Voce S.*, Iacumin L.*, Comuzzo P.*

*Università degli Studi di Udine, Dipartimento di Scienze Agroalimentari, Ambientali e Animali, via Sondrio 2/A, 33100, Udine 

 
Corresponding Author: sabrina.voce@uniud.it

Article extracted from the presentation held during Enoforum Web Scientists (March 13, 2023).

Abstract

Non-Saccharomyces yeasts (NSY) represent a consistent part of natural microflora of grape must. Recently, there was an increasing interest in their use for winemaking, since they show a good ability to release higher amounts of polysaccharides compared to Saccharomyces spp. during alcoholic fermentation; furthermore, a non-negligible contribution in increasing glutathione content in sequential or mixed fermentations was also reported, as well as an improvement of wine quality in terms of color stability and aroma profile. However, few studies have been carried out till now about their contribution during wine aging on lees. 

For this reason, twenty yeast strains were isolated from must and pomace of red grapes cv. Merlot, supplied by a local producer (Friuli Venezia Giulia, North-East Italy). The identification was carried out by colony morphology (on differential WL Nutrient Agar plate), cell morphology (100x optical microscopy) and by genetic characterization. The ability of the isolated strains to produce compounds of enological interest was assessed after growth in standardized conditions (48h at 30°C under anaerobic conditions) and after enzyme-assisted lysis, performed by addition of β-glucanase (5% w/v) and incubation at 45°C for 24h. Biomass production (by weighting), cell viability (by serial dilution and counting of viable cells after 48h at 30°C), the release of polysaccharides (by ethanol precipitation and SE-HPLC analysis), amino acids, thiol compounds (determined spectrophotometrically) and total glutathione (by enzymatic assay) were analyzed. 

Cell viability (about 107 CFU/mL) was quite similar among the strains tested, whereas for all the other parameters evaluated, differences were observed not only among the genera, but also intra-genus. Basically, strains that produced higher content of soluble compounds, in particular polysaccharides, thiol compounds and total glutathione during the growth phase, also showed the highest release of such molecules after lysis treatment. Among these, interesting results were generally obtained for the strains belonging to Hanseniaspora spp., with the highest content of polysaccharides and antioxidant compounds released both after growth and lysis, together with a biomass production comparable to that obtained by commercial Saccharomyces and Torulaspora strains used as reference. 

These results might suggest that a correct management of wild microflora might impact on wine composition and quality not only during fermentation but also during aging on lees. The most promising NSY might be also interesting to produce fermentation starters or inactive dry yeast preparations, e.g., autolysates, naturally rich of compounds of enological interest.

INTRODUCTION 

Non-Saccharomyces yeasts (NSY) represent a non-negligible part of natural microflora of grapes and must; [1]; the increasing interest for winemaking application is mainly related to their ability to enhance the varietal characteristics, improve color stability and aroma complexity of wine, especially when used in sequential or mixed fermentations with S. cerevisiae [2,3]. 

An interesting feature that characterizes NSY is the higher release of polysaccharides during alcoholic fermentation compared to S. cerevisiae [4,5]. The polysaccharidic fraction mainly consists on mannoproteins with high mannose/glucose ratio [5] or galactomannans [6,7]; a considerable release of such compounds is also observed during aging on lees, with differences that are strain-dependent, probably due to the different structure and composition of cell walls or different autolytic susceptibility [6,8]. The release of polysaccharides and mannoproteins in shorter time and in higher amount compared to S. cerevisiae might allow to reduce the conventional period of aging. 

Concerning the role of NSY towards protection against wine oxidation, several authors have observed a positive effect on stability of phenolic fractions during red winemaking, mainly related to the high production of pyruvic acid or acetaldehyde, with subsequent formation of more stable pigments (i.e., vitisins) [9,10]. Few evidence is till now reported about the antioxidant activity during white winemaking and, above all, the potential release of antioxidant molecules (i.e., glutathione) during aging on lees. Some authors have recently evaluated the production and release of glutathione by some strains of NSY, during the growth phase [11], after pure or sequential fermentations [12], as well as during the production process of active dry yeasts to be used as fermentation starters [13]. 

