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Article

Influence of Saccharomyces pastorianus and Saccharomyces bayanus Inoculation Ratio to Oenological Characteristics of Sauvignon Blanc Wine

by
Maria Dimopoulou
1,*,
Vicky Troianou
2,
Chrisavgi Toumpeki
2,
Despina Lola
3,
Elli Goulioti
3,
Aikaterini Tzamourani
1,
Etienne Dorignac
4,
Spiros Paramithiotis
5,* and
Yorgos Kotseridis
3
1
Department of Wine, Vine and Beverage Sciences, School of Food Science, University of West Attica, 28 Ag. Spyridonos St., 12243 Athens, Greece
2
Innovino Research & Development, 21 Meg. Alexandrou St., 15351 Pallini, Greece
3
Laboratory of Oenology, Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos St., 11855 Athens, Greece
4
Fermentis, 137 Rue Gabriel Péri, 59703 Lille, France
5
Laboratory of Food Process Engineering, Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos St., 11855 Athens, Greece
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(6), 3393; https://doi.org/10.3390/app13063393
Submission received: 9 February 2023 / Revised: 28 February 2023 / Accepted: 6 March 2023 / Published: 7 March 2023
(This article belongs to the Special Issue Yeast Fermentation and Biotechnology)
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Versions Notes

Abstract

:
The aim of the present study was to assess the effect of the inoculation ratio between Saccharomyces pastorianus strain SP2 and S. bayanus strain BCS103 on the enological properties and aroma profile of Sauvignon Blanc wines. For that purpose, a total of eight different fermentation trials on Sauvignon Blanc must took place. These included spontaneous fermentation as well as inoculation with pure and mixed cultures of the S. pastorianus and S. bayanus strains. The mixed cultures contained different proportions of the two strains (S. pastorianus SP2 to S. bayanus BCS103: 99–1%, 97–3%, 95–5%, 90–10% and 70–30% w/w). Classical oenological analyses were employed to assess the course of fermentation and classical microbiological enumeration combined with inter-delta sequence profile analysis was used for yeast population dynamics estimation. The volatile compounds of each wine were analyzed with GC/MS. The fermentation was completed between 11 and 13 days, while the inoculation ratio significantly affected the chemical composition and the sensorial evaluation of the resulting wines. Based on the sensory evaluation, the least-appreciated Sauvignon Blanc wine was the one resulting from spontaneous fermentation, and the higher the ratio of the S. bayanus strain in the inoculum, the higher the level of appreciation of the wine.

1. Introduction

Wine quality depends on the inherent characteristics of the grape varieties, the pedoclimatic environment, the agricultural and winemaking practices as well as their interaction [1,2,3,4,5,6,7,8]. The outcome of the fine balance between these factors is the production of wines with varietal typicity expressing the unique character of each region. This is also the case of Sauvignon Blanc, a variety that is among the most preferred for the production of aromatic white wines. Sauvignon Blanc wines are characterized by a very wide palette of aromas and flavors. Among the most distinctive ones are grapefruit, passion fruit and box tree notes, the occurrence of which has been attributed mainly to the presence of aromatic volatile thiols, namely 3-mercaptohexan-1-ol, 3-mercaptohexyl acetate and 4-mercapto-4-methylpentan-2-one, respectively [9,10,11]. Especially regarding the first two, their presence and concomitantly, the intensity of the respective aromas seems to be largely dependent upon the yeast strain carrying out alcoholic fermentation [12,13,14].
In recent years, two additional factors have emerged that demanded specific attention and proper adjustment of the winemaking practices, namely the modification of the climatic conditions and consumer preferences [15,16,17]. The first resulted in a series of effects on vine phenology and grape ripening, which significantly affected the microbial ecology of the must and ultimately yeast metabolism and alcoholic fermentation [15]. At the same time, a consumer trend towards increased aromatic complexity and modified varietal typicity has also been noted [16,17]. Research has initially addressed this demand through the use of non-Saccharomyces yeasts in combination with S. cerevisiae. However, such coexistence is not always feasible due to the antagonistic relationship that may develop [12,18,19,20,21,22,23,24,25,26]. Interestingly, such an issue has not yet been reported when strains within the Saccharomyces sensu stricto group are combined.
Saccharomyces pastorianus and S. bayanus are interesting alternatives that have been successfully employed to modulate Sauvignon Blanc typicity [27]. More specifically, the wines fermented with S. pastorianus monoculture contained less acetic and malic acids, while those fermented with the coculture of S. pastorianus and S. bayanus, at a ratio of 70/30 (w/w), were characterized by higher citrus fruit notes, compared to the wines fermented with S. cerevisiae. In addition, the significance of the inoculation ratio between S. pastorianus and S. cerevisiae was also recently highlighted [28]. A series of inoculation ratios were assessed and each of them significantly affected the enological properties and the aroma profile of the produced Sauvignon Blanc wines. Therefore, the aim of the present study was to further assess the effect of the inoculation ratio between S. pastorianus and S. bayanus on the enological properties and aroma profile of Sauvignon Blanc wines. For that purpose, five different inoculation ratios were employed for alcoholic fermentation and the resulting wines were thoroughly assessed from a chemical and organoleptic perspective.

