Introduction

Alcoholic fermentation (AF) is one of the most important processes during winemaking and the management of this is key to obtaining a good quality product.

Mannoproteins (MPs) are macromolecules released by yeasts during AF and during the autolysis of cells in wine ageing on lees (Dupin et al., 2000a; Chalier et al., 2007). The release of MPs depends on yeast strain (González-Ramos et al., 2008), must turbidity (Boivin et al., 1998), temperature (Llaubères et al., 1987), nutritional conditions (sugar, nitrogen) (Ribéreau-Gayon et al., 2006) and the chemical composition of the wine (pH). These molecules are glycoproteins with a high sugar content, mainly mannose (Quirós et al., 2010), and are, together with fibrous β-1,3-glucan, one of the major components of the yeast cell wall, representing an important part of total polysaccharides in wine (Vidal et al., 2003).

In recent years, MPs have become one of the most interesting yeast molecules for the improvement of wine quality, due to their oenological properties such as the enhancement of mouthfeel and the improvement of wine protein and tartaric stabilities (Escot et al., 2001; Moine-Ledoux and Dubourdieu, 2002; Caridi, 2006; González-Ramos et al., 2008; Comuzzo et al., 2011). In 2005, the European Community authorised the addition to wine of MPs obtained from the hydrolysis of yeast cell walls, for improving tartaric and protein stabilisation (EU Regulation 2165/2005).

One of the first oenological properties described for yeast MPs is the partial protection against protein precipitation in white wines. Ledoux et al. (1992) showed that a Sauvignon blanc wine aged on yeast lees has lower haze potential and lower bentonite requirements for stability than wine aged without lees. The compound responsible for improving protein stability in white wine aged on its lees has been identified as a 31.8 kDa mannoprotein (MP32), which was a yeast invertase fragment released from yeast cell walls (Moine-Ledoux and Dubourdieu, 1999).

Waters et al. (1993) isolated from Muscat of Alexandria wine a high molecular weight mannoprotein that is able to prevent visible wine protein haze formation. This molecule, called haze protective factor (HPF), was characterised and a putative structural gene was identified in the Saccharomyces cerevisiae genome (Dupin et al., 2000b).

Another property attributed to MPs is the protection against tartaric instability. MPs inhibit the crystallisation of tartrate salts by lowering the crystallisation temperature (Gerbaud et al., 1996; Moine-Ledoux and Dubourdieu, 2002), preventing thus the occurrence of precipitates in wine. Furthermore, MPs improve the foam of sparkling wines. The hydrophobicity, high glycosylation and high molecular mass of MPs allow them to surround, and thus stabilise, the gas bubbles of the foam. It has been demonstrated that both the production and the stabilisation of foam also depend on other proteins; however, the main contributors to the foam formation are MPs (Coelho et al., 2011; Vincenzi et al., 2014).

Some authors have proposed recombinant S. cerevisiae wine yeasts engineered to overproduce MPs (Gonzalez-Ramos and Gonzalez, 2006; Brown et al., 2007; Gonzales-Ramos et al., 2008). To bypass various countries’ regulations on the use of GMOs in food, Quirós et al. (2010) developed a non-recombinant method to select wine yeasts overproducing MPs, consisting in a random mutagenesis using UV light as a physical agent, followed by a direct selection on YPD (yeast extract-peptone-dextrose medium) plates containing killer toxin from Williopsis saturnus.

