevisiae population (containing more than 190 molecular patterns among 590 S. cerevisiae isolates). According to the S. cerevisiae strain diversity found in vineyards, they were classified as vineyards with high and low polymorphic S. cerevisiae populations. Six vineyards showed a high polymorphic population, with more than 20 different S. cerevisiae molecular patterns. S. cerevisiae populations in these vineyards were diverse and irregularly distributed, with different strains in each vineyard site. Low polymorphic S. cerevisiae population vineyards displayed very low yeast diversity, with only 9 to 10 different S. cerevisiae strains and presence of two commercial strains widely distributed. Population diversity estimators were calculated to determine the population structure of S. cerevisiae in the ZARM vineyards. The obtained values support the hypothesis that the eight sampled subpopulations come indeed from a larger population.     

Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.     

Yeasts found in vineyards and wineries

Wine is a complex beverage, comprising thousands of metabolites that are produced through the action of a plethora of yeasts and bacteria during fermentation of grape must. These microbial communities originate in the vineyard and the winery and reflect the influence of several factors including grape variety, geographical location, climate, vineyard spraying, technological practices, processing stage and season (pre‐harvest, harvest, post‐harvest). Vineyard and winery microbial communities have the potential to participate during fermentation and influence wine flavour and aroma. Therefore, there is an enormous interest in isolating and characterising these communities, particularly non‐Saccharomyces yeast species to increase wine flavour diversity, while also exploting regional signature microbial populations to enhance regionality. In this review we describe the role and relevance of the main non‐Saccharomyces yeast species found in vineyards and wineries. This includes the latest reports covering the application of these species for winemaking; and the biotechnological characteristics and potential applications of non‐Saccharomyces species in other areas. In particular, we focus attention on the species for which molecular and genomic tools and resources are available for study. Copyright © 2016 John Wiley & Sons, Ltd.     

The microbial ecology of wine grape berries

Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing.The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect.     

Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine

Over the last few decades, there has been a progressive increase in the ethanol content in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. This increased ethanol content can have negative consequences on the sensory properties of the wines, human health, and economic aspects. Several microbiological approaches for decreasing the ethanol content have been suggested, such as strategies based on genetically modified yeasts, the adaptive evolution of yeasts, and the use of non-Saccharomyces yeast. In the present study, we investigated the interspecies and intraspecies variability of some non-Saccharomyces wine yeast species under anaerobic fermentation conditions. Across different grape juices and fermentation trials, Hanseniaspora uvarum, Zygosaccharomyces sapae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus promoted significant reductions in ethanol yield and fermentation efficiency in comparison with Saccharomyces cerevisiae. The diversion of alcoholic fermentation and the abundant formation of secondary compounds might explain the marked reduction in ethanol yield, as determined through the segregation of the majority of the strains according to their species attributes observed using principal component analysis. These data suggest that careful evaluation of interspecies and intraspecies biodiversity can be carried out to select yeast that produces low-ethanol yields.     

沙城产区酿酒酵母多样性研究

对沙城产区龙眼葡萄相关环境中分离筛选的酿酒酵母进行多样性研究。在连续3年(2008、2009、2010年)的实验中,共从葡萄园土壤、葡萄酒厂设备表面、接触过葡萄酒厂设备的葡萄汁和自然发酵过程中采集菌源样品227份,共分离得到1358株酵母菌。用5.8S-ITS区域RFLP方法进行分子水平的分类鉴定及赖氨酸培养基复筛,得到了270株酿酒酵母。再利用Interdelta PCR指纹图谱法将酿酒酵母区分为16类,其中土壤5类,自然发酵过程中第2、3、4期分别得到4类、10类和11类;酒厂设备表面3类;接触酒厂设备的葡萄汁3类。酿酒酵母的种类因样品采集时间、采集地点等不同有明显区别。自然发酵过程中得到的酿酒酵母被认为是本土酵母的可能性最大。     

Effects of the origins of Botrytis cinerea on earthy aromas from grape broth media further inoculated with Penicillium expansum

Earthy “off” aromas from wine and grape juice are highly detrimental to the production of quality grape products. These volatile compounds are produced on grapes by Botrytis cinerea, Penicillium expansum and/or a combination of P. expansum and B. cinerea strains. B. cinerea strains were isolated from different (a) vineyards in Spain and Portugal, (b) grape varieties (c) bunches (i.e., sound and botrytized) and (d) positions in the botrytized bunch (i.e., interior or exterior). A novel Headspace-Phase Microextraction (SPME) followed by Gas Chromatrography/Mass Spectrometry (GC-MS) dedicated to analyze geosmin, methylisoborneol (MIB), 1-octen-3-ol, fenchone and fenchol in grape broth medium was used. Approximately 50% of the B. cinerea strains induced detectable geosmin. One strain accumulated significant amounts of anisoles, demonstrating that this contamination might already occur in the vineyard. Strains from the interior of Cainho grape bunches induced more geosmin and hence it may be possible to reduce this volatile in wine by avoiding using these grapes in case of B. cinerea attack.     

Yeast interactions and wine flavour

Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.     

Biodiversity and safety aspects of yeast strains characterized from vineyards and spontaneous fermentations in the Apulia Region,Italy

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy). Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. The polymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twenty different species belonging to 9 genera were identified. Predominant on the grape surface were Metschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrima and H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates were identified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eight fermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able to produce histamine and cadaverine, during must fermentation. The production of BAs in wine must was different than that observed in the synthetic medium. This feature indicate the importance of an “in grape must” assessment of BAs producing yeast. Overall, our results suggest the importance of microbiological control during wine-making to reduce the potential health risk for consumer represented by these spoilage yeasts.     

