Dissemination and survival of commercial wine yeast in the vineyard: a large-scale, three-years study

The use of commercial wine yeast strains as starters has been extensively generalised over the past two decades. In this study, a large-scale sampling plan was devised over a period of three years in six different vineyards to evaluate the dynamics and survival of industrial yeast strains in the vineyard. A total of 198 grape samples were collected at various distances from the wineries, before and after harvest, and yeast strains isolated after spontaneous fermentation were subsequently identified by molecular methods. Among 3780 yeast strains identified, 296 isolates had a genetic profile identical to that of commercial yeast strains. For a large majority (94%), these strains were recovered at very close proximity to the winery (10-200 m). Commercial strains were mostly found in the post-harvest samples, reflecting immediate dissemination. Analysis of population variations from year to year indicated that permanent implantation of commercial strains in the vineyard did not occur, but instead that these strains were subject to natural fluctuations of periodical appearance/disappearance like autochthonous strains. Our data show that dissemination of commercial yeast in the vineyard is restricted to short distances and limited periods of time and is largely favoured by the presence of water run-off.     

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.     

Analysis of yeast diversity during spontaneous and induced alcoholic fermentations

The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae . In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

A molecular genetic study of natural strains of Saccharomyces isolated from Asturian cider fermentations

AIMS: To analyse the genetic diversity and the dynamics of Saccharomyces strains in spontaneous fermentation in ciders. The effect of the cellar, harvest and cider-making technology were evaluated. METHODS AND RESULTS: The ecology of spontaneous cider fermentations in the same cellar (Asturias) was studied for two consecutive harvests (2000 and 2001) by using mtDNA restriction analysis. Our results showed that there was a succession of genetically different strains of Saccharomyces during cider production. In general, strains of Saccharomyces bayanus species predominated at the early fermentation steps (begining and/or tumultuous fermentations), while Saccharomyces cerevisiae yeasts were the most abundant at the end of the fermentation. Five S. bayanus strains (patterns III, VII, VIII, XV and XVII) were present at significant frequencies in all the experimental tanks during the two consecutive years. The results of the cluster analysis (unweighted pair group method using average linkage) showed higher similarities for the patterns III, XV, VII and VIII. Therefore, these strains should be considered associated with the microbiota of this cellar. CONCLUSIONS: A high polymorphism within populations of Saccharomyces was found throughout the different stages of Asturian production of cider. In all the cider fermentations, a variable number of S. bayanus and S. cerevisiae strains was always present. Our results indicate, over the period of time studied, the existence of the natural microbiota in the cellar. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of wild Saccharomyces yeast in Asturian cider fermentations.     

Dynamics of Saccharomyces cerevisiae populations in controlled and spontaneous fermentations for Franciacorta D.O.C.G. base wine production

The evolution of the yeast populations was investigated during controlled and spontaneous fermentations of Chardonnay must in two Franciacorta wineries (A and B) that used the same starter culture. Two hundred and three isolates were collected and identified as Saccharomyces cerevisiae (97.5 %), Pichia membranifaciens (2.0 %) and Hanseniaspora vinae (0.5 %) through the analyses of ITS rDNA region by RFLP, D1/D2 of 26S rDNA partial sequence and scHO gene. A high intraspecific diversity of S. cerevisiae isolates was detected by means of the inter-delta sequence PCR analysis: 117 profiles corresponding to different strains were distinguished (at level of similarity <90.5 %) and monitored to follow the dynamics of cell populations. In winery A, the commercial strain maintained the predominance since its δ-PCR profile constituted most of the colonies recovered at different times of sampling (from 44 to 100 % of plate counts), in this case only 18 different genotypes out of 74 isolates were recognized. In winery B, where spontaneous fermentations were performed in the same environment, the starter culture never took control and a succession of indigenous populations overcame without one prevailed on the others; actually, 40 genotypes out of 53 isolates can be identified. The highest level of biodiversity was observed in spontaneous fermentation (winery B) where 59 genotypes out of 71 S. cerevisiae isolates were discriminated; a continuous change in cell populations was noticed with the simultaneous presence from 6 to 10 different genotypes. The management of the starter culture and the environmental hygiene was shown to be fundamental to control the inoculated fermentations.     

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.     