To avoid the use of these microorganisms, likely to be viewed as releasing components not normally present in wine, Domizio et al. (2014) proposed the use of non-Saccharomyces wine yeasts, found in grape and winemaking environments, as novel sources of MPs in wine. Giovani et al. (2012) firstly demonstrated that non-Saccharomyces yeasts can release polysaccharides from their cell wall during AF, as with S. cerevisiae. They found out that the amount released depends on yeast species and cell vitality. Non-Saccharomyces yeasts, once defined as a spoilage microorganism, predominate in the early stages of must AF and recently their role has been reconsidered as some were found to enhance the analytical composition and aroma profile of the wine (Jolly et al., 2014; Ciani and Comitini, 2015; Padilla et al., 2016; Vilela, 2019). Several studies proposed the use of controlled mixed fermentations combining the inoculation of non-Saccharomyces yeasts contemporarily or before Saccharomyces cerevisiae to improve wine complexity and quality (Ciani and Comitini, 2011; Jolly et al., 2003). Mixed fermentations with Metschnikowia pulcherrima and S. cerevisiae significantly decreased volatile acidity and total acidity of the final wines and showed a positive correlation with medium-chain fatty acids, 2-phenyl ethanol and isoamyl acetate production (Comitini et al. 2011). Mixed and sequential fermentations (first the non-Saccharomyces yeast then S. cerevisiae) carried out with Lachancea thermotolerans and an S. cerevisiae starter showed a reduction in the volatile acidity and an increase in total acidity, glycerol, 2-phenyl ethanol, ethyl lactate, diacetyl and polysaccharides content (Comitini et al., 2011; Gobbi et al., 2013; Del Fresno et al., 2017). The use of Torulaspora delbrueckii in mixed fermentations with S. cerevisiae resulted in an enhancement of the 2-phenyl ethanol and polysaccharides content and a significant reduction in the volatile acidity (Comitini et al. 2011). Other authors found that sequential fermentations with T. delbrueckii and S. cerevisiae increase the complexity of wine. In fact, the presence of T. delbrueckii increased the concentration of some positive esters, such as ethyl lactate or 2-phenylethyl acetate, and of ethyl acetate, diacetyl and isoamyl acetate in wines, keeping volatile acidity and acetaldehyde at suitable levels (Loira et al., 2014; Taillandier et al., 2014; González-Royo et al., 2015; Del Fresno et al., 2017). Moreover, the effect of non-Saccharomyces yeasts on wine foam has been studied, showing that sequential fermentations with S. cerevisiae may be a useful tool to improve wine characteristics and quality. Sequential inoculation with T. delbrueckii and S. cerevisiae improved wine foamability and foam persistence, while sequential inoculation with M. pulcherrima and S. cerevisiae increased both the foam persistence and the smoky and flowery notes in the wine aromatic profile (González-Royo et al., 2015; Medina-Trujillo et al., 2017).

Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast commonly found in oenological environments (Bovo et al., 2011). Recently, it has shown to be of oenological interest in mixed AF with S. cerevisiae, improving the content of compounds related with wine quality. Many studies indicate that the use of S. bacillaris, together with S. cerevisiae, enhance the glycerol content of wines and cider, with moderate volatile acidity production, and, due to the low ethanol yield, reduce ethanol content (Rantsiou et al., 2012; Bely et al., 2013; Wang et al., 2014; Lemos Junior et al., 2016; Lemos Junior et al., 2017a; Lemos Junior et al., 2017b; Nadai et al., 2018). Moreover, Lemos Junior et al. (2019) proposed the potential use of S. bacillaris as a fermentation starter for the production of low-alcohol beverages obtained from unripe grapes.

In this study, seven strains of the non-conventional yeast S. bacillaris were investigated for their ability to release MPs during AF in synthetic must, both in single-strain and in sequential AF together with S. cerevisiae. These molecules were quantified, characterised in terms of carbohydrate composition, and tested to assess their ability to reduce protein and tartrate instabilities and to affect the foaming properties of wine.

Materials and methods

Results and discussion

1. MPs release during fermentation

Fermentation performances of seven strains of S. bacillaris, both in single-strain and in sequential AF together with S. cerevisiae EC1118, were evaluated in synthetic must. All the fermentations trials were stopped after 624 h, when the AF of S. cerevisiae EC1118 was completed. Fermentation vigour, in terms of CO₂ production after 48 h of incubation, CO₂ production after 312 h and when AF was stopped (624 h), together with sugars consumption and secondary metabolite production, were considered to assess AF performances (Table A.1 and Table A.2). S. bacillaris single-strain fermentation evidenced a high glycerol production and a fermentation rate slower than that of S. cerevisiae EC1118. When sequential fermentations were performed in synthetic must the S. bacillaris strains significantly increased glycerol content and reduced ethanol concentration, while no significant differences were found in acetic acid concentrations that were comparable to the S. cerevisae EC1118 tested strain.