2. Materials and Methods

2.1. Microorganisms and Culture Conditions

Saccharomyces bayanus strain BCS103 (Fermentis, Marcq-En-Baroeul, France) and S. pastorianus strain SP2 [28] were used throughout this study. The first was stored in its commercially available dry form, while the latter was stored in Nutrient Broth supplemented with 20% glycerol at −20 °C. Both strains were activated by cultivating twice in YM broth (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract) at 25 °C for 48 h.

2.2. Winemaking Conditions

Grapes of the Sauvignon Blanc variety, grown in Asprokampos (Nemea, Greece), were harvested, manually destemmed and crushed, and 50 mg/L sodium metabisulfite (Scharlab S.A, Barcelona, Spain) was added. The initial must density and total acidity were determined at 11.87 °Be and 6 g tartaric acid/L, respectively. After cold clarification, the must was decanted into 16 glass fermenters (30 L each) and inoculated with the yeast strains at final populations of 106 CFU/mL. The cases that were assessed are presented in Table 1.
The different inoculation ratios were achieved by mixing the appropriate serial dilutions of the two yeast strains. Two fermentation vessels were inoculated for each case. The addition of 200 mg/L SpringFerm™ (Fermentis, France) took place 24 h after inoculation. Fermentation took place at 18 °C.

2.3. Chemical Analyses

2.3.1. Standard Enological Parameters

Alcoholic fermentation was monitored in daily intervals through the determination of glucose and fructose; their depletion (less than 2 g/L) signified the end of fermentation. Analysis of glucose/fructose, along with malic acid, acetic acid and glycerol as well as initial and final pH value and total acidity were determined using enzymatic kits adapted for Biosystems Auto-analyser (Barcelona, Spain). Determination of free and total SO2 was performed by titrimetric methods. NIR spectrometry was employed for ethanol quantification [29].

2.3.2. Volatile Compounds Quantification

Head-Space Solid Phase Micro Extraction (HS-SPME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) was employed to analyze wine volatile compounds. The analysis was performed according to Dimopoulou et al. [27]. In brief, DVB/CAR/PDMS, 75 μm fiber was used for volatile compounds absorption. Analysis was performed with an Agilent 7890A GC (Santa Clara, CA, USA) equipped with an Agilent 5873C MS detector. The DBWAX capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was employed for the separation of the volatile compounds using helium as a gas carrier at a flow rate of 1.2 mL/min. The temperature of the injector and the MS-transfer line was 250 °C and 260 °C, respectively. The oven temperature was maintained at 30 °C for 5 min, then raised to 220 °C at 4 °C/min and held at this temperature for 20 min.

2.3.3. Quantification of Varietal Thiols

The method described by Tominaga et al. [10] was employed for the quantification of varietal thiols, namely 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA) and 4-methyl-4-methylpentan-2-one (4MMP).

2.4. Microbiological Analyses

Microbiological analyses were performed 48 h after inoculation, after completion of approximately 2/3 of fermentation and at the end of fermentation. Since the speed of fermentation differed between samples, the latter two corresponded to different days. The enumeration of Saccharomyces and non-Saccharomyces yeasts population was performed by plating serial dilutions on Wallerstein Laboratory Nutrient (WLN) agar and Lysine medium agar, respectively. The latter was incubated at 28 °C for 48–72 h. Phenotypic differentiation between S. bayanus and S. pastorianus was achieved by incubating the WLN plates at elevated temperatures. More specifically, incubation at 37 °C for 24 h resulted in the enumeration of the S. bayanus colonies due to the inability of S. pastorianus to provide adequate growth at temperatures above 30 °C. Conversely, incubation of the WLN plates at 28 °C for 48 h will result in the enumeration of colonies of both yeast strains. Verification of the yeast identity was performed by the inter-delta sequence profile analysis according to Dimopoulou et al. [28].

2.5. Sensory Analysis

Sensory analysis was performed by a panel of eight trained and experienced judges, according to Dimopoulou et al. [27]. The trained judges belonged to the age range from 25 to 55 years old, all working in the academic departments of the present study (affiliations 1 and 3). The descriptors assessed were grouped into four categories: 1. Visual; 2. Olfactory (aroma intensity, freshness feeling, floral, citrus, amylic, tropical fruits, vegetal/herbaceous, reduction, complexity); 3. Gustatory (acidity, body/roundness, sweetness, balance, aftertaste/persistence); 4. Overall quality.

