Wild Yeast Research Articles

Characterization of wild indigenous yeasts that can withstand different stresses from traditional fermented beverages in the Amhara region, Ethiopia

Characterization of wild indigenous yeasts that can withstand different stresses from traditional fermented beverages in the Amhara region, Ethiopia

Comparative modeling reveals the molecular determinants of aneuploidy fitness cost in a wild yeast model

Although implicated as deleterious in many organisms, aneuploidy can underlie rapid phenotypic evolution. However, aneuploidy will be maintained only if the benefit outweighs the cost, which remains incompletely understood. To quantify this cost and the molecular determinants behind it, we generated a panel of chromosome duplications in Saccharomyces cerevisiae and applied comparative modeling and molecular validation to understand aneuploidy toxicity. We show that 74%-94% of the variance in aneuploid strains' growth rates is explained by the cumulative cost of genes on each chromosome, measured for single-gene duplications using a genomic library, along with the deleterious contribution of small nucleolar RNAs (snoRNAs) and beneficial effects of tRNAs. Machine learning to identify properties of detrimental gene duplicates provided no support for the balance hypothesis of aneuploidy toxicity and instead identified gene length as the best predictor of toxicity. Our results present a generalized framework for the cost of aneuploidy with implications for disease biology and evolution.

Evaluating the role of nitrate reductase and the manipulation of the culture conditions on the biogenesis of silver nanoparticles by the wild yeasts

Evaluating the role of nitrate reductase and the manipulation of the culture conditions on the biogenesis of silver nanoparticles by the wild yeasts

Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas 'cutting' combined with homologous recombination of a 'repair' template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo , which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic ( HIS3, URA3 ) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.

Cryptococcus laurentii: a wild yeast for xylanase production from agricultural by-products.

The development of technologies that allow the production of enzymes at a competitive cost is of great importance for several biotechnological applications, and the use of agro-industrial by-products is an excellent alternative to minimize costs and reduce environmental impacts. This study aimed to produce endo-xylanases using agro-industrial substrates rich in hemicellulose as sources of xylan in culture media. For this purpose, the yeast Cryptococcus laurentti and five lignocellulosic materials (defatted rice bran, rice husk, corn cob, oat husks, and soybean tegument), with and without pretreatment, were used as a source of xylan for enzyme production. To insert the by-products in the culture medium, they were dried and treated (if applicable) with 4% (w.v-1) NaOH and then added in a concentration of 2% (w.v-1). The cultures were agitated for 96h, and the aliquots were removed to determine the enzymatic activities. Among the by-products studied, the maximum activity (8.7 U. mL-1 at pH 7.3) was obtained where rice bran was used. In contrast, corn cob was the by-product that resulted in lower enzyme production (1.6 U.mL-1). Thus, the defatted rice bran deserves special attention in front of the other by-products used since it provides the necessary substrate for producing endo-xylanases by yeast.

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance

BackgroundOrganisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated “cross protection” mechanisms, where mild ‘primary’ doses of one stress can enhance tolerance to severe doses of a different ‘secondary’ stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature.ResultsDuring the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general.ConclusionsUltimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

Wild yeast isolation by middle school students reveals features of North American oak populations of Saccharomyces cerevisiae and Kluyveromyces lactis.

Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO, which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle school students in Denver, Colorado, USA to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. By contrast, we isolated the "dairy yeast", Kluyveromyces lactis, from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state, pointing to high genetic diversity. The outreach activity partnered middle school, high school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus.

Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.

Creation of Single Cell Protein-Producing Mutants of Phaffia Rhodozyma

Recently, there has been an increasing demand for sustainable protein sources for aquaculture. In aquaculture, sustainable protein includes sources that can be produced with a minimal environmental footprint, such as single-cell proteins, proteins from algae, insects, and by-products from the agricultural industry. Single-cell proteins that are derived from microorganisms show a sustainable alternative to traditional protein sources due to their high protein content and rapid growth. This research focuses on creating mutants of the yeast Phaffia rhodozyma to increase single-cell protein production. This yeast has significant industrial potential and biological features. The yeast Phaffia rhodozyma is already known for its unique ability to synthesize astaxanthin, so this case will focus on its ability to produce protein. In the study, we used wild strain yeast and previously obtained white mutants on inorganic nitrogen media for further random mutagenesis methods to introduce specific mutations into the genome aimed at improving their ability to biosynthesize protein. Amino acid inhibitor was used to select potential protein-producing mutants. The protein content of the biomass obtained from the experiments was analyzed in detail using BCA (Protein Assay Reagent) analysis, which confirmed their potential as a source of nutrients for aquaculture. This method is based on the reaction between bicinchoninic acid and the peptide bonds of proteins, which results in the formation of a color whose intensity is proportional to the amount of protein in the sample. The results obtained allow further research into the development of cost-effective and environmentally friendly alternatives for feed in aquaculture.