The AF was conducted in MS300 synthetic must (i.e. without protein or polysaccharide), so every macromolecule present at the end of the AF was exclusively produced and released by yeast cells (Figure 1). S. cerevisiae strain produced a significantly (p<0.001) higher content of macromolecules (119 mg/L) than S. bacillaris single-strain AFs (from 40.51 to 57.56 mg/L).

These results are different from those obtained by Domizio et al. (2014) who found that S. bacillaris #22 released a level of total polysaccharides similar to S. cerevisiae EC1118 (around 200 mg/L). However, this effect could be explained by the different AF conditions. In fact, Domizio et al. (2014) performed AFs in a “minimal must medium” at 28°C under continuous agitation at 150 rpm, while in the present study the fermentation conditions are mimicking a must in static fermentation.

Figure 1. Mannoproteins content in S. cerevisiae (EC1118) and S. bacillaris single-strain fermentation (mean of all strains) and sequential fermentation.

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The sequential AFs released much higher quantities of macromolecules (151–270 mg/L) than the S. cerevisiae single-strain AF (119 mg/L). This higher macromolecules production in sequential AFs cannot be ascribed solely to the presence of two microorganisms in the same medium, as, in some cases (in particular FRI751 and FRI779), the total macromolecules content is higher than the sum of the macromolecules released by the two microorganisms individually cultured. This behaviour clearly indicates that the interaction existing between the two microorganisms during AF leads to an increase in the macromolecules release into the culture medium. The interaction between different yeasts in multi-starter wine AF has been reviewed by Ciani and Comitini (2015), and has been shown to have an effect on both metabolite production (glycerol, ethanol, esters, etc.) and microbial growth. Regarding cell-wall polysaccharides, even Comitini et al. (2011) found a higher release of polysaccharides by S. bacillaris in sequential AF together with S. cerevisae than S. cerevisiae single-strain AF in natural grape juice. This could be explained by a modulation of S. bacillaris death due to the direct interaction with S. cerevisiae cells, as recently suggested by Englezos et al. (2019).

2. Polysaccharides characterisation

The different macromolecules obtained from the filtered media were suspended in water at 10 mg/mL and then used for further characterisation.

The analysis of sugar composition confirmed that these polysaccharides released in the media are of cell-wall origin, and therefore essentially MPs. As a matter of fact, only mannose and glucose were found after acidic hydrolysis, with a predominance of mannose, which accounts for about 90% of the total sugar in the polysaccharides isolated from S. cerevisiae (Figure 2). This result is in agreement with data from the literature reporting similar values of mannose content (Escot et al., 2001; Vidal et al., 2003; Domizio et al., 2014). Conversely, the mean content of mannose in MPs isolated from single-strain AFs of S. bacillaris is 82.23±1.43%, according to the data from Domizio et al. (2014). These data confirm the existence of a chemical difference between the MPs released by the two microorganisms, which can influence their technological properties, such as the ability to decrease the protein instability in white wines (Ribeiro et al., 2014). Finally, in sequential AFs the mannose percentage of MPs was intermediate (with the exception of sequential AF of FRI728 strain), but still significantly lower (from 85.28 to 87.43%) when compared to single-strain AF of EC1118 (Figure 2).

Figure 2. The composition with regards to sugars represented by a coefficient (mannose/glucose) of the polysaccharides released by each yeast strain at the end of alcoholic fermentation.

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3. Tartaric stabilisation

First, the MPs were added to a very unstable wine at a final concentration of 200 mg/L to test for their ability to decrease the tartaric instability.