Regarding nitrogen compounds, it is well known their influence on yeast metabolism and growth, with different utilization and consumption that are strain dependent [14]; during the growth phase, such compounds subsequentially influence biomass production, fermentation rate and production of volatile compounds. On the other hand, during aging on lees, the release of nitrogen compounds may lead to undesirable, spontaneous fermentations, and formation of biogenic amines.  

The aim of the present work was to assess the ability of wild NYS isolated from red grape pomace and must, to produce polysaccharides and antioxidant compounds after growth and induced lysis, aimed at evaluating i) their potential use as fermentation co-starters; ii) their contribution during aging on lees; iii) their potential use for yeast derivatives production. The aptitude of the strains to be used for winemaking purposes was evaluated in terms of cell viability and biomass production, and release of polysaccharides, antioxidant compounds and amino acids after growth and lysis induced by β-glucanase addition, in comparison with two commercial yeasts – S. cerevisiae e T. delbrueckii. 

MATERIALS AND METHODS 

Growth conditions and lysis treatment
For each isolated strain, single pure colony was transferred in sterile tubes containing 10 mL of YPD broth, then incubated at 30°C for 48 hours. At the end of growth phase, yeast suspensions were centrifuged; the supernatant was used for analytical evaluations concerning the growth phase whereas the biomass was washed twice and resuspended in sterile water; commercial preparation with β-glucanase activity 5 % (w/v) was then added and suspensions were incubated at 45°C for 24 hours. As concern the growth phase, the culture medium (YPD broth) was also subjected to the same analyses because of the presence of yeast extracts in its formulation, and the results were used for correcting data referred to the growth. 

Cell viability and biomass production
Cell viability was evaluated by plating serial dilutions on Malt Extract agar plate, and microbial counts were carried out after incubation at 30°C for 48 hours in aerobic conditions; the results were expressed as logarithm of colony forming units (log CFU/mL).
The production of biomass was evaluated by filtering under vacuum 10 mL of yeast suspensions on pre-weighed cellulose membranes (pore size 0.45 µm) and the results were expressed as g/L. 

Soluble polysaccharides
The amount of polysaccharides released after growth and induced lysis was evaluated by alcoholic precipitation and SE-HPLC, as reported in Voce et al. [15]. The concentrations were calculated by a calibration curve made with mannan (0-1000 mg/L) and the results were expressed as mg/109 cells. 

Thiol compounds and total glutathione
Antioxidant molecules were determined on the soluble fraction of yeast suspensions after growth and induced lysis. Thiol compounds were evaluated as reported by Gallardo-Chacon et al. [16], whereas total glutathione (both oxidized and reduced) by enzymatic assay [17]. The concentrations were determined by a calibration curve made with glutathione (0-650 mM) and the results were expressed as µmol/109 cells. 

Free amino acids
The amount of free amino acids released after growth and induced lysis was evaluated by spectrophotometric assay [18]. Concentrations were determined by a calibration curve made with isoleucine (0-10 mM) and the results were expressed as mg/109 cells. 

Statistical analysis
Data were means and standard deviations of three replicates. Homogeneity of variance, one-way ANOVA and Tukey HSD test (p < 0.05) were carried out for all the parameters evaluated; the statistical elaboration was carried out by the software Statistica for Windows version 8.

