Worldwide, two main phenomena are increasingly being reported for wine fermentation, first is the occurrence of stuck fermentations, as fructose becomes the main carbohydrate during the late stages of alcoholic fermentation, and the yeasts have to ferment under conditions of high ethanol concentration and nitrogen limitation. And second, a higher alcohol content and less aroma complexity [1
]. Some authors have used the positive response to a certain stress as a selection tool for yeast with potential for use in wine production. For example, Zuzuarregui and del Olmo [3
] analyzed the resistance to oxidative, osmotic and ethanol stresses among a collection of commercial (winery) and non-commercial S. cerevisiae
strains, and correlating fermentative behavior with resistance to oxidative stress (by exposure to H2
) and to ethanol stress as the most relevant. García and collaborators [4
] analyzed the tolerance to osmotic pressure, ethanol, and pH stresses in a warm climate region DO ‘Vinos de Madrid’ (Spain). These authors identified some Saccharomyces
strains adapted to these fermentation stresses, concluding that these yeasts are important in the quality wine in these warm areas.
The use of non-conventional yeast as inoculum for wine making has increased in importance in the last decades, as it has been observed that some strains can increase the aroma complexity of the fermented products [5
]. Our research has explored the potential use of novel yeast strains obtained from mezcal, which is a traditional Mexican liquor that involves a very stressful alcoholic fermentation [6
]. These yeasts are proposed since the musts of cooked agave plants contain a high fructose content (around 90% of fermentable sugars), Maillard compounds, furfural and even toxic saponins, and mezcal fermentation is carried out without temperature control, making this a very stressful fermentation system [2
]. In addition, these yeasts are part of a different domestication event as compared to other S. cerevisiae
wine strains [7
], and their phenotypic characteristics may be different, particularly, when submitted to high fructose concentration [6
]. Novel yeast applications to increase the aroma complexity in wine may be supported by the used of mixed starters. As an example, co-inoculation of S cerevisiae
strains changes wine composition regarding to monoculture [1
] and mixed starters with Saccharomyces
) strains enhances aromatic profile compared to simple mixed inoculation and increased the wine quality [10
In the current study, fermentative profiles of mezcal yeasts (Saccharomyces and non-Saccharomyces) in terms of primary and volatile metabolites production were compared when cultivated in a semi-synthetic medium (M3) to simulate wine fermentation. The results were used to choose Saccharomyces and non-Saccharomyces strains to be cultivated in grape juice individually, or as a mixed inoculum, and their fermentative performance and potential as starters for wine production were evaluated.
2. Materials and Methods
2.1. Yeast Strains and Inoculum Growth Conditions
The 24 yeast strains used belong to the mezcal LCBG yeast collection (which comprises 96 different strains, belonging to ten different yeast genera) and are conserved in 60% glycerol at −70 °C. The commercial wine strain Saccharomyces cerevisiae
Fermichamp (DSM Food Specialties B.V., The Netherlands) was used as a control for its fructophilic character, which is used to reactivate stuck fermentations as indicated by the manufacturer. The strains used were selected based on both their level of stress tolerance [6
] and to be representative of the yeast diversity found in the fermentation of mezcal from Tamaulipas (Mexico). For all the strains, their 26S nucleotide sequences are available in the GenBank and are presented on Table 1
, along with the fermentation stage from where they were originally isolated.
An initial preculture of the tested yeasts was grown on yeast extract-peptone-dextrose agar YPD, Difco Laboratories, France) agar plates containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L D-glucose, plus 20 g/L bacteriological agar (Difco Laboratories, France), and incubated at 30 °C for 48 h. A loop of this preculture was used as inoculum for liquid YPD broth incubated for 24 h at 30 °C with shaking at 200 rpm, and final yeast concentrations (total and viable) were quantified using a Neubauer chamber, adjusting if needed using sterile isotonic solution (9 g/L NaCl solution) and used immediately as inoculum at in the fermentation experiments carried out as described below.