Synergistic Fermentation of Pichia kluyveri and Saccharomyces cerevisiae Integrated with Two-Step Sugar-Supplement for Preparing High-Alcohol Kiwifruit Wine.

Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the kiwifruit wine. Pichia kluyveri was isolated from kiwifruit pulp by lineation separating, screened by morphological characteristics in Wallerstein Laboratory Nutrient Agar (WL) medium and microscope observation, and further identified by 26S rDNA D1/D2 domain sequence analysis. Taking alcohol content and sensory evaluation as two indexes, the fermentation condition for kiwifruit wine was optimized by single factor and response surface experiment. The optimal fermentation conditions were optimized as follows: the fermentation temperature was at 24 °C, the initial pH was 3.8, the sugar dosage in second step was 8% (w/w), and the inoculating quantity of Pichia kluyveri and Saccharomyces cerevisiae was 0.15 g/L at equal proportion. Under these optimal conditions, the maximum estimated alcohol content was 15.6 vol%, and the kiwifruit wine was light green in color with strong kiwifruit aroma and mellow taste.

Molecular Mechanisms of Genotype-Dependent Lifespan Variation Mediated by Caloric Restriction: Insight from Wild Yeast Isolates.

Caloric restriction (CR) is known to extend lifespan across different species and holds great promise for preventing human age-onset pathologies. However, two major challenges exist. First, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. Second, genetic differences causing variations in response to CR and genetic factors contributing to variability of CR response on lifespan are largely unknown. Here, we took advantage of natural genetic variation across 46 diploid wild yeast isolates of Saccharomyces species and the lifespan variation under CR conditions to uncover the molecular factors associated with CR response types. We identified genes and metabolic pathways differentially regulated in CR-responsive versus non-responsive strains. Our analysis revealed that altered mitochondrial function and activation of GCN4-mediated environmental stress response are inevitably linked to lifespan variation in response to CR and a unique mitochondrial metabolite might be utilized as a predictive marker for CR response rate. In sum, our data suggests that the effects of CR on longevity may not be universal, even among the closely related species or strains of a single species. Since mitochondrial-mediated signaling pathways are evolutionarily conserved, the dissection of related genetic pathways will be relevant to understanding the mechanism by which CR elicits its longevity effect.

Comparative Study of the Stilbenes and Other Phenolic Compounds in Cabernet Sauvignon Wines Obtained from Two Different Vinifications: Traditional and Co-Inoculation

From grape cultivation to ripening and harvest timing to processing, each step of the winemaking process can be a critical point when it comes to wine quality and phenolic composition. In this study, the influence of winemaking technology on resveratrol and quercetin content, as well as other polyphenolic compounds, was investigated. Resveratrol is a non-flavonoid polyphenolic stilbene synthesized by grape skin when damaged by infectious diseases or ionizing radiation. Quercetin is a phenol found in grape skins and stems and is produced to protect grapes from UV light damage. Trans-resveratrol and quercetin are known to act as antioxidants, reduce the risk of atherosclerosis and type 2 diabetes, inhibit the growth of cancer cells, and prevent the release of allergic and inflammatory molecules. However, the question was whether red wine could be enriched with these phenols using a co-inoculation winemaking technology. The main new idea was to completely replace the cold maceration process with maceration with the addition of wild yeast (Torulaspora delbrueckii, Td). Maceration with the addition of wild yeast (Td) offers the following advantages over traditional cold maceration: (1) higher concentrations of trans-resveratrol (&gt;35–40%) and quercetin (&gt;35–40%) in the final wine, (2) the new wine has a higher potential for human health, (3) the wine has better aroma and stability due to the higher mannoprotein content, and (4) better energy efficiency in the production process. The study of stability during storage and aging also included derivatives of benzoic acid and hydroxycinnamic acid, piceid, catechin, naringenin, rutin, kaempherol, hesperetin, and anthocyanins. This study found that younger wines had higher phenolic content, while storage of the wine resulted in a decrease in total phenolic content, especially monomeric stilbenes and quercetin. This study represents a small part of the investigation of the influence of non-Saccharomyces yeasts on the phenolic profile of wine, which still requires extensive research with practical application. In addition, non-Saccharomyces yeasts such as Kluyveromyces thermotolerans, Candida stellata, and Metschnikowia pulcherrima could also be used in future studies.