2.6. Statistical Analysis

Statistical analysis was performed using JMP version 3.1.5 software (SAS Institute Inc., Cary, NC, USA). The differences between the chemical parameters and sensorial descriptors were assessed with a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test (p < 0.05).

3. Results

Table 2 shows the concentration of glucose and fructose (in g/L) during the alcoholic fermentation of Sauvignon Blanc must under the different inoculation cases assessed. Alcoholic fermentation was considered as completed (glucose + fructose < 2 g/L) after 10 days, when fermentation was carried out by S. bayanus BCS103 as a monoculture and in coculture with S. pastorianus SP2, with the population of the latter up to 95% of the total inoculum. The fermentation was completed after 12 days when it was carried out spontaneously by S. pastorianus SP2 as a monoculture and in coculture with S. bayanus BCS103 at a ratio of 97/3. Finally, the fermentation was completed after 13 days when it was carried out by the coculture of S. bayanus BCS103 with S. pastorianus SP2 at a ratio of 1/99.

3.1. Microbiological Analyses

A total of 250 colonies were randomly selected from the agar plates used for the enumeration of the S. bayanus BCS103 and S. pastorianus SP2 strains and subjected to inter-delta fingerprinting in order to verify their identity. The inability of the S. pastorianus strain to grow adequately at 37 °C was verified in all cases; therefore, the temperature-based differentiation that was employed in the present study was deemed accurate.
The evolution of Saccharomyces and non-Saccharomyces yeast populations during the alcoholic fermentation of Sauvignon Blanc must under the different inoculation cases assessed is presented in Figure 1. In all cases, alcoholic fermentation was driven by Saccharomyces yeasts while the non-Saccharomyces kept a low population along the fermentation process. Worth to mention the exception of the spontaneous one, in which the non-Saccharomyces population prevailed at the 48th hour of fermentation (Figure 1A) while the indigenous Saccharomyces dominated the rest of the process but in a lower population compared to the inoculated conditions. (Figure 1B,C).
In Figure 2, the evolution of S. bayanus BCS103 and S. pastorianus SP2 populations during the alcoholic fermentation of Sauvignon Blanc must is shown. Despite the different initial populations that were used when co-inoculation of S. bayanus BCS103 and S. pastorianus SP2 was employed, at the 48th hour of fermentation, no statistically significant differences between their populations were observed, and the fermentation seemed to be driven by both strains (Figure 2A). This was also the case at the 2/3 of fermentation and at the end of fermentation with the exception of the 90/10 and the 99/1 co-inoculation experiments, in which the population of the S. bayanus strain was higher than the respective of the S. pastorianus strain (p < 0.05) (Figure 2B,C).

3.2. Chemical Analyses

In Table 3, the standard enological parameters after the completion of alcoholic fermentation are presented. The pH value ranged between 3.28 and 3.39, with the lowest value observed in the wine made with the monoculture of S. bayanus BCS103 and the highest observed in the wine made with the monoculture of S. pastorianus SP2. The total acidity of the wines ranged between 5.78 and 6.04 g tartaric acid/L, without statistically significant differences between them. The total SO2 ranged between 18 and 40 mg/L. The lowest concentration was observed in the wine made by spontaneous fermentation as well as the one made with the monoculture of S. bayanus BCS103. The highest amount of total SO2 was observed in the wine made with the co-culture of the two strains at a ratio of 99/1. No free SO2 was detected in any wine. In all cases, the ethanol produced ranged between 12.5 and 12.8 % vol.
The volatile and non-volatile compounds quantified at the end of alcoholic fermentation of Sauvignon Blanc must, under the different inoculation cases studied, are exhibited in Table 4. The wine produced by spontaneous fermentation presented significant differences from those produced by inoculation; it contained the highest amounts of malic acid, acetic acid and hexyl acetate and the lowest amounts of glycerol, ethyl 2-methyl butyrate, ethyl isobutyrate, 2-phenyl ethanol, propanol and isoamyl alcohol. Similarly, the inoculation strategy affected the concentration of the volatile and non-volatile compounds. More specifically, the wine made with the monoculture of S. pastorianus SP2 had more ethyl octanoate, ethyl hexanoate, ethyl butyrate, ethyl hydroxy propanoate, ethyl 3-hydroxy butanoate, isobutanol, butanol, isoamyl alcohol, 3-sulfanylhexan-1-ol and 3-sulfanylhexan-1-ol acetate and less ethyl 2-hydroxy isobutyrate, hexyl acetate, isoamyl acetate, 2-phenylethyl acetate and 4-methyl-4-sulfanylpentan-2-one than the wine made with the monoculture of S. bayanus BCS103. Co-inoculation of the two strains affected significantly the volatile and non-volatile content of the produced wines, with the exception of glycerol and propanol, the amount of which presented no statistically significant differences between the wines produced with the monoculture of S. pastorianus SP2 and the ones produced with any co-culture employed. The wines produced with any co-culture had less ethyl hexanoate, ethyl butyrate, ethyl hydroxy propanoate and 3-sulfanylhexan-1-ol acetate than those produced with the monoculture of S. pastorianus SP2. Such reduction was also observed in the case of malic acid, ethyl octanoate, ethyl 3-hydroxy butanoate, hexyl acetate, isobutanol, butanol and 3-sulfanylhexan-1-ol, but under different co-culture ratios. Conversely, an increase in the amount of acetic acid, ethyl decanoate, ethyl 2-methyl butyrate, ethyl isobutyrate, ethyl 2-hydroxy isobutyrate, isoamyl acetate, 2-phenyl ethanol and isoamyl alcohol was observed, but not in all co-culture ratios. Interestingly, the amount of 2-phenylethyl acetate and 4-methyl-4-sulfanylpentan-2-one increased at co-culture ratios of 70/30 and 90/10 but decreased at ratios 97/3 and 99/1.
The diversity of the volatile compounds produced under the different inoculation schemes is illustrated by the Principal Component Analysis in Figure 3. The PCA analysis of the volatile compounds clearly distinguished in different quadrants the monoculture fermentation schemes and the spontaneous fermentation while the co-inoculation schemes were distributed mainly on the upper part of the biplot. The co-inoculation modality of SP2 90%-BCS103 10% was the closest to the monoculture condition BCS103, both characterized by the production of ethyl 2-hydroxyisobutyrate, isoamyl acetate, 2-phenylethyl acetate and 4-methyl-4-sulfanylpentan-2-one. Diametrically opposed, the fermentation schemes of S. pastorianus as monoculture, as well as the coculture with S. bayanus at a ratio of 70/30 and 99/1, were positioned to the right part of the biplot. At this side of the plot, the majority of the ethyl esters as well as 3-sulfanylhexan-1-ol, butanol and isobutanol were grouped. Finally, the spontaneous fermentation is clearly distinguished by the rest fermentation conditions and positioned on the lower left quadrant, characterized by high hexyl acetate production.