2.2. Setup of Minifermentation Conditions
All fermentation experiments were carried out in minibioreactor tubes of 50 mL with 4-hole vent caps (Corning Science de México, Reynosa, TAM, Mexico), but covering 3 of the 4 holes available in the cap with cellotape just before inoculation, to allow semianaerobic fermentation conditions and also to diminish loss of water. The minibioreactors contained 20 mL of either the semi-synthetic medium M3 (Oliva-Hernández et al., 2013) or grape juice medium. Medium M3 contained 200 g/L of total sugars (glucose/fructose, 1:1), 1 g/L of yeast extract, 2 g/L of (NH4)2SO4, 0.4 g/L of MgSO4 7H2O, 5 g/L of KH2PO4, dissolved distilled water and with the pH adjusted to 5 before autoclaving. For wine-type fermentations, pasteurized red grape juice was used (Carrefour, Toulouse, France), which was typically around 200 g/L of total sugars, and also adding a small volume of ammonium sulfate sterile solution to have a final concentration of 2 g/L of (NH4)2SO4 in the grape juice, to avoid nitrogen limitation during fermentation. The inoculum used was 3 × 106 cells/mL, either when using individual or a mixed inoculum. In the latter case, a ratio of 1:9 of S. cerevisiae/non-Saccharomyces strains was used. Incubation was performed at 30 °C using an agitated Minitron HG incubator (Infors AG, Switzerland) at 75 rpm. Each experiment was run in triplicate, hence withdrawing and analyzing three different tubes per sampling time, and measurements for each minibioreactor were performed at least two times. Average values and standard deviation are reported accordingly.
2.3. Mixed Yeasts Populations Quantification
Quantification of the yeasts populations during fermentation was performed on Wallerstein Differential Agar WLD (Sigma-Aldrich, St. Louis, MO, USA) for following the non-Saccharomyces populations, as S. cerevisiae is unable to grow on such media. This allowed an easy verification of the viable count of the non-Saccharomyces species. Colony counts for S. cerevisiae were obtained by subtracting the WLD count number to the count obtained on on Wallerstein Nutrient Agar (WL Sigma-Aldrich, St. Louis, MO, USA) for whole yeasts population counts, and are reported as colony forming units per milliliter CFU/mL. Total and viable cell counts were determined by counting on a Neubauer chamber, using methylene blue staining as an indicator of viability of the whole population (Saccharomyces and non-Saccharomyces). All the samples were analyzed in triplicate.
2.4. Biomass and CO2 Production Quantification
Biomass was quantified as dry weight by centrifuging 2 mL of each sample in dry and pre-weighed 2 mL Eppendorf tubes for 10 min at 14,000 rpm. The supernatant was recovered and filtered for further high performance liquid chromatography HPLC analysis, and tubes containing the biomass pellet were dried half-open at 60 °C overnight, placed in a desiccator for at least 4 h, and then weighed. Biomass production was calculated as the difference in the weight of the tube divided by the volume of the centrifuged sample. Duplicate samples were taken from each of the three minibioreactor tubes per sampling time.
The release of carbon dioxide was used as an indicator of fermentation progress and to decide when to stop the experiments; hence, weight loss was followed for each minibioreactor every 24 h. At the experimental conditions tested, both in the semi-synthetic medium M3 and in grape juice, the rate of water loss in the minibioreactors per open hole in the cap was measured to be 0.0034 gwater/h per hole (R2 = 0.999), and this value was used as a correction factor to assess the CO2 liberated per liter of medium.
2.5. Sugar Consumption and Metabolite Quantification by HPLC
The consumption of sugars and the production of metabolites (ethanol, glycerol and acetic acid) in the centrifuged (15 min at 10,000 rpm at 4 °C) and filtered (Millex-GV13 0.22 µm pore size, Millipore Sigma, Burlington, MA, USA) sample supernatants were measured with an Accela HPLC (Thermo Scientific, France) coupled to an auto sampler and using a Phenomenex ROA-Organic acid column (250 mm × 4.6 mm; 8-µm diameter beads). The mobile phase was 5 mM H2SO4. The volume of the injection loop was 25 µL with each run lasting around 30 min with a flow rate of 0.17 mL/min at 30 °C. The peaks were detected by infra red IRD and/or ultra violet UVD, depending on the compound measured. Calibration curves were constructed using ethanol, glycerol, acetic acid, fructose, and glucose standards ranging from 0.125 to 5 g/L.
2.6. Volatile Compound Quantification by GC-MS
The production of volatile metabolites relevant for the organoleptic profile characterization of each strain was assessed by GC-MS in a TraceGC machine (Thermo Finnigan, Villebon Sur Yvette, France). Fermentation samples were centrifuged at 7000 rpm (5697× g) for 15 min at 10 °C in a Sigma 6K15 centrifuge, and 10 mL were taken and extracted by SPME (PDMS fiber assembly, SUPELCO, Bellefonte, PE, USA) at 40 °C and adding 3 g of NaCl, and the volatile compounds were measured by using a ZB-5ms Phenomenex column (30 m length × 0.25 mm internal diameter, 0.25 µm film thickness). The carrier gas was helium at a flow rate of 1 mL/min, the injector was set at 240 °C, and the following temperature program was used: 10 min at 35 °C, first ramp of 2 °C/min up to 60 °C, isothermal at 60 °C for one minute, second ramp of 2.5 °C/min up to 90 °C, third ramp of 10 °C/min up to 130 °C, isothermal at 130 °C for 2 min, and fourth ramp of 20 °C/min up to 240 °C. The transfer line was set at 250 °C. Internal standard was 3-octanol. The MS was performed in a PolarisQ ion trap machine (Thermo Finnigan, Villebon Sur Yvette, France) with a source temperature of 200 °C, ionization of 70 eV, and the multiplier offset was 0 volts.