The tendency of tartaric acid to precipitate was determined by measuring the conductivity drop after addition of potassium hydrogen tartrate (crystallisation nuclei) at 0°C. The effect of MPs on this parameter was measured by comparing the conductivity drop of treated samples with that of untreated wine (Figure 3). On average, the MPs produced by S. bacillaris showed low inhibition capacity (from 6.00 to 31.00 µS) when compared with those of EC1118 alone (41 µS) (p<0.001). However, the MPs recovered after the sequential AF showed, in some cases (FRI719, FRI751 and FRI779), a significant higher activity compared to single-strain AF of EC1118, indicating a better capacity to inhibit the salt crystallisation. This behaviour confirms an interaction between the two yeast species, as not only the quantity, but also the intrinsic characteristics of MPs were modified when recovered after a sequential AF. We can hypothesise that these MPs modifications could depend on the activity of hydrolytic enzymes released by one or both species able to specifically act on the MPs of the co-fermenting species.

Figure 3. Tartaric stabilisation in S. cerevisiae (EC1118) and S. bacillaris single-strain fermentation (mean of all strains) and sequential fermentation.

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4. Protein stabilisation

The MPs were also tested for their ability to affect the protein instability, by adding them to an unstable wine at a final concentration of 200 mg/L. Then the wines were heated at 80°C in order to induce the aggregation and precipitation of grape unstable proteins (Pocock and Rankine, 1973).

The induced turbidity was measured by nephelometry and the difference between the initial turbidity and that obtained after heating was noted. The wine used for the experiment was very unstable, giving a value of 225 NTU, and all the results were expressed in comparison to this value. In all cases the addition of MPs caused a reduction of haze risk (Figure 4), confirming the ability of these molecules to decrease the protein precipitation (Waters et al., 1993; Waters et al., 1994).

Figure 4. Protein stabilisation in S. cerevisiae (EC1118) and S. bacillaris single-strain fermentation (mean of all strains) and sequential fermentation. The difference in ΔNTU from the untreated wine (225 NTU) was reported.

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Considering the MPs released by the S. bacillaris strains when cultured alone, the protective activity (obtained considering the mean value of all the single-strain S. bacillaris) was always significantly higher than the MPs produced by the EC1118 strain (p<0.001). This is in disagreement with the data reported by Ribeiro et al. (2014), which showed a higher stabilisation effect for MPs with higher mannose to glucose ratio. These data suggest that mannose content is not the only factor affecting the stabilising effect of MPs, and that other factors such as the nature of glycosyl linkages among sugars or the aminoacidic composition of the peptidic fraction could have an important effect on the chemical-physical behaviour of these macromolecules. When the same S. bacillaris strains were cultured in sequential inoculum with EC1118, the protein stabilisation effect of the obtained MPs was reduced, but in general, the stabilising effect is comparable to that of the commercial strain alone, and only in one case (FRI754) significantly higher (Figure 4). This strain requires further study, especially in real grape juice, as its use in combination with S. cerevisiae should decrease the bentonite demand for protein stabilisation, with wine qualitative improvement, and this could be interesting for winemakers. In addition, the particular stabilising ability of S. bacillaris MPs could also be of great interest for the wine products industry, as the most interesting yeast strains could be used for the manufacturing of new oenological products.

5. Foamability

The effect on wine foamability of the collected MPs, isolated from Saccharomyces and Starmerella strains in single or in sequential AF, was studied by adding them to a Prosecco base wine.

When added to the wine all at the same concentrations (50 mg/L), the MPs obtained from Saccharomyces single-AF and sequential AFs increased the foamability (Figure 5), causing an average increase of the maximum height (HM) of about 40%. This is in agreement with the data reported by several studies (Martínez-Rodríguez et al., 2001; Núñez et al., 2006; Vincenzi et al., 2014; González-Royo et al., 2015) which showed that yeast MPs are important for the wine foamability. The foam volumes were significantly higher than that of the original wine (CTRL), but no significant differences were found among the MPs produced by the different strains when cultured in sequential AF, except for strain FRI729. Apparently, the modifications induced by S. bacillaris on MPs produced and released in sequential AF did not influence their effect on wine foamability, at least at the tested concentration (50 mg/L). Therefore, it could be hypothesised that the MPs released by S. cerevisiae in the presence or absence of Starmerella have a similar effect in terms of wine foamability, and that the effect of the different strains of Starmerella is reflected in the quantity of released MPs. However, different concentrations of MPs, such as 100 mg/L, 200 mg/L or more, must be tested to verify the effect on wine foamability.