3.3. Sensory Analysis

In Figure 4, the sensory analysis of the wines produced from the Sauvignon Blanc variety is presented. In general, no statistically significant differences were observed in the grades received in the visual descriptor ‘color’, the olfactory descriptors ‘floral’ and ‘citrus’ as well as the gustatory descriptor ‘sweetness’ between the wines produced. The wines produced by spontaneous fermentation received lower grades in the olfactory descriptor ‘aroma intensity’, the gustatory descriptor ‘balance’ as well as the overall quality, compared to the wines produced by inoculation. The different inoculation strategies also affected the sensory analysis of the wines to some extent. The wines produced with the monocultures of the strains under study received different grades (p < 0.05) in the overall quality and the olfactory descriptor ‘aroma intensity’. More specifically, the grades received by the wine made with the monoculture of the S. bayanus BCS103 were higher than the respective received by the wine made with the monoculture of the S. pastorianus SP2. Co-inoculation with both strains had also a significant effect on the sensory analysis of the wines. More specifically, the olfactory descriptor ‘aroma intensity’ of the wine made with the inoculum ratio 90/10 and the overall acceptance of the wines made with the inoculum ratios 70/30 and 90/10 were improved compared to the wine made by the monoculture of the S. pastorianus SP2. Conversely, the olfactory descriptors ‘aroma intensity’, amylic’ and ‘complexity’ as well as the gustatory descriptors ‘body/roundness’ and ‘aftertaste/persistence’ and the overall quality of the wine made with the inoculum ratio 99/1 received lower grades than the respective made with the monoculture of the S. bayanus BCS103. Similarly, the wine made with the inoculum ratio 99/1 received a higher grade in the olfactory descriptor ‘reduction’ compared to the one made with the monoculture of the S. bayanus BCS103.