RESULTS AND DISCUSSION  

Cell viability and biomass production
Concerning cell viability, the values ranged from 6.99 log CFU/mL (observed for one strain of Hanseniaspora spp.) to 7.85 log CFU/mL (Pichia spp); even if some statistical differences emerged, they may be considered negligible from the practical point of view, with values quite similar among all the strains (about 107 cells/mL), highlighting a good growing ability in the conditions tested.
Regarding biomass, differences were observed among genera and intra-genus. The highest production of biomass was observed for Pichia spp. strain (5.34 g/L), whereas Candida spp., some strains of Hanseniaspora spp. and Starmerella spp. showed the lowest recovery (≤ 0.5 g/L). By considering the same genus, for example in the case of Hanseniaspora spp., the strain (encoded as H5) produced about 0.50 g/L, lower than produced by strains H3 or H4, with an amount of 3.41 e 3.32 g/L, respectively.
Nevertheless, some wild Saccharomyces spp. and Hanseniaspora spp. have showed a good ability to produce biomass in the conditions here tested, being potentially used as starting material for producing fermentation starters or yeast derivatives. The amount obtained in the present study in terms of cell viability and biomass production are similar to those previously reported by other authors in winemaking condition [5,19]. 

Release of polysaccharides after growth and induced lysis
The amount of polysaccharides released after growth and induced lysis by the different yeast strains was reported in table 1. 

Table 1. Polysaccharides (as mg/109 cells) released by different strains after growth (48 hours in YPD broth, 30°C) and lysis induced by β-glucanase enzyme. Data were means and standard deviations (SD) of three replicates. Different letters within the same columns marked statistical differences among the strains, according to ANOVA and Tukey HSD test (p < 0.05)S_COMM and T_COMM: commercial active dry yeast preparations. Concerning data referred to “growth”, negative (red box) and positive (blue box) values indicated a decrease (consumption) and an increase (release) respectively, in relation to the initial composition of YPD medium.

For all the strains belonging to Hanseniaspora spp. (except for H2), a considerable production of polysaccharides during the growth phase was observed, with the highest amount obtained by strain H1 (121 mg/109 cellule), thus resulting statistically different from all the others. Even the strains H3 and H4 (67 and 68 mg/109 cells) and commercial yeasts (about 40 mg/109 cells) showed a good release of such compounds during growth. The ability of Hanseniaspora spp. to release higher amount of polysaccharides compared to Saccharomyces spp. was also confirmed after the lysis treatment, with concentration ranging from 0.6 mg/109 cells (strain H5) to 3 mg/109 cells for strains H2, H3 e H4, followed by commercial Saccharomyces spp. (1.7 mg/109 cells). The remaining strains determined a release of polysaccharides after the lysis treatment lower than 1 mg/109 cells. The ability of Hanseniaspora spp. to release notable amount of polysaccharides might be due to the higher production of cell wall components (mannoproteins and glucans) during the growth phase, as well as the higher autolytic susceptibility might explain the higher release of such compounds even after β-glucanase addition. The result here obtained agreed with those reported by several authors in winemaking conditions [4,20,21] 

This interesting feature of Hanseniaspora spp. makes this strain suitable to be used as fermentation co-starter, thus potentially enriching wine of polysaccharides even during the first steps of winemaking; consequentially, the lees composition might be managed and modulated for the following aging on lees. The release of such compounds in higher amount and, possibly in shorter period, might determine an improvement of wine quality and stability in faster time. Furthermore, the potential use of this strain for producing yeast derivatives might be also useful for obtaining products naturally rich of mannoproteins and polysaccharides, to be used during aging as wine quality enhancers.

Release of antioxidant compounds after growth and lysis
The amount of antioxidant molecules (thiol compounds and total glutathione) released after growth and induced lysis by the different yeast strains was reported in table 2. 

Table 2. Thiol compounds and total glutathione (as µmol of glutathione /109 cells) released by different strains after growth (48 hours in YPD broth, 30°C) and lysis induced by β-glucanase enzyme. Data were means and standard deviations (SD) of three replicates. Different letters within the same columns marked statistical differences among the strains, according to ANOVA and Tukey HSD test (p < 0.05)S_COMM e T_COMM: commercial active dry yeast preparations. Concerning data referred to “growth”, negative (red box) and positive (blue box) values indicated a decrease (consumption) and an increase (release) respectively, in relation to the initial composition of YPD medium. 

