Antonio MORATA1*, Juan Manuel del Fresno1, Iris Loira1, Rafael Cuerda2, Carmen González1, José Antonio Suárez Lepe1
1 enotecUPM, Universidad Politécnica de Madrid, Madrid, Spain
2 Comenge Cellars, Curiel de Duero, Valladolid, Spain
*Email: antonio.morata@upm.es
Article extracted from the presentation held during Enoforum Web Conference (23-25 February 2021)
Introduction
Ageing on lees (AoL) is a powerful technique to enhance the quality of red wines because it produces an improvement of the softness, thus favouring a better integration and a reduction of the astringency. The release of polysaccharides and mannoproteins increases the body and structure producing more palatable wines. AoL is normally associated with the release of nitrogen reduced compounds as glutathione that helps to control oxidation producing a reductive ageing with positive effects on the protection of color and the preservation of varietal aroma (Figure 1). Therefore, it is an interesting technique to produce high quality wines. Additionally, AoL is complementary to the barrel ageing, producing really complex wines with the integration of the spicy wood aromas and the varietal aromas in a well-balanced body (Loira et al, 2013, del Fresno et al, 2018). However, it is a very slow process, usually taking one year or longer to reach a suitable aroma and polysaccharide content.
The AoL starts at the end of fermentation with the death of the yeasts and the autolysis processes. Autolysis is the depolymerization of the cell wall envelopes (cell wall and membrane) by the own enzymes of the yeasts (Figure 2). Autolysis is a slow process taking more than 7 months to produce a significant release of cell wall components such as polysaccharides and mannoproteins. For that the recommended ageing on lees time in bottle for most of high quality natural sparkling wines is 9-15 months (Figure 2). Also we have observed that autolysis is a yeast strain-dependent process. Some yeast strains are