4. Discussion

Enhancing aromatic complexity without altering varietal typicity is the key to effectively addressing the recent consumer trends toward new sensorial challenges. The strategy that has been employed for that purpose includes the utilization of yeast strains that unfold the typical varietal aromas and at the same time supplement them through the production of fermentation aromas. Thus, novel combinations are created, at least from the varietal typicity perspective. S. pastorianus is not that common in winemaking while S. bayanus is used more frequently, however, usually for difficult fermentations, i.e., for high levels of sugar musts or stuck fermentations; however, they have provided very interesting results regarding the alcoholic fermentation of Sauvignon Blanc must, especially under co-inoculation mode [27]. The aim of the present study was to expand our knowledge of the interactions between two strains of the aforementioned species and their effect on the sensorial quality of Sauvignon Blanc wine. Thus, the chemical composition and the sensorial properties of wines made by the co-culture of these strains at different ratios were evaluated and compared with the ones made by each strain separately and through spontaneous fermentation.
Controlled fermentation through the utilization of suitable strains is the strategy employed in order to obtain reproducible results. In the case of alcoholic fermentation, strains belonging to the Saccharomyces genus are employed for ethanol production, and strains belonging to other genera, such as Hanseniaspora, Metschnikowia, Issatchenkia, Pichia, Schizosaccharomyces, Torulaspora and Lachancea, for the production of aroma compounds [23,30,31,32,33,34,35,36]. Spontaneously fermented wines are usually characterized by higher sensorial complexity and richer body, which has been attributed to the larger variety of metabolites produced by the larger yeast consortium that contributes to alcoholic fermentation [16,37,38]. However, this consortium is uncontrolled, and, therefore, the outcome of the fermentation is unpredictable. Thus, spontaneously fermented wines may be characterized as highly acceptable [39,40], or, as in the present study, sensorially unsatisfactory [41]. Additionally, according to our results, the volatile and sensory profiles of the spontaneously fermented wines were well distinguished compared to the inoculated ones. It seems that the dominance of non-Saccharomyces yeast after 48h of alcoholic fermentation clearly has an impact on the final produced wines [42].
Even if the impact of non-Saccharomyces yeast in wine has been well documented in the literature [43,44], the prediction of their intra- and inter-species interaction along the winemaking process is not yet established. The main obstacle of both spontaneous and co-inoculation strategies in wine production is the compatibility of the microbial strains, in terms of population growth and metabolite production. The antagonistic interactions that possibly develop may have a negative impact on wine quality [45,46,47]. Regarding the relationship between the S. bayanus and the S. pastorianus strains used in the present study, good compatibility was observed between the strains since no negative oenological attributes were detected. In terms of coexistence during fermentation, S. bayanus seems to grow more efficiently in the must environment, since it managed to grow at populations equal to or higher than the respective population of S. pastorianus, even though it was inoculated at a lower initial population. Therefore, it can be concluded that there are no evident indirect interactions of mutualism or commensalism between the two strains as their co-existence didn’t affect their growth compared to the pure culture conditions [47,48].
Interestingly, the inoculation of Sauvignon Blanc must with different ratios of the two strains resulted in the production of wines with different chemical compositions and concomitantly sensory evaluation. These differences could not be correlated with the population of the two strains during fermentation, since in the majority of the cases no statistically significant differences were observed. Our findings suggest that the interaction of the two strains could be based mainly on the presence of metabolites through physical contact between these microorganisms. Wine yeasts have been reported to secrete small quorum-sensing molecules to assess their population density and adapt their behavior under stress conditions [49,50]. For instance, the aromatic alcohol 2-phenylethanol has been reported to play the role of a signaling molecule in S. cerevisiae [51]. In our case, the production of 2-phenylethanol was significantly higher when the inoculation rate of S. pastorianus was higher than 90% compared to the monoculture conditions. It would be interesting to study in the future the kinetics of 2-phenylethanol under a co-inoculation mode of the two species and also study more metabolites through non-targeted metabolomics analysis.

5. Conclusions

The present study aimed to assess the effect of different S. pastorianus and S. bayanus inoculation ratios on the sensory profile of Sauvignon Blanc wines, towards the modification of the varietal typicity. The good compatibility of the strains employed was exhibited by the absence of oenological attributes. The S. bayanus strain seemed to grow more efficiently in the must environment, and the higher the ratio of the S. bayanus strain in the inoculum, the higher the level of appreciation of the Sauvignon Blanc wine.