2.7. Statistical Analyses
Statistical analysis was performed using the Analyze-it software for Microsoft Excel (version 2.20) and the JMP routine of the SAS software for ANOVA analysis.
One of the main objectives of this work was to compare the capabilities of production of aromatic volatile (flavor) compounds of the different mezcal strains when fermenting on a wine-type synthetic medium (M3), and in real grape juice, also, with a single strain inoculum or co-inoculated in a mixed fashion. Semi-synthetic medium M3 allowed us to compare the individual productive behavior of all the 24 strains. We found that ethanol was produced by all the strains, being maximal (but variable amongst strains) for S. cerevisiae as expected, but it also was produced in good quantities by some of the non-Saccharomyces strains belonging to Kluyveromyces, Torulaspora, and Zygosaccharomyces genera, which made them candidates to be tested in the grape juice medium as part of mixed inoculum with different S. cerevisiae strains, in terms of their displayed natural tolerance to this alcohol.
Concerning specifically to S. cerevisiae
strains, Camarasa et al. [11
] analyzed in a high glucose (240 g/L) synthetic medium the phenotypic variability, including the production of aromatic compounds, of a collection of 72 S. cerevisiae
strains obtained from seven different ecological niches: bakery, laboratory, natural isolates (plants and soil), clinical isolates, fermentative processes (beer, sake, palm wine), vineyard, and commercial wine. They observed that the larger differences amongst the strains are in their biomass production and formation of by-products but, interestingly, not in their ethanol production levels, different to what was observed in this work (Table 2
), specifically for ethanol. These authors concluded that commercial wine strains are characterized by high biomass concentration and good fermentative performance, low acetate production, and low ethyl butyrate synthesis. More recently, and similar to the work presented here for S. cerevisiae
mezcal strains, Franco-Duarte et al. [12
] established that, for their 24 S. cerevisiae
strains, ethanol and organic acids (in particular acetic acid) concentration explained most of the metabolic differences among strains. The S. cerevisiae
strains studied in more detail here produce comparable amounts of ethanol as the commercial strain Fermichamp, and the selected strains also led to high glycerol levels and were able to almost completely consume glucose and fructose during fermentation. In general, primary metabolites were produced in higher amounts in the grape juice medium than in the semi-synthetic medium M3.
For the mixed inocula fermentations, we observed an increased glycerol and acetic acid productions in the mixed cultures as compared with data obtained in pure cultures as reported by Reference [13
]. In mixed cultures, the S. cerevisiae
strains and T. delbrueckii
1AN9 reached their maximal populations at 24 h, similar to what was also reported by Reference [13
]. The comparison between the cell concentrations obtained in both nutrient WL (non-selective) and differential WL (no growth of S. cerevisiae
) agar media clearly shows that at the beginning of the fermentation process the majority of the population belongs to the non-Saccharomyces
strain, as inoculated in higher amounts, but as time proceeds, S. cerevisiae
becomes dominant up to the end of fermentation, similar to that reported by Reference [13
]. However, unlike these authors, who report a low percentage (<1%) of viability for their non-Saccharomyces
strains (C. zemplinina
and H. uvarum
), our strain Td1AN9 have a viability between 10 and 15% at the end of fermentation, and it is most affected by co-inoculation with control strain Fermichamp, being not due to ethanol concentration, as it was similar in all inoculum combinations. We observed the same phenomenon of a major inhibition due to the presence of control strain Fermichamp for the K. marxianus
and Z. bailli
strains (data not shown).
