Edited by: Victor Ladero, Spanish National Research Council (CSIC), Spain
Reviewed by: Jean-luc Legras, Institut National de la Recherche Agronomique Centre Montpellier, France; Isak Stephanus Pretorius, Macquarie University, Australia
This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
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Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although
Sherry wines are distinctive wines elaborated in the southern Spanish areas of Jerez–Xerez–Sherry, Montilla–Moriles, and Condado de Huelva. In these regions, Sherry wines are made using a traditional method termed “criaderas-solera,” which involves a dynamic biological aging process carried out by flor yeast, which are specific strains of
The criaderas–solera system consists of numerous rows (criaderas) of 600 L oak barrels with wine in various stages of aging. The rows are numbered from the floor to the top where the first lies on the ground, so called “solera,” and contains the most aged wine; the second row above is the “first criadera” and subsequently the second, third, etc. Before the criadera–solera system, the wine is kept in ∼2,000 L clay jars known as “sobretablas.” Glycerol and volatile acidity levels decrease, while acetaldehyde increases as the wine approaches solera (
Flor formation is induced by the lack of non-fermentable carbon sources that trigger the migration of yeast cells to the wine surface where oxygen is more abundant and the biological aging takes place. During this process, yeast cells oxidize ethanol to acetaldehyde and acetate, while glycerol is gradually catabolized (
Until now, several identification techniques have been aimed to characterize the flor microbiota. Flor yeast strains
The study of wine microbiota by next-generation sequencing (NGS) has ushered in a new era of biodiversity surveillance without the need of microorganism cultures between sampling and identification, enabling a high-throughput analysis of complex microbial communities
Besides NGS, microbial identification through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has recently gained popularity due to its cheaper costs and its application in many different areas such as medicine, food, military science, and ecological research. Microbial identification using MALDI-TOF MS compares peptide mass fingerprint (PMF) from the unknown organism from an axenic culture, with the PMF entries in the database, or by comparing the mass of the biomarker in the unknown organism with the reference proteome database (
In this study, we analyze for the first time the flor velum microbiota in criaderas–solera Sherry wine barrels in Montilla-Moriles by using novel culture-independent (ITS-metabarcoding) and -dependent techniques (MALDI-TOF MS). We also characterized the chemical profiles of the wine samples to see whether there were any possible correlations between the wine and the microbiota identified.
Flor velum samples were taken from 13 wine barrels at the Gracia winery in Montilla-Moriles region between September 2020 and April 2021 at various stages of the criaderas–solera system: 7 from solera and 6 from the first criadera. To collect flor yeast samples, a special sterile steel net was used. The net was sterilized by flame with 96% (v/v) ethanol before and after every sampling. The flor velum sampled was resuspended in 25 ml wine from same barrel in sterile tubes. Once in the laboratory, samples were treated in different ways (
Workflow of material and methods/experimental design.
To obtain higher biomass prior to identification, half of each sample was inoculated into a 300 ml media consisting of 1:1 yeast extract peptone dextrose (YPD) broth (1% yeast extract, 2% bacteriological peptone, 2% glucose) and wine in 500 ml Erlenmeyer flasks. This treatment was called “regrowth,” and the samples were incubated at 28°C and 175 rpm for 5 days and later under static conditions at 22°C for 5 days. The reason for this treatment was that untreated samples had a poor viability rate, due to the demanding conditions in the winery. Further, flor velum samples were cultivated at 22°C in wine from the same barrel in Erlenmeyer flasks to assess the development of the biofilm under laboratory conditions.
For ITS-metabarcoding identification, samples were directly processed, but for MALDI-TOF, in order to obtain isolates, the flor velum from 13 samples (7 from solera and 6 from first criadera) with and without regrowth were plated in YPD agar plates after serial dilutions and incubated for 5 days at 28°C. Then, 10 colonies randomly selected from each plate were isolated to obtain axenic cultures and subsequently identified by MALDI-TOF. Every sample was plated in triplicate.