Author Contributions

Conceptualization, Y.K., V.T. and E.D.; methodology, V.T., C.T., D.L., E.G., A.T., M.D. and Y.K.; formal analysis, M.D., S.P. and Y.K.; investigation, V.T.; data curation, V.T., C.T., D.L., E.G. and A.T.; writing—original draft preparation, M.D., S.P. and Y.K.; writing—review and editing, M.D. and S.P.; visualization, M.D. and S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 03393 g001 550
Figure 1. Evolution of Saccharomyces and non-Saccharomyces populations (in log CFU/mL) during alcoholic fermentation of Sauvignon Blanc must after 48 h (A), 2/3 of the process (B) and at the end of fermentation (C). The must was fermented under different inoculation schemes using S. bayanus BCS103 (BCS103) and S. pastorianus SP2 (SP2) as monoculture and co-inoculation of S. pastorianus SP2 and S. bayanus BCS103 at a ratio of 90/10, 95/5, 97/3, 99/1 and 70/30. The condition with no inoculation scheme was also tested (spontaneous).
Figure 1. Evolution of Saccharomyces and non-Saccharomyces populations (in log CFU/mL) during alcoholic fermentation of Sauvignon Blanc must after 48 h (A), 2/3 of the process (B) and at the end of fermentation (C). The must was fermented under different inoculation schemes using S. bayanus BCS103 (BCS103) and S. pastorianus SP2 (SP2) as monoculture and co-inoculation of S. pastorianus SP2 and S. bayanus BCS103 at a ratio of 90/10, 95/5, 97/3, 99/1 and 70/30. The condition with no inoculation scheme was also tested (spontaneous).
Applsci 13 03393 g001
Applsci 13 03393 g002 550
Figure 2. Evolution of S. bayanus BCS103 and S. pastorianus SP2 populations (in log CFU/mL) during alcoholic fermentation of Sauvignon Blanc must after 48 h (A), 2/3 of the process (B) and at the end of fermentation (C). The must was fermented under different inoculation schemes using S. bayanus BCS103 (BCS103) and S. pastorianus SP2 (SP2) as monocultureς as well as co-inoculation of S. pastorianus SP2 and S. bayanus BCS103 at a ratio of 90/10, 95/5, 97/3, 99/1 and 70/30.
Figure 2. Evolution of S. bayanus BCS103 and S. pastorianus SP2 populations (in log CFU/mL) during alcoholic fermentation of Sauvignon Blanc must after 48 h (A), 2/3 of the process (B) and at the end of fermentation (C). The must was fermented under different inoculation schemes using S. bayanus BCS103 (BCS103) and S. pastorianus SP2 (SP2) as monocultureς as well as co-inoculation of S. pastorianus SP2 and S. bayanus BCS103 at a ratio of 90/10, 95/5, 97/3, 99/1 and 70/30.
Applsci 13 03393 g002
Applsci 13 03393 g003 550
Figure 3. Principal component analysis of 20 volatile compounds of Sauvignon blanc wines fermented with monocultures of S. bayanus (BCS103) and S. pastorianus (SP2), mixed cultures of both species in different inoculation ratio as well as the spontaneous fermentation condition.
Figure 3. Principal component analysis of 20 volatile compounds of Sauvignon blanc wines fermented with monocultures of S. bayanus (BCS103) and S. pastorianus (SP2), mixed cultures of both species in different inoculation ratio as well as the spontaneous fermentation condition.
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Applsci 13 03393 g004 550
Figure 4. Heatmap analysis representation corresponding to Sauvignon Blanc wine sensory evaluation fermented under different inoculation schemes. The relative content of each sensory attribute is illustrated through a color scale (from dark blue, minimum, to dark red, maximum). The asterisk denotes a statistically significant difference (p < 0.05) between each attribute and the fermentation schemes.
Figure 4. Heatmap analysis representation corresponding to Sauvignon Blanc wine sensory evaluation fermented under different inoculation schemes. The relative content of each sensory attribute is illustrated through a color scale (from dark blue, minimum, to dark red, maximum). The asterisk denotes a statistically significant difference (p < 0.05) between each attribute and the fermentation schemes.