Application of essential oils as natural antimicrobials in lactic acid bacteria contaminating fermentation for the production of organic cachaça

Sugarcane-based fermentation is an essential process for different sectors of economic importance, such as the food industry with fermented and distilled beverages. However, this process can suffer from high contamination by wild yeasts and bacteria, especially lactic acid bacteria (LAB). This makes it necessary to use decontamination strategies and search for new methods that have a low environmental impact and contribute to the production of organic products. Among the options, oregano and thyme essential oils stand out for their antibacterial compounds. The aim of this study was to use oregano and thyme essential oils as natural antimicrobials in the alcoholic fermentation of sugar cane juice. Initially, the minimum inhibitory concentration of the essential oils in the fermentation was assessed through turbidity in the sensitivity test, which allowed us to determine which concentrations of essential oils would inhibit the contaminants, 3 morphologically selected LAB strains, as well as assessing the viability of CA-11. For LAB, 3 concentrations of each essential oil were tested, ranging from 0.1 to 0.4 μl/mL, while for CA-11 it was 0.06, to 0.1 μl/mL. The results indicated a maximum total value of essential oils per ml of 0.06 μl. Based on this result, a 24-1 fractional factorial was established, with 8 conditions +3 central points, with 4 variables, oregano essential oil (0, 0.03 and 0.06 μl/ml), thyme (0, 0.03 and 0.06 μl/ml), initial soluble solids (14, 16 and 18°Brix) and initial yeast concentration (2.5, 3 and 3.5 g/l), with the temperature set at 32 °C for a period of 12 h. The results showed that the center point condition with 0.03 μl/ml of oregano EO, 0.03 μl/ml of thyme controlled the proliferation of contaminating bacteria compared to the control condition. In the experimental validation, the treatment with essential oils had a lower final population of LAB (5.95 log) than the final population of the control treatment (6.53 log), and it was also observed that the treatment with EOs had an alcohol production around 3 % higher than the treatment without antimicrobials. The experimental validation phase confirmed the synergistic action of oregano and thyme essential oils in controlling the proliferation of contaminating bacteria. In conclusion, it was possible to determine the synergistic antimicrobial action of essential oils against LAB during alcoholic fermentation based on organic sugar cane.

Field-isolated brewing yeast produces wheat beer flavor without wheat ingredients

This study explored the potential of 120 wild yeasts obtained from eight different fruits, with particular emphasis on Saccharomyces cerevisiae S-1. This strain demonstrated remarkable attributes, notably its superior alcohol tolerance when compared to the conventional yeast, US-05. Significantly, the S-1 yeast unveiled its ability to produce three distinct aroma compounds—isoamyl acetate, β-phenylethyl acetate, and ethyl decanoate— not typically found in beer brewed with US-05 stain. These compounds are essential for enriching the sensory experience of beer drinkers. Further investigation revealed that the diminsihed expression of Leu2 in US-05 hindered metabolite synthesis, resulting in the absence of these aroma compounds. In contrast, S-1 exhibited approximately three times higher Leu2 expression, which may explain the observed differences in aroma compounds production. This sheds light on the potential mechanism behind the disparity in aroma profiles between the two strains. Overall, the study highlighted S-1's capacity to enhance beer flavor and aroma. It suggested that wild yeast strains, such as S-1 yeast isolated from grapes, present innovative opportunities for the brewing industry. The hightened Leu2 expression and synthesis of unique aroma compounds in S-1 yeast underscore the latent capabilities of wild yeast strains in elevating beer characteristics.

Alert for microbreweries – a case study on unpasteurized and unfiltered beer

Beer stability, influenced by physical, chemical, and biological factors, poses a considerable challenge for breweries. Biological stability, as a major concern, is effectively managed through practices such as good hygiene, pasteurization, and filtration. Microbiological contamination, often recognizable by increased turbidity or off-flavours, is influenced by temperature, with optimal conditions promoting the growth of specific microorganisms. Lower temperatures slow down flavour changes and chemical reactions in beer, impacting its non-biological stability. This case study explores the impact of storage temperature on autolysis off-flavour development in 10 different unfiltered and unpasteurized beers. The results showed that autolysis off-flavour occurred at both low and higher storage temperatures and that its dependence on the temperature was minimal. This unexpected fact was discovered mainly due to uncovered significant contamination of the commercial beers tested (wild yeasts, lactic acid bacteria, enterococci and coliform bacteria). For this reason, the concentration of cells in the beer appeared as a more influential factor in the development of off-flavours, including autolysis, than the storage temperature. The partial results need to be studied in more detail, nevertheless, we felt it was important to publish the revelation of a strong contamination of beers purchased from regular stores for our experiments. At this point it can already be concluded that the low storage temperature did not help to prevent the development of the contamination and the associated sensory deterioration of the beers.

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.