It is interesting to note how, during the growth phase, almost all the strains belonging to Hanseniaspora spp. (except for H5) are characterized by the higher release of thiol compounds, with concentrations ranging from 1.3 to 3.3 µmol /109 cells, followed by St2 (Starmerella spp.) and commercial yeasts. On the other hand, wild strains belonging to Saccharomyces spp., Candida spp. and Starmerella spp. (St1 and St4) showed a consumption of molecules containing thiol groups (i.e., glutathione and cysteine). By considering the concentration of total glutathione determined on yeast suspensions after growth, even in this case, Hanseniaspora spp. strains – in particular strains H1, H2 and H3 – have determined the highest release of such compound (with values from 0.2 to 0.5 µmol/109 cells), together with Candida spp., whereas the remaining strains, also including commercial yeasts, showed the tendency to consume it. After the lysis treatment, Hanseniaspora spp. strains are confirmed to be the highest producers of antioxidant molecules: H1 and H2 strains determined the highest release of thiol compounds with an amount of 1.1 and 1.7 µmol /109 cells, respectively; the strain H3 showed the highest release of total glutathione, thus resulting the only statistically different from all the other strains tested. The ability of NSY to produce a non-negligible amount of glutathione during the growth phase and also under winemaking conditions was previously reported [11], with a potential increase of glutathione content into the wine up to 10 mg/L at the end of alcoholic fermentation [12]. 

This feature of Hanseniaspora spp. strains is particularly interesting, confirming the great potential of such strain for winemaking purpose. The release of antioxidant compounds during fermentation and aging on lees might be useful for protecting wine against oxidation, especially in white winemaking. The natural contribution of such molecules by using these strains as fermentation co-starter, by managing the natural microflora of grapes and must, or even by adding yeast derivatives obtained by these strains of NSY, might allowed to increase the concentration of antioxidant compounds already during the first steps of winemaking, and potentially reduce the amount of sulfur dioxide to be added. 

Release of free amino acids after growth and induced lysis
The amount of free amino acids released after growth and induced lysis by the different yeast strains was reported in table 3. 

Table 3. Free amino acids (as mg/109 cells) released by different strains after growth (48 hours in YPD broth, 30°C) and lysis induced by β-glucanase enzyme. Data were means and standard deviations (SD) of three replicates. Different letters within the same columns marked statistical differences among the strains, according to ANOVA and Tukey HSD test (p < 0.05)S_COMM e T_COMM: commercial active dry yeast preparations. Concerning data referred to “growth”, negative (red box) and positive (blue box) values indicated a decrease (consumption) and an increase (release) respectively, in relation to the initial composition of YPD medium. 

By considering the growth phase, the strains showed a different behavior towards amino nitrogen, with differences among the genera and intra-genus. Almost all the strains belonging to Saccharomyces spp. tendentially showed a consumption of nitrogen compounds, except for S2 and S3 strains that, on the contrary, determined a release of such compounds (2 and 6 mg//109 cells, respectively). Both commercial yeasts showed a consumption, as well as a poor release of amino acids was observed in Pichia spp. and Starmerella spp..The highest producers of amino acids were Candida spp. (23 mg/109 cells), followed by H3 and H4 strains (Hanseniaspora spp.), with a release of 20 and 12 mg/109 cells, respectively. For the latter genus, some differences emerged among the strains: H3 and H4 strains released amino acids during the growth phase, whereas the others showed an opposite trend, with a variable consumption of amino nitrogen, ranging from 1 mg/109 cells (H2) to – 24 mg/109 cells (H1). 

As expected, after the lysis treatment, a release of amino acids was observed for all the strains tested. Commercial yeasts and strains belonging to Pichia spp. showed the poorest content, lower than 10 mg/109 cells. The highest concentrations were detected in Candida spp., followed by H3, resulting the two strains with high production of amino acids both after growth and induced lysis.  

It is interesting to note how, some strains that during growth have determined a poor release (S2, St3, St4) or have consumed amino acids (S5, St1), have showed a good ability to release such compounds after lysis treatment, with a concentration of about 24-25 mg/109 cells. 