Applsci 13 03393 g004
Table
Table 1. Inoculation strategies employed in the present study.
Table 1. Inoculation strategies employed in the present study.
CodeInoculum
SP2S. pastorianus strain SP2
BCS103S. bayanus strain BCS103
70/30S. pastorianus SP2—S. bayanus strain BCS103 70%-30% (w/w)
90/10S. pastorianus SP2—S. bayanus strain BCS103 90%-10% (w/w)
95/5S. pastorianus SP2—S. bayanus strain BCS103 95%-5% (w/w)
97/3S. pastorianus SP2—S. bayanus strain BCS103 97%-3% (w/w)
99/1S. pastorianus SP2—S. bayanus strain BCS103 99%-1% (w/w)
spontaneousno inoculum
Table
Table 2. Concentration of glucose and fructose (in g/L) during alcoholic fermentation of Sauvignon Blanc must.
Table 2. Concentration of glucose and fructose (in g/L) during alcoholic fermentation of Sauvignon Blanc must.
Time (Days)SpontaneousInoculum
BCS103SP270/3090/1095/597/399/1
0210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a
1210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a210.0 (0.0) a
2204.0 (0.8) g182.0 (1.5) c202.0 (0.9) g169.0 (1.3) a173.0 (1.5) b194.0 (1.4) e188.0 (1.3) d199.0 (1.0) f
3189.0 (1.2) g150.0 (2.9) c186.0 (1.3) g125.0 (2.7) a134.0 (2.0) b173.5 (2.1) e164.0 (1.7) d179.0 (1.2) f
4135.0 (2.7) c120.0 (5.4) b145.0 (6.6) d112.0 (6.3) a116.0 (2.6) ab137.0 (2.8) c138.0 (2.7) c152.0 (2.9) e
575.0 (7.4) a78.0 (2.3) a110.0 (1.0) c79.0 (3.8) a83.0 (6.9) a97.0 (7.1) b108.0 (5.3) c116.0 (6.3) c
645.0 (2.2) c37.6 (1.4) a69.0 (1.2) f40.4 (2.1) b48.0 (1.1) d61.8 (0.3) e73.0 (1.6) g78.0 (1.3) h
727.3 (1.4) c18.5 (0.8) a38.4 (0.2) e19.3 (0.2) a24.1 (1.2) b35.1 (0.9) d45.1 (0.8) f47.3 (1.4) g
815.5 (0.3) d7.6 (0.1) b29.4 (0.1) f6.7 (0.1) a10.5 (0.1) c21.1 (0.1) e31.8 (0.1) g37.1 (0.2) h
102.2 (0.3) c<0.18.4 (0.0) e<0.10.1 (0.0) a1.5 (0.2) b6.9 (0.1) d12.9 (0.1) f
120.2 (0.0) a<0.11.6 (0.0) d<0.1<0.10.4 (0.1) b1.3 (0.0) c2.1 (0.1) e
130.1 (0.0) a<0.10.3<0.1<0.1<0.1<0.10.4 (0.0) b
The average values are presented. Standard deviation is given in parenthesis. Different letters in each row designate statistically significant differences (p < 0.05).
Table
Table 3. Standard enological parameters at the beginning and the end of alcoholic fermentation of Sauvignon Blanc must.
Table 3. Standard enological parameters at the beginning and the end of alcoholic fermentation of Sauvignon Blanc must.
InoculumpHTotal SO2 (mg/L)Total Acidity
(g Tartaric acid/L)		Ethanol (% vol)
uninoculated must3.23 (0.02) a24 (1.8) bc6.18 (0.26) a-
BCS1033.28 (0.05) ab18 (2.5) a5.89 (0.32) a12.7 (0.01) bc
SP23.39 (0.02) d39 (2.3) f5.96 (0.33) a12.6 (0.00) ab
70/303.30 (0.06) bc21 (1.7) ab5.78 (0.41) a12.8 (0.02) c
90/103.30 (0.05) bc26 (2.4) cd5.89 (0.29) a12.6 (0.02) ab
95/53.33 (0.03) bcd28 (1.9) de5.96 (0.35) a12.6 (0.03) ab
97/33.34 (0.04) bcd31 (2.1) e6.00 (0.51) a12.6 (0.00) ab
99/13.36 (0.02) cd40 (2.6) f6.04 (0.21) a12.6 (0.01) ab
spontaneous3.35 (0.04) cd18 (3.2) a6.04 (0.57) a12.5 (0.03) a
The average values are presented. Standard deviation is given in parentheses. Different letters in each column designate statistically significant differences (p < 0.05).
Table
Table 4. Volatile and non-volatile compounds at the end of alcoholic fermentation of Sauvignon Blanc must.
Table 4. Volatile and non-volatile compounds at the end of alcoholic fermentation of Sauvignon Blanc must.
MonoculturesCo-Cultures of SP2 with BCS103 at RatioSpontaneous Fermentation
BCS103SP270/3090/1095/597/399/1
Polyols (g/L)
glycerol7.03 (0.34) b7.25 (0.32) bc7.36 (0.27) bc7.42 (0.23) bc7.85 (0.42) c7.71 (0.33) c7.67 (0.18) c5.82 (0.58) a
Organic acids (g/L)
Malic acid 11.21 (0.02) c1.20 (0.02) c1.21 (0.04) c1.10 (0.04) a1.12 (0.03) ab1.16 (0.03) bc1.19 (0.02) c1.30 (0.05) d
Acetic acid0.01 (0.00) a0.02 (0.00) a0.03 (0.01) b0.07 (0.01) c0.1 (0.01) d0.18 (0.00) e0.03 (0.00) b0.35 (0.02) f