Revalorization of agave bagasse in the obtention of type II sourdough inoculated with Lactococcus lactis NRRL B-50307

Sourdough is an ancient culture comprising flour and water, and fermented to allow leavening capacity from wild yeasts, producing bread that is preferred by the consumers. Agave bagasse (AB) is an abundant by-product in Mexico, rich in fiber and carbohydrates, but mostly wasted. This work aimed to revalorize AB as an ingredient for wheat sourdough. Different ratios (5%, 10%, and 15%, by weight) of AB powder were mixed with wheat flour, hydrated with 10% (w/v) water, and incorporated with Lactococcus lactis (NRRL B-50307). The fermentation was monitored for 6 days; physicochemical parameters (pH, titratable acidity [TTA], leavening capacity) were analyzed, and sourdough bread was produced. The population of lactic acid bacteria, yeasts, and molds, and adaptation of the added strain were monitored. Leavening capacity increased during fermentation, and L. lactis grew about 2 log CFU/day. Analysis of the sourdough showed a pH range of 3.6–4.9, and TTA ranged from 8 mg/mL to 11 mg/mL of lactic acid. Proximate analysis (%, w/w) revealed 28.95 ± 0.4 protein, 12.33 ± 0.98 fat, 37.68 ± 0.65 total carbohydrates, and 1.04 ± 0.32 ash. Sourdough bread added with AB + L. lactis showed significantly higher (p < 0.05) firmness (35.42 N ± 0.034 N) and volume (70.24 mL ± 4.67 mL) than using wheat only (20.48 N ± 4.04 N, and 49.10 mL ± 6.35 mL, respectively). AB is a good source of fiber and other compounds like minerals, suggesting its potential as a food ingredient, and encouraging the agave industry towards more sustainable development.

Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Sourdough starter culture microbiomes influence physical and chemical properties of wheat bread.

Sourdough fermentation is an ancient leavening method that uses wild yeasts to produce carbon dioxide, contributing to bread rise, and bacteria which produce organic acids. Sourdough starter cultures are known to be diverse in terms of the microorganisms they comprise and while specific genera and species of microorganisms have been identified from starters and associated with specific attributes, overarching relationships between sourdough starter culture microbiomes and bread quality are not well understood. The objective of this study was to characterize differences in the physical and chemical properties of breads produced with sourdough starter cultures with unique microbiomes. Sourdough starter cultures (n=20) of known microbial populations were used to produce wheat-based dough and bread, which were analyzed for chemical and physical properties then compared to their microbial populations in order to identify relationships between microbial profiles and dough/bread qualities. All samples were also compared to bread produced only with Saccharomyces cerevisiae (baker's yeast). Significant differences among pH, titratable acidity, loaf volume, crumb firmness, crust color, free amino acids, and organic acids were observed when comparing sourdough breads to the yeast-only control (p≤0.05). Furthermore, bacterial diversity of sourdough starter cultures was correlated with lactic acid and free amino acid in the dough and loaf volume and crumb firmness of baked breads. No significant correlations were found between fungal diversity and measured outcomes. These data demonstrate the importance of considering sourdough starter microbiomes as an ingredient in baked goods and they contribute to quality and safety outcomes in bread production. PRACTICAL APPLICATION: Sourdough starter cultures have diverse and dynamic populations of bacteria and yeasts, which contribute to the production of bread products. These populations can influence the physical and chemical properties of sourdough fermentation and final breads. Understanding of the relationship between sourdough starter microbiomes and bread quality parameters can lead to targeted development of sourdough bread products with specific physical and chemical properties.

Impact of selected wild yeasts starters on the volatilome and phenolic contents of Gordal, Manzanilla and Hojiblanca naturally fermented green olives

The impact of wild yeast starters previously isolated from table olive fermentations and selected for their volatile profile (Pichia kluyveri L13, Kluyveromyces lactis L39 and Wickerhamomyces anomalus L25) and/or its β-glucosidase activity (W. anomalus L25) on the volatilome and phenolic content of natural-style green table olives was investigated. For this, the selected yeast strains were inoculated in brines of natural-style green olives from Gordal, Manzanilla and Hojiblanca cultivars and fermentations were carried out for 6 months. In each case, spontaneous fermentations were carried out in parallel and served as controls. Metagenomic studies showed that the three strains were able to survive along the fermentations and affect the microbial composition of the brines. In all the three olive cultivars, the use of K. lactis L39 as starter resulted in a fermented product with a higher concentration of volatiles associated to pleasant aromas compared to that from the corresponding uninoculated fermentation. Despite W. anomalus L25 was a β-glucosidase-positive strain, its use as starter did not manage to reduce the total phenol content or the oleuropein content in pulp compared to the corresponding uninoculated control, demonstrating that the presence of β-glucosidase activity is not necessarily related to the ability to hydrolyze oleuropein.