Genomic DNA was extracted from 1 to 1.5 ml of flor velum resuspended in wine or YPD broth, containing yeasts by using a quick yeast genomic DNA extraction kit (Bio Knowledge Lab, S.L., Córdoba, Spain), following the guide provided by the manufacturer. The DNA from three samples (solera 1–2 and criadera 1) with and without regrowth were extracted.
The fungal ITS region was amplified using specific primers (ITS5-1737F and ITS2-2043R) with barcodes. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, United States).
To visualize PCR amplification, an equal volume of 1 × loading buffer (with SYB green) and PCR products were loaded on 2% (w/v) agarose gel for detection. Samples with bright main strip between 400 and 450 bp were chosen for further experiments. PCR products were mixed at equal density ratios and then purified with Qiagen Gel Extraction Kit (Qiagen, Germany).
Purified amplicons were prepared for Illumina sequencing by constructing a library using NEBNext Ultra™ DNA Library Prep Kit for Illumina and quantified
Analysis was carried out using QIIME2 v2020.8 (
After denoising, forward and reverse reads are merged into one sequence, dereplicated and assigned to an ID, considering them ASVs (amplicon sequence variants).
Amplicon sequence variants were organized in operational taxonomic units (OTUs) by using
A qualitative analysis of yeast isolates was performed with MALDI-TOF mass spectrometry (Bruker Daltonics, Bremen, Germany). A colony from each isolate was placed in 300 μl distilled water and 900 μl ethanol and vortexed until homogenization. Then, samples were pelleted at 13,000 rpm for 2 min, and the pellet was dried at room temperature. Lastly, 50 μl of 70% formic acid and 50 μl of acetonitrile were added to the pellet. Samples were centrifuged for 2 min at 13,000 rpm, obtaining a supernatant with proteins.
About 1 μl of supernatant was dried at room temperature in a matrix-assisted laser desorption/ionization (MALDI) plate, and each sample was coated with 1 L of HCCA matrix (α-cyano-4-hydroxycinnamic acid) prepared in a mixture of 50% acetonitrile and 2.5% trifluoroacetic acid. Samples were again dried at room temperature.
Dried samples were analyzed with MALDI-TOF/TOF “ULTRAFLEXTREME” (Bruker Daltonics, Bremen, Germany) equipment. Generated spectra were treated with MALDI Biotyper compass (MBT Compass; Bruker, Billerica, MA, United States) software, which calibrates the spectra and automatizes the measures and identifications before searching and matching the results. Obtained spectra were compared with reference profiles from the MBT Compass Library (Bruker). Scores ≥2.0 were used as a selection criterion for identifications at species level (
Wines with different microbiota were chemically analyzed in order to assess differences between them. The most important chemical analysis (CA) parameters from an oenological point of view are relative density, ethanol, titratable acidity, pH, volatile acidity, and free SO2. These parameters were quantified in accordance with the European Union Official Methods (
Considering the most abundant alcohols (methanol, 1-propanol, isobutanol, isoamylic, and 2-phenylethanol), 3 carbonyl compounds (acetaldehyde, 1,1-diethoxyethane, and acetoin), 3 ethyl esters (ethyl acetate, ethyl lactate, and ethyl succinate), 2 polyols (glycerol and 2,3-butanediol), and 13 wine aroma compounds were quantified by gas-chromatographic analysis (GCA) using the method of
The multiple comparison analysis (MCA) for each chemical variable using the Bonferroni’s test at a confidence level of 95% (i.e., α = 0.05 significance level) was carried out in order to identify those variables showing significant differences in the wines sampled. MCA groups samples that show significant differences in homogeneous groups (HGs). Averages with different HGs show statistically significant differences at the 95.0% confidence level. The method currently being used to discriminate among the averages is Fisher’s least significant difference (LSD) procedure. With this method, there is a 5.0% risk of calling each pair of averages significantly different when the actual difference equals 0. The variables obtained in major volatile compounds and polyols analysis were subjected to a analysis of clusters (AC) in order to identify differences in the groups of wine based on the presence or absence of non-
The flor velum samples for yeast identification were treated directly and after the “regrowth medium” (cultivated in 1:1 YPD:wine) in laboratory conditions. After 5 days of static incubation at 22°C, a flor velum or biofilm formation was observed in regrown samples on the surface of the medium. The biofilms were generally thick at the beginning and less consistent in the following days due to a progressive precipitation (
Biofilm formation in regrowth medium after 10 days (5 days at 28°C and 175 rpm and later under static conditions at 22°C for 5 days).