赤霞珠葡萄酒自然发酵中酿酒酵母的菌株区分

采用Interdelta指纹图谱分析, 对分离自宁夏地区赤霞珠葡萄自然发酵过程中的45个酿酒酵母单菌落进行菌株区分, 研究发酵过程中酿酒酵母菌株的变化, 为发酵的有效控制及选育优良酿酒酵母菌株提供依据。结果发现, 本研究分离到的45个酿酒酵母单菌落中, 产生5种指纹图谱, 代表5种不同的基因型, 基因型I-V分别占所分离单菌落的71%、13%、9%、5.0%、2.0%, 基因型I是发酵过程中的优势菌株。本研究中, 二氧化硫处理影响自然发酵过程中酿酒酵母菌株的类型、数目及比例, 但其影响不是很大。     

Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of 'Malvasia delle Lipari'

AIMS: Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'. METHODS AND RESULTS: A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity. CONCLUSIONS: It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from different grape varieties and winemaking regions

We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8-43 strains per fermentation) was associated with high percentage (60-100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0-40%) corresponded to a rather low strain diversity (1-10 strains per fermentation).For the majority of the populations, observed heterozygosity (Ho) was about two to five times lower than the expected heterozygosity (He). The inferred ancestry showed a very high degree of admixture and divergence was observed between both grape variety and geographical region. Analysis of molecular variance showed that 81-93% of the total genetic variation existed within populations, while significant differentiation within the groups could be detected. Results from AMOVA analysis and clustering of allelic frequencies agree in the distinction of genetically more dispersed populations from the larger wine region compared to the less extended region. Our data show that grape variety is a driver of populational structures, because vineyards with distinct varieties harbor genetically more differentiated S. cerevisiae populations. Conversely, S. cerevisiae strains from vineyards in close proximity (5-10 km) that contain the same grape variety tend to be less divergent. Populational similarities did not correlate with the distance between vineyards of the two wine regions. Globally, our results show that populations of S. cerevisiae in vineyards may occur locally due to multi-factorial influences, one of them being the grape variety.     

Yeast diversity and persistence in botrytis-affected wine fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by SACCHAROMYCES: In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of CANDIDA: Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

Effect of must characteristics on the diversity of Saccharomyces strains and their prevalence in spontaneous fermentations

Aims: The aim of this study was to investigate whether grapevine variety and must characteristics influence the diversity of Saccharomyces strains and their prevalence during spontaneous fermentations. Methods and results: Musts from different grapevine varieties, all of them autochthonous from Galicia, were used to perform spontaneous fermentations. Yeasts were isolated from the must and at the beginning, in the middle and at the end of fermentations. Those yeasts identified as Saccharomyces were characterized at the strain level by analysis of mtDNA‐RFLP. The results showed a low diversity of Saccharomyces strains, which was related to must sugar content and total acidity. Moreover, from a total of 44 different Saccharomyces strains, only eleven of them appeared at frequencies higher than 20% and were able to lead fermentations. A significant correlation between yeast strains and must acidity was observed, with the predominance of certain strains at high acidity values. Conclusions: Must characteristics, such as sugar content and acidity, influence the Saccharomyces strains diversity and the leader strains during fermentation. Significance and Impact of the Study: These results showed the adaptation of certain Saccharomyces strains to must with specific characteristics; this may be considered by winemakers for yeast inocula selection. Our findings have special relevance because this is the first study carried out in Galicia dealing with the influence of must properties on yeast strains that control fermentations.     

Survival of commercial yeasts in the winery environment and their prevalence during spontaneous fermentations

Inoculation of active dry yeasts during the wine-making process has become a common practice in most wine-producing regions; this practice may affect the diversity of the indigenous population of Saccharomyces cerevisiae in the winery. The aim of this work was to study the incidence of commercial yeasts in the experimental winery of Estación de Viticultura e Enoloxía de Galicia (EVEGA) and their ability to lead spontaneous fermentations. To do this, 64 spontaneous fermentations were carried out in the experimental cellar of EVEGA over a period of 7 years. Samples were taken from must and at the beginning, vigorous and final stages of fermentation. A representative number of yeast colonies was isolated from each sample. S. cerevisiae strains were characterised by analysis of mitochondrial DNA restriction patterns. The results showed that although more than 40 different strains of S. cerevisiae were identified, only 10 were found as the dominant strain or in codominance with other strains in spontaneous fermentations. The genetic profiles (mtDNA-RFLPs) of eight of these strains were similar to those of different commercial yeasts that had been previously used in the EVEGA cellar. The remaining two strains were autochthonous ones that were able to reach implantation frequencies as high of those of commercial yeasts. These results clearly indicated that commercial wine yeasts were perfectly adapted to survive in EVEGA cellar conditions, and they successfully competed with the indigenous strains of S. cerevisiae, even during spontaneous fermentations. On the other hand, autochthonous dominant strains that presented desirable oenological traits could be of interest to preserve wine typicity.     

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.     

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...     

Ethanol Production and Maximum Cell Growth Are Highly Correlated with Membrane Lipid Composition during Fermentation as Determined by Lipidomic Analysis of 22 Saccharomyces cerevisiae Strains

Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R² = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R² = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.     

Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.