The content of amino acids is an important parameter to be considered in winemaking, especially in relation to the utilization: NSY high-producers of amino acids might be used as co-starter in sequential or mixed fermentations or for producing yeast derivatives to be used as fermentation enhancers (both alcoholic and malolactic fermentation); on the contrary, high concentration of amino acids might cause microbial instability during aging on lees, especially in presence of low amount of sulfur dioxide. 

CONCLUSION   

The results obtained in the present study may contribute to clarify the role of NSY in winemaking, both during alcoholic fermentation and aging on lees. Fermentation lees contain a non-negligible part of NSY; the ability of some strains to release polysaccharides and antioxidant compounds might contribute to improve wine quality, already during the first steps of winemaking process, by correctly managing natural microflora or by using them as fermentation co-starters. 

Strains belonging to Hanseniaspora spp. have showed a good ability to produce and release amino acids, and above all polysaccharides and antioxidant compounds, both after growth and lysis treatment, with a biomass production and cell viability comparable to commercial yeasts used as reference. The aptitude of this strain to produce compounds of enological interest makes it suitable for the potential application as fermentation starter and for producing yeast derivatives, naturally rich of polysaccharides and antioxidants.

REFERENCES
  1. Pallmann, C.L.; Brown, J.A.; Olineka, T.L.; Cocolin, L.; Mills, D.A.; Bisson, L.F. Use of WL Medium to Profile Native Flora Fermentations. Am. J. Enol. Vitic. 2001, 52, 198–203.
  2. Loira, I.; Vejarano, R.; Bañuelos, M.A.; Morata, A.; Tesfaye, W.; Uthurry, C.; Villa, A.; Cintora, I.; Suárez-Lepe, J.A. Influence of Sequential Fermentation with Torulaspora Delbrueckii and Saccharomyces Cerevisiae on Wine Quality. LWT – Food Sci. Technol. 2014, 59, 915–922.
  3. Mylona, A.E.; Del Fresno, J.M.; Palomero, F.; Loira, I.; Bañuelos, M.A.; Morata, A.; Calderón, F.; Benito, S.; Suárez-Lepe, J.A. Use of Schizosaccharomyces Strains for Wine Fermentation-Effect on the Wine Composition and Food Safety. Int. J. Food Microbiol. 2016, 232, 63–72.
  4. Giovani, G.; Rosi, I.; Bertuccioli, M. Quantification and Characterization of Cell Wall Polysaccharides Released by Non-Saccharomyces Yeast Strains during Alcoholic Fermentation. Int. J. Food Microbiol. 2012, 160, 113–118.
  5. Domizio, P.; Liu, Y.; Bisson, L.F.; Barile, D. Use of Non-Saccharomyces Wine Yeasts as Novel Sources of Mannoproteins in Wine. Food Microbiol. 2014, 43, 5–15.
  6. Palomero, F.; Morata, A.; Benito, S.; Calderón, F.; Suárez-Lepe, J.A. New Genera of Yeasts for Over-Lees Aging of Red Wine. Food Chem. 2009, 112, 432–441, doi:10.1016/j.foodchem.2008.05.098.
  7. Domizio, P.; Liu, Y.; Bisson, L.F.; Barile, D. Cell Wall Polysaccharides Released during the Alcoholic Fermentation by Schizosaccharomyces Pombe and S. Japonicus: Quantification and Characterization. Food Microbiol. 2017, 61, 136–149.