Internal transcribed spacer region analysis was carried out in the velum samples of solera 1, solera 2, and criadera 1 barrels. In those flor samples in which DNA was extracted directly,
Species identified by ITS-metabarcoding, localization, and frequency (%) in each sample.
Solera 1 regrowth | 22.90 | 74.72 | 2.38 |
Solera 1 | 100.00 | 0.00 | 0.00 |
Solera 2 regrowth | 13.20 | 83.88 | 2.90 |
Solera 2 | 100.00 | 0.00 | 0.00 |
Criadera 1 regrowth | 100.00 | 0.00 | 0.00 |
Criadera 1 | 100.00 | 0.00 | 0.00 |
All yeast isolates from solera or criadera without regrowth treatment were identified as
Species identified by MALDI-TOF MS, localization, and frequency (%) in each sample.
Solera 1 | 20.00 | 0.00 | 0.00 | 0.00 | 80.00 | 0.00 | 0.00 |
Solera 2 | 20.00 | 72.00 | 7.00 | 0.00 | 0.00 | 1.00 | 0.00 |
Solera 3 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Solera 4 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Solera 5 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Solera 6 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Solera 7 | 0.00 | 0.00 | 0.00 | 100.00 | 0.00 | 0.00 | 0.00 |
Criadera 1 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Criadera 2 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Criadera 3 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Criadera 4 | 13.30 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 86.70 |
Criadera 5 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Criadera 6 | 40.00 | 60.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
It was observed that the isolated and identified non-
Biofilm formation by different axenic yeast cultures at 10× once the biofilm covered the whole medium surface.
Colony of a putative non-
Multiple comparison analysis analysis identifies significant differences with Bonferroni’s test at a confidence level of 95%.
Means and standard deviations for oenological variables.
Solera w/o non-S |
Solera w non- |
Criadera w/o non- |
Criadera w non- |
|
Acetaldehyde | 235.25 ± 30.13c | 145.5 ± 7.67b | 58.48 ± 0.89a | 72.94 ± 14.60a |
Ethyl acetate | 21.89 ± 0.31a | 23.13 ± 0.56a | 73.79 ± 1.04b | 74.08 ± 3.014b |
1,1-Diethoxyethane | 19.20 ± 2.19c | 5.32 ± 4.61b | 0.00a | 0.00a |
Methanol | 100.18 ± 2.6a | 93.42 ± 41.8a | 79.55 ± 5.4a | 83.41 ± 13.29a |
1-Propanol | 63.15 ± 2.14d | 60.21 ± 3.82cd | 55.44 ± 1.26a | 56.86 ± 0.69ab |
Isobutanol | 79.73 ± 0.57c | 78.89 ± 1.71c | 45.87 ± 0.13a | 48.38 ± 0.19b |
Isoamyl alcohol | 394.76 ± 4.78b | 401.04 ± 7.68b | 322.79 ± 2.38a | 328.72 ± 0.66a |
Acetoin | 68.9 ± 11.8c | 55.88 ± 4.71b | 14.77 ± 1.56a | 14.75 ± 2.01a |
Ethyl lactate | 112.87 ± 19.27b | 48.60 ± 3.62a | 206.69 ± 9.93c | 215.03 ± 15.69c |
2,3-butanediol (l + m) | 1571.02 ± 359bc | 1694.21 ± 56.38c | 1215.94 ± 112.49ab | 1191.86 ± 103.63b |
Diethyl succinate | 102.85 ± 37.79b | 95.17 ± 8.22b | 52.36 ± 4.83a | 52.97 ± 3.64a |
2-phenylethanol | 66.89 ± 11.01b | 71.97 ± 4.00b | 47.56 ± 11.03a | 41.16 ± 3.44a |
Glycerol | 1425.08 ± 178.80a | 1146.67 ± 11.18a | 8646.03 ± 641.07b | 8029.87 ± 304.77b |
Ethanol (% v/v) | 15.17 ± 0.2a | 14.54 ± 0.2a | 14.99 ± 0.2a | 14.89 ± 0.2a |
Relative density | 0.9867 ± 0.0007a | 0.9867 ± 0.00b | 0.9877 ± 0.00b | 0.9877 ± 0.00b |
Volatile acidity (g/L) | 0.13 ± 0.01a | 0.18 ± 0.00b | 0.34 ± 0.01c | 0.36 ± 0.01c |
Titratable acidity (g/L) | 4.01 ± 0.27a | 4.01 ± 0.00a | 4.49 ± 0.06b | 4.74 ± 0.21b |
pH | 3.16 ± 0.03a | 3.2 ± 0.00b | 3.21 ± 0.01b | 3.21 ± 0.00b |
Free SO2 (mg/L) | 7.8 ± 0.77a | 10 ± 0.00ab | 12.3 ± 1.52bc | 14.00 ± 2.00c |
AC analysis identifies those samples showing a significant difference by means of the squared Euclidean distance, as a measure of the proximity between two samples and the method of Ward as a clustering rule. Considering that the chemical composition of the samples is affected by the aging time (criadera and solera) and by the presence/absence of non-
Cluster analysis of wines with different aging times and the presence of non-
Principal component analysis analysis agrees with the AC (
Principal component analysis of wines with different aging times and the presence of non-
Ethanol, ethyl lactate, pH, and titratable acidity were the parameters that contribute most to the variance.
Internal transcribed spacer-metabarcoding techniques are useful to study the microbiota diversity in winemaking processes such as alcoholic or malolactic fermentation without the risk of ignoring microorganisms that are not cultivable under laboratory conditions (
On the other hand, MALDI-TOF MS has been very useful to identify most of yeast isolates in a quick and economical manner; however, as it is observed in
Regrowth process through wine + YPD broth (1:1) seems to be a good step to conduct before identification. This will reveal the presence of non-
The presence of non-
The metabolomic study revealed no significant differences between wines with non-
It is the first time that
Further, this is the first report of
The main source of non-
Solera barrels had not been refilled for more than a year, and it is a six criaderas wine, so the wine used to refill these barrels it is an old wine too; for this reason, we agree with
The high concentration of acetaldehyde and ethanol (and maybe other compounds) could have caused mutations in the genome of these non-
Further studies are needed to understand if these yeasts have an impact on the quality of Sherry wines, but it seems that they have higher ethanol tolerance than other strains isolated from different environments. Therefore, these yeasts may be of interest in wine production because they would produce important metabolites for longer times due to their ethanol tolerance. Killer toxin production by the non-
In conclusion, ITS-metabarcoding and MALDI-TOF MS investigations reveal the existence of eight different yeast species in Montilla-Moriles Sherry wine, two of which are completely new in the criaderas and solera system. These two technologies were able to identify non-
The data presented in the study are deposited in the Sequence Read Archive (SRA) repository (NCBI), accession numbers
JC-P conducted the study, analyzed the experimental data, and drafted the manuscript along with JM-G. JuM helped to analyze and review the experimental data, participated in the selection and interpretation of the statistical analysis applied, and coordinationed the study. JCM and TG-M coordinated the work, critically reviewed the manuscript prior to submission, and secured the acquisition of funds. All authors approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
This research was co-funded by the Programa Operativo FEDER 2014-2020 and Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía (Reference: 1380480-R; TG-M and JuM) and by the contract ART. 83 OTRI with company Perez Barquero associated with the CDTI 2020 (Reference: 12020062; JCM).
We thank the staff at the Central Research Support Service (SCAI) of the University of Córdoba for kind help with the yeast identification from MALDI Biotyper and BKL by metataxonomic analysis, as well as the Perez Barquero Winery and PAUDIRE company.