Figure 5. Maximum foam height (HM) obtained after the addition of 50 mg /L of mannoproteins isolated from single-strain fermentations of Saccharomyces and from sequential fermentations with the seven strains of Starmerella bacillaris. Samples with different letters are statistically different (p=0.05).

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However, among the MPs produced in single-strain AF by Starmerella bacillaris, three (FRI729, FRI751 and FRI779) showed a much higher foaming capacity compared to those produced by the same strains in sequential AFs (Figure 6). This is clearly linked to a different composition of the MPs produced by Starmerella in single-strain AF in respect to those produced in sequential AF with S. cerevisiae, as reported above.

Figure 6. Maximum foam height (HM) obtained after the addition of 50 mg/L of mannoproteins isolated from different strains of Starmerella bacillaris in single-strain (■) and sequential (■) fermentation. For each strain, samples with different letters are statistically different (p<0.05).

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A PCA analysis on all the collected data (MPs release, FH (HM), tartaric and protein stabilisation) for single-strain and sequential AFs (Figure 7) showed that two main factors (F1 and F2) account for 97.40% of total variance, with F1 and F2 accounting for 76.31% and 21.09%, respectively. The analysis confirmed the high level of differences between MPs of the single-strain AF of S. bacillaris, grouped on the right side of the graphic, and that of EC1118 (both in single-strain and in sequential AF), grouped on the left side.

Figure 7. Principal component analysis (PCA) biplot showing fermentation performances (variables: mannoprotein production, foam height (HM), tartaric and protein stabilisation) of single-strain fermentations of S. bacillaris strains (observations from FRI719 to FRI7100) and strain EC1118 (observation EC1118) and of sequential fermentation of S. bacillaris strains together with EC1118 (observations from FRI719S to FRI7100S) in MS300. Only variables that showed significant correlations are reported.

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It is interesting to note that the ability to decrease the tartaric instability in general does not coexist with the ability to improve the protein stability, in fact the two vectors are opposite, with a correlation coefficient of -0.887. This behaviour confirms that the tartaric and protein stabilisation are based on different mechanisms and, consequently, differently affected by the MPs structure.

The foaming properties of these MPs (HM), on the contrary, are not correlated to the other two parameters.

Conclusions

Even though they are not able to complete the AF, S. bacillaris strains can be used in sequential AF with S. cerevisae to improve some organoleptic properties. In addition, the data here reported demonstrate that the interaction between the two microorganisms affect differently the MPs production in terms of both quantity and quality. All the tested strains in sequential AF increased the total MPs production, but the strains affected in different ways the MPs properties in terms of tartaric and protein stabilities. This is interesting from the technological point of view, as it suggests a possibility to use different selected strains to obtain specific characteristics. It will be interesting to check the results in a real grape juice, as there may be differences in respect to synthetic must. In fact, a direct relationship between the degree of grape must clarification and the quantity of yeast macromolecules recovered in the wine has been described (Guilloux-Benatier et al., 1995). Furthermore, Boivin et al. (1998) reported that yeast cell-wall porosity increased in clarified must, but that macromolecule production decreased. The most interesting aspect is that the improvement in stability can be achieved during the AF, reducing the extent of stabilising treatments at the end of winemaking process. This is converted in a greater sustainability as it leads to a reduction of costs and environmental pollution and in an improvement of organoleptic properties (i.e. a reduction of aroma loss associated with bentonite fining).

In addition, the particular ability of MPs released in single-strain AF by some S. bacillaris strains (i.e. a high effect on protein stabilisation or on foam volume) could be interesting as a source for the manufacturing of new oenological products.

Further studies in natural grape juice, aimed to verify the S. bacillaris MPs properties discovered in synthetic must, are needed, because in the latter medium yeasts could over-release mannoproteins in comparison with fermentations in grape must.

Acknowledgements

The authors wish to thank CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

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