Characterization of the yeast ecosystem in grape must and wine using real-time PCR

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.     

山地酿酒葡萄园土壤养分与葡萄品质的关系

对陕西省耀县山地不同海拔高度3个酿酒葡萄园0～60cm土层土壤的养分及葡萄果实质量进行了研究。结果表明:各葡萄园土壤pH均在8.0左右,属石灰性土壤;土壤有机质和N、P、K等大量元素含量偏低,各种微量元素除了B含量很低外,其它元素均含量丰富。葡萄糖酸比随海拔升高而降低,总酚和单宁含量以海拔1096m的葡萄园含量最高。营养元素与葡萄各指标的相关性除了有效Mn与含糖量达显著水平外,其它均未达显著水平。     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Selection of indigenous Saccharomyces cerevisiae strains for Nero d'Avola wine and evaluation of selected starter implantation in pilot fermentation

The present research studied Saccharomyces cerevisiae yeasts isolated from Nero d'Avola grapes, collected in different areas of the Sicily region. RAPD-PCR analysis with M13 primer was used for preliminary discrimination among 341 S. cerevisiae isolates. Inoculated fermentations with S. cerevisiae strains, exhibiting different RAPD-PCR fingerprinting, revealed the impact of selected strains on volatile compound concentration. Two selected strains were used in fermentation at cellar level and the restriction analysis of mtDNA on yeast colonies isolated during fermentation was used to control strain implantation. This study represents an important step to establish a collection of indigenous S. cerevisiae strains isolated from a unique environment, such as Nero d'Avola vineyards. Different starter implantation throughout inoculated fermentation represents an additional character, which might be considered during the selection program for wine starter cultures.     

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.     

葡萄园土壤中铜、铁、锌、锰四种金属元素的分析与研究

采用湿法消解处理样品,火焰原子吸收光谱法检测河北昌黎葡萄酒产区5 个葡萄园土壤中的铜、铁、锌、锰4 种金属元素。并对比研究3 种葡萄、葡萄酒与葡萄园土壤中铜、铁、锌、锰4 种金属元素含量的关系。对不同葡萄园和同一葡萄园种植不同品种葡萄,其土壤中金属元素含量进行差异性分析。结果显示：葡萄园地理位置和微环境及所栽培的葡萄品种、树龄和架势不同,土壤中4 种金属元素含量不同,差异性显著。铁、锌、锰3 种元素在土壤中的含量与葡萄中的含量有一定关系。赤霞珠、美乐和西拉3 种葡萄的土壤中铜、铁、锌的含量与葡萄及其葡萄酒中相对含量规律相同,而锰不同。     

Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.)

Non-Saccharomyces yeast species assume an important role in wine flavor. Notwithstanding, the chemical basis for the flavor characteristics of wines from some grape varieties is not yet defined. The value of this work lies in the use of Malvar white grape, an autochthonous variety from Madrid (Spain) winegrowing region to conduct spontaneous fermentations. This is the first time that a comparative characterization of a wide range of non-Saccharomyces species and a comprehensive analysis of these yeast-derived volatiles has been carried out in this grape variety. β-glucosidase and pectinase (polygalacturonase) extracellular activities were tested on agar plates as primary selection criteria among the 504 non-Saccharomyces isolated from Malvar spontaneous fermentations during four consecutive harvests. Analysis of the wines obtained after fermentation using the selected yeast strains indicates that non-Saccharomyces yeasts isolated along the fermentative process seem that could have a positive impact, showing a high variability in the volatile compounds contributing to the organoleptic characteristics of Malvar wines. Torulaspora delbrueckii CLI 918 was defined as the yeast strain with potential interest for its contribution to the aromatic wine profile with flowery and fruity aromas and could be used in mixed starter cultures with Saccharomyces cerevisiae. However, Hanseniaspora guilliermondii increased the volatile acidity and ethyl acetate, but this species along with the genus Pichia and Candida seem to provide a high quantity of extracellular enzymes which may be beneficial in wine making.     

Yeasts isolated from three varieties of grapes cultivated in different locations of the Dolenjska vine-growing region, Slovenia

The number and diversity of yeasts on grape berry surfaces are influenced by several factors, such as grape variety, degree of grape maturity at harvest, climatological conditions, geographic location, physical damage of grapes, the intensity of pest management etc. Cvicek is a typical Slovene wine, which has obtained a special protection under the Slovene Wine Law for its geographical origin. This blended red wine is produced from different grape varieties (Vitis vinifera L.), mostly from red grapes of Zametovka and Modra frankinja and from white grapes of Kraljevina. The aim of this study was to evaluate the impact of geographical locations in the Dolenjska vine-growing region and to obtain precise information about the influence of different grape varieties on the composition of yeast community on grape berries. The restriction fragment length polymorphism of PCR-amplified fragments from the rDNA gene cluster (PCR RFLP of rDNA) has been used for the differentiation of yeast species. The standard identification procedure has been performed on representative strains that shared identical RFLP profiles. The number of yeasts and yeast species isolated varied according to different grape varieties, Zametovka, Modra frankinja and Kraljevina (V. vinifera L.) and according to different sampling location. On the surface of grape berries 13 different yeast species have been identified. Saccharomyces cerevisiae has not been found.     

Survey of hydrogen sulphide production by wine yeasts

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.