  8. Kulkarni, P.; Loira, I.; Morata, A.; Tesfaye, W.; González, M.C.; Suárez-Lepe, J.A. Use of Non-Saccharomyces Yeast Strains Coupled with Ultrasound Treatment as a Novel Technique to Accelerate Ageing on Lees of Red Wines and Its Repercussion in Sensorial Parameters. LWT – Food Sci. Technol. 2015, 64, 1255e1262-1262.
  9. Medina, K.; Boido, E.; Farina, L.; Dellacassa, E.; Carrau, F. Non-Saccharomyces and Saccharomyces Strains Co- Fermentation Increases Acetaldehyde Accumulation: Effect on Anthocyanin-Derived Pigments in Tannat Red Wines. Yeast 2016, 33, 339–343.
  10. Escribano-Viana, R.; Portu, J.; Garijo, P.; López, R.; Santamaría, P.; López-Alfaro, I.; Gutiérrez, A.R.; González-Arenzana, L. Effect of the Sequential Inoculation of Non- Saccharomyces / Saccharomyces on the Anthocyans and Stilbenes Composition of Tempranillo Wines. Front. Microbiol. 2019, 10, 1–10.
  11. Lemos Junior, W.J.F.; Binati, R.L.; Bersani, N.; Torriani, S. Investigating the Glutathione Accumulation by Non-Conventional Wine Yeasts in Optimized Growth Conditions and Multi-Starter Fermentations. LWT – Food Sci. Technol. 2021, 142.
  12. Binati, R.L.; Lemos Junior, W.J.F.; Torriani, S. Contribution of Non-Saccharomyces Yeasts to Increase Glutathione Concentration in Wine. Aust. J. Grape Wine Res. 2021, 27, 290–294.
  13. Torrellas, M.; Rozès, N.; Aranda, A.; Matallana, E. Basal Catalase Activity and High Glutathione Levels Influence the Performance of Non-Saccharomyces Active Dry Wine Yeasts. Food Microbiol. 2020, 92.
  14. Gobert, A.; Tourdot-Maréchal, R.; Morge, C.; Sparrow, C.; Liu, Y.; Quintanilla-Casas, B.; Vichi, S.; Alexandre, H. Non-Saccharomyces Yeasts Nitrogen Source Preferences: Impact on Sequential Fermentation and Wine Volatile Compounds Profile. Front. Microbiol. 2017, 8, 1–13.
  15. Voce, S.; Iacumin, L.; Comuzzo, P. Characterization of Non-Saccharomyces Yeast Strains Isolated from Grape Juice and Pomace: Production of Polysaccharides and Antioxidant Molecules after Growth and Autolysis. Fermentation 2022, 8, doi:10.3390/fermentation8090450.
  16. Gallardo-Chacón, J.J.; Vichi, S.; Urpí, P.; López-Tamames, E.; Buxaderas, S. Antioxidant Activity of Lees Cell Surface during Sparkling Wine Sur Lie Aging. Int. J. Food Microbiol. 2010, 143, 48–53, doi:10.1016/j.ijfoodmicro.2010.07.027.
  17. Adams, D.O.; Liyanage, C. Modification of an Enzymatic Glutathione Assay for Determination of Total Glutathione in Grapevine Tissues. Am. J. Enol. Vitic. 1991, 42, 137–140.
  18. Dukes, B.C.; Butzke, C.E. Rapid Determination of Primary Amino Acids in Grape Juice Using an O-Phthaldialdehyde/N-Acetyl-L-Cysteine Spectrophotometric Assay. Am. J. Enol. Vitic. 1998, 49, 125–134.
  19. Aplin, J.J.; White, K.P.; Edwards, C.G. Growth and Metabolism of Non-Saccharomyces Yeasts Isolated from Washington State Vineyards in Media and High Sugar Grape Musts. Food Microbiol. 2019, 77, 158–165.
  20. Romani, C.; Domizio, P.; Lencioni, L.; Gobbi, M.; Comitini, F.; Ciani, M.; Mannazzu, I. Polysaccharides and Glycerol Production by Non-Saccharomyces Wine Yeasts in Mixed Fermentation. Quad. di Sci. Vitocole ed Enol. 2010, 31, 185–189.
  21. Del Fresno, J.M.; Escott, C.; Loira, I.; Carrau, F.; Cuerda, R.; Schneider, R.; Bañuelos, M.A.; González, C.; Suárez-Lepe, J.A.; Morata, A. The Impact of Hanseniaspora Vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety. Int. J. Mol. Sci. 2021, 22, 1–14.