Ethyl esters (mg/L)
Ethyl decanoate557 (90.6) ab401 (100.3) a540 (104.7) ab543 (95.8) ab715 (106.3) bc802 (110.6) c799 (107.3) c639 (102.8) bc
Ethyl octanoate1533 (67.3) a2077 (89.4) e1598 (47.1) a1717 (26.9) b1827 (54.9) bc1934 (85.2) cd1978 (56.9) de1867 (63.1) cd
Ethyl hexanoate1147 (27.6) a1617 (41.5) e1201 (32.5) ab1239 (37.1) bc1304 (36.8) c1419 (44.2) d1457 (39.3) d1266 (54.2) bc
Ethyl butyrate529 (6.3) d585 (7.2) f501 (9.6) c445 (8.1) a461 (8.4) b501 (9.7) c547 (6.3) e504 (8.2) c
Ethyl hydroxy propanoate9561 (215.3) a15024 (309.4) e10034 (388.2) a12162 (406.2) b13159 (370.9) c13875 (502.4) d14345 (429.7) d12358 (417.3) b
ethyl 3-hydroxy butanoate323 (13.5) a397 (21.6) c345 (33.2) ab368 (30.5) bc334 (26.9) ab357 (24.7) abc354 (23.1) abc360 (25.3) abc
ethyl 2- methyl butyrate11.3 (0.62) b13.2 (1.23) bc13.4 (1.87) c16.1 (0.66) de17.2 (1.75) e17.0 (1.24) e14.8 (1.06) cd5.2 (0.44) a
ethyl isobutyrate105 (3.1) b109 (2.8) b109 (3.6) b139 (4.5) d129 (4.9) c141 (5.2) d123 (4.4) c72 (3.2) a
ethyl 2-hydroxyisobutyrate38 (2.2) d24 (1.5) a35 (3.5) cd34 (2.4) cd28 (2.8) ab25 (1.7) a24 (3.2) a31 (2.4) bc
Acetate esters (mg/L)
hexyl acetate302 (11.7) e198 (12.3) cd220 (21.5) d179 (20.6) c150 (13.6) b120 (14.2) a110 (11.5) a338 (23.3) f
Isoamyl acetate6026 (956.2) d2959 (885.3) ab5123 (1023.1) cd4257 (982.3) bc3036 (907.6) ab2154 (709.4) a1988 (752.2) a3481 (937.3) ab
2-phenylethyl acetate385 (25.3) e264 (24.2) c358 (31.6) de331 (30.8) d248 (28.1) bc209 (20.5) ab199 (24.3) a273 (21.8) c
Higher alcohols (mg/L)
2- phenyl-ethanol18.61 (0.36) bc18.23 (0.24) b18.98 (0.51) c19.81 (0.36) d20.5 (0.49) e19.89 (0.44) de18.78 (0.37) bc15.72 (0.28) a
propanol27.9 (1.84) b28.2 (1.62) b27.0 (2.67) b28.1 (2.35) b27.8 (2.53) b31.0 (3.17) b28.0 (2.31) b22.0 (1.69) a
isobutanol14.1 (4.36) a30.0 (5.21) c16.7 (3.78) ab22.5 (6.07) bc27.1 (5.05) c28.0 (5.12) c26.0 (4.68) c21.7 (3.69) abc
butanol0.73 (0.036) a1.24 (0.027) e0.81 (0.085) bc1.08 (0.124) d1.14 (0.058) de0.91 (0.069) c1.18 (0.074) de1.08 (0.044) d
isoamyl alcohol166.4 (0.69) b170.2 (0.75) cd170.0 (0.87) cd171.5 (0.90) d168.6 (0.96) c173.3 (1.08) e168.6 (1.41) c141.4 (0.92) a
Thiols (ng/L)
3-sulfanylhexan-1-ol206 (7.3) ab295 (6.5) d295 (9.2) d281 (8.2) d224 (8.5) c211 (8.7) bc195 (9.2) a192 (7.2) a
3-sulfanylhexan-1-ol acetateND5 (0.6)NDNDNDNDNDND
4-methyl-4-sulfanylpentan-2-one24.2 (1.14) e6.7 (0.87) b17.1 (1.21) d12.2 (1.69) c5.5 (0.71) ab4.8 (0.63) a4.6 (0.47) a4.7 (0.39) a
1 Initial malic acid concentration was 1.89 g/L. The average values are presented. Standard deviation is given in parentheses. Different letters in each column designate statistically significant differences (p < 0.05).
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Dimopoulou, M.; Troianou, V.; Toumpeki, C.; Lola, D.; Goulioti, E.; Tzamourani, A.; Dorignac, E.; Paramithiotis, S.; Kotseridis, Y. Influence of Saccharomyces pastorianus and Saccharomyces bayanus Inoculation Ratio to Oenological Characteristics of Sauvignon Blanc Wine. Appl. Sci. 2023, 13, 3393. https://doi.org/10.3390/app13063393

AMA Style

Dimopoulou M, Troianou V, Toumpeki C, Lola D, Goulioti E, Tzamourani A, Dorignac E, Paramithiotis S, Kotseridis Y. Influence of Saccharomyces pastorianus and Saccharomyces bayanus Inoculation Ratio to Oenological Characteristics of Sauvignon Blanc Wine. Applied Sciences. 2023; 13(6):3393. https://doi.org/10.3390/app13063393

Chicago/Turabian Style

Dimopoulou, Maria, Vicky Troianou, Chrisavgi Toumpeki, Despina Lola, Elli Goulioti, Aikaterini Tzamourani, Etienne Dorignac, Spiros Paramithiotis, and Yorgos Kotseridis. 2023. "Influence of Saccharomyces pastorianus and Saccharomyces bayanus Inoculation Ratio to Oenological Characteristics of Sauvignon Blanc Wine" Applied Sciences 13, no. 6: 3393. https://doi.org/10.3390/app13063393

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