Overall, at the conditions tested in this work, the presence of a S. cerevisiae
strain reduces the growth capacity of Td1AN9 when it is mixed from 48 h of culture. At this time, the concentration of ethanol is around 65 g/L (Figure 2
), which is lower to the maximum ethanol production capability by the pure Td1AN9 inoculum fermentation (Figure 1
) and where the cell viable count is the same as the individual S. cerevisiae
strains, around 1 × 108
cells/mL. Hence, we cannot attribute solely to ethanol the inhibition/damaging effect over T. delbrueckii
cells at this time, although we know that concentrations above 8% ethanol are stressing on solid media as previously determined for this strain in YPD [6
], and that the S. cerevisiae
strain is taking advantage of the cellular contents leaked by the non-Saccharomyces
strain. This is in contrast to what has been reported for H. guilliermondii
]. These authors tested not only different ratio of species in the mixed inocula but also aerobic conditions and different media and S. cerevisiae
strains (data not shown by the authors) and concluded that inhibition and death of their non-Saccharomyces
strain was due to some unknown compound present and accumulated in the supernatant of S. cerevisiae
cultures, different than the killer toxins already reported for S. cerevisiae
. Similarly, it was reported that the main inhibitory mechanism towards their K. thermotolerans
and T. delbrueckii
strains was the physical presence of S. cerevisiae
]. Our results seem to support this latter explanation, although it is clear that such effect most probably is species-specific, as in the work of Kosel et al. [16
]. These authors did not find any effect of the cell-to-cell contact of the commercial S. cerevisiae
EC1118 over the growth kinetics of the cultures of Dekkera bruxellensis
, which is a spoilage yeast of low growth and a low tolerance to high sugar concentration, as well a weak producer of ethanol. All this indicates that more work is needed to clarify the specific properties of S. cerevisiae
with T. delbrueckii
Concerning volatile metabolites production, we found that different esters and higher alcohols were produced, and there were differences between pure and mixed cultures, being strain dependent as observed also by Reference [17
] and, in general, being higher for the mixed rather than for the individually inoculated fermentations. Pure cultures of S. cerevisiae
strains showed the highest total ester content, except for phenyl ethyl acetate, as compared to the non-Saccharomyces
strain Td1AN9. Maturano et al. [17
] obtained, for phenyl ethyl acetate, values from 30 to a maximum of 310 µg/L for the varieties of wine analyzed, while, in the results presented here, the non-Saccharomyces
yeasts showed values ranging from 130 to 5069 µg/L and, when used a mixed inocula, values were high, ranging from 450 to 540 µg/L, higher than those reported by Reference [17
]. This is a positive feature due to the great importance of this compound for its very pleasant floral aroma. As reported by Reference [1
], which also worked in wine-type synthetic medium, the production of certain volatile compounds that influence wine aroma was strain-dependent, and they observed that the concentrations of the measured compounds (except acetic acid) varied significantly in function of the inoculated strain, to the point that it permitted their identification, concluding that the combined use of two or more yeast strains or species is an interesting alternative for improving wine quality [19
]. In the work of Kosel et al. [16
], the non-contact (cultures separated by a membrane) co-culture of S. cerevisiae
with D. bruxellensis
in a synthetic wine must resulted in a higher production of aromatic ethyl ester compounds, as compared with the pure cultures of the two yeasts. These authors propose a hybrid computational pheno-metabolomic approach to classify and select those S. cerevisiae
strains with an increased performance on wine making, correlating this selection with a good growth on cycloheximide, on iprodion, and a temperature of 18 °C, the presence of two homozygous alleles (ScAAT6-256 and ScAAT5-256), and a high production of 2-phenylethyl acetate, ethyl butanoate, ethyl hexanoate, and ethyl octanoate.
The co-inoculation of Td1AN9 with any of the two S. cerevisiae
mezcal strains prevented/inhibited the production of isoamyl acetate but not when mixed with control strain Fermichamp, which is also capable of producing it. This may indicate a level of recognition and/or compatibility amongst mezcal strains, Saccharomyces
, as this compound (and isoamyl alcohol) has been recently reported as possessing a wide antimicrobial feature when present in a fermented (sake) beverage [20
], but also evidenced here by the relatively higher viability of the Td1AN9 strains at the end of the fermentation, when in presence of the two S. cerevisiae
mezcal strains, but not with control, wine strain Fermichamp. Overall, isoamyl alcohol concentrations varied between S. cerevisiae
yeasts as reported in other studies in wine [17
] and other fermented beverages [20
In winemaking, the use of pure yeast cultures allows a better control of the fermentations; however, it can also reduce the production of some desired metabolites, both from the yeast’s metabolism itself and from transformation of precursors present in the grape must. For this purpose, it is increasingly seen more convenient to use different yeast genera and species, which can contribute or influence the chemical composition and the flavor of wines [4
]. The volatile compounds produced by the strains analyzed in this study are of great aromatic value, especially the production of ethyl hexanoate and ethyl octanoate (apple note), isoamyl acetate (banana note), and phenyl ethyl acetate (fruity, floral notes), compounds which could render (in the appropriate amounts) good organoleptic characteristics to a wine.