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This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
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The non-
As most non-
The objective of the present work was to evaluate the use of new killer
EX85, EX85R, and E7AR1 are prototrophic and homothallic
Yeast strains used.
Strain | Genotype/Relevant phenotype | Origin |
---|---|---|
M. Ramíreza (from wine) | ||
M. Ramíreza | ||
M. Ramíreza | ||
This study (from wine) | ||
This study (from EX1180) | ||
This study (from EX1180) | ||
This study (from wine) | ||
This study (from EX1257) |
YEPD + cycloheximide (cyh) is YEPD-agar (1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose, 2% Bacto-agar) supplemented with cyh, prepared in a concentrated ethanol solution to a final concentration of 2 μg/mL (
Killer activity was tested on low-pH (pH 3.3 or 4.0) methylene blue plates (3.3MB or 4.0MB;
Must fermentation was carried out in 5-L Erlenmeyer flasks with 3.5 L of Cigüente grape must (18.0°Brix, pH 3.5, 50 mg/L SO2, and 0.3 g/L Actimax nutrients from Productos Agrovin S.A.) sterilized by membrane filtration through a Millipore system (0.45-μm membrane). Yeast cells were cultured in YEPD broth for 2 days at 30°C, washed twice (by centrifugation) with sterile water, and suspended in the must at the desired concentration. Fermentations were conducted at 18°C for 20 days. Yeast growth (determination of total yeast cells by counting with a Neubauer chamber, and viable cells by counting the yeast colonies that arose on YEPD-agar plates), and the °Brix were monitored. All experiments were done in triplicate.
The yeast inocula were obtained in a pilot plant of the company Heral Enología SL following its industrial procedure. Cells were cultured in beet molasses broth [5% beet molasses, 0.2% Bacto-yeast extract, 0.075% (NH4)2HPO4, 0.1% MgSO4⋅7H2O, adjusted to pH 3.5 with HCl] for 18 h at 30°C with strong aeration, washed twice (by centrifugation) with sterile distilled water, and inoculated in 350-L stainless steel tanks with cold-settled white Cigüente (19.0–19.8°Brix, pH 3.42, 80–250 NTU, 50 mg/L SO2, and 0.3 g/L Actimax) or Macabeo (20.4–20.8°Brix, pH 3.29–3.55, 80–250 NTU, 50 mg/L SO2, and 0.3 g/L Actimax) grape must to a final concentration of 2–4 × 106 cells/mL for
Determination of the percentage of genetically marked yeasts was done by the replica-plating method (
The percentage of wild parent yeasts, or genetically marked yeasts for the replica-plating results validation, was mostly determined by analyzing the mtDNA restriction pattern as previously described (
The yeast spore (after yeast growth on sporulation medium for 7–30 days at 25°C) or vegetative cell morphology were also eventually analyzed for validation of the previous results obtained by the replica-plating or mtDNA restriction pattern analyses. This morphology analysis was done by microscopic observation in a Nikon Eclipse 600 microscope equipped with a Nomarski 60× objective.
Density, °Brix, pH, total acidity, volatile acid, reducing sugars, alcohol, and malic acid were determined according to the EC recommended methods (
The wine aroma compounds were analyzed by gas chromatography coupled to a mass detector. The minor aroma compounds were isolated and pre-concentrated following a solid-phase extraction (SPE) procedure (
Data were analyzed for statistical significance by a one-way analysis of variance (ANOVA,
The influence of any given yeast on winemaking will mostly depend on its ability to dominate the must fermentation while reducing the influence of the other participating yeasts. Complementary and reliable methods to monitor the different wine yeasts in the fermenting must are required to accurately determine the degree of domination of each yeast strain. We isolated and characterized new spontaneous cyh-resistant (cyhR) mutants from new
To determine whether the
Once we had determined the
In view of these disappointing results, new vinification trials were performed using single inoculation with
Must fermentation parameters and white wine analysis results of independent winery vinifications made with Cigüente and Macabeo musts and of an ANOVA to study the effect of single initial inoculation with
Parameter | Yeast species |
||
---|---|---|---|
T15 (days) | 1.75 ± 0.23 | 5.13 ± 0.60 | 0.000 |
T100 (days) | 10.0 ± 3.87 | 20.7 ± 3.08 | 0.043 |
Preference (%) | 64.4 ± 4.67 | 56.7 ± 3.79 | 0.128 |
Frequency in TF (%) | 100 ± 0.00 | 96.1 ± 2.23 | 0.180 |
Frequency in EF (%) | 100 ± 0.00 | 86.8 ± 7.49 | 0.172 |
Alcohol (% v/v) | 11.3 ± 0.58 | 11.2 ± 0.56 | 0.967 |
Glycerol (g/L) | 6.1 ± 0.20 | 5.65 ± 0.37 | 0.315 |
pH | 3.07 ± 0.07 | 3.20 ± 0.05 | 0.165 |
Total acidity (g/L) | 7.21 ± 0.23 | 6.89 ± 0.23 | 0.362 |
Volatile acidity (g/L) | 0.26 ± 0.05 | 0.36 ± 0.07 | 0.366 |
Density (g/L) | 990.7 ± 0.41 | 994.6 ± 1.25 | 0.026 |
Reducing sugars (g/L) | 1.24 ± 0.15 | 5.98 ± 2.15 | 0.091 |
Mannoproteins (mg/L) | 58.8 ± 4.74 | 123.3 ± 32.6 | 0.086 |
Malic acid (g/L) | 1.47 ± 0.12 | 1.45 ± 0.16 | 0.926 |
Lactic acid (g/L) | 0.07 ± 0.01 | 0.14 ± 0.06 | 0.363 |
None of these wines underwent malolactic fermentation, even those inoculated with
The wines made with
The total (summatory) amount of ethyl esters, acetate esters, organic acids, alcohols, monoterpenes, lactones, and carbonyl compounds was greater in the
The new
Sequential yeast inoculation with
Single
The main fermentation aroma of the
Overall, our results are partially in agreement with those previously reported for the influence of
In sum, it seems that
Notwithstanding this finding of variability in the
MR conceived the project. MR, RV, MÁ, and EZ designed and performed the experiments. MR, RV, EZ, and LH analyzed the data. MR wrote and edited 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.
This work was funded by grants GR10088 from the Extremadura Regional Government and AGL2011-25711 from the Spanish Ministry of Education and Science. RV was the recipient of a studentship from the Extremadura Regional Government. We are grateful for the technical and human support provided by the SAIUEx Facility of Elemental and Molecular Analysis (financed by UEX, Junta de Extremadura, MICINN, FEDER, and FSE) for the analyses of the wine aroma compounds.
The Supplementary Material for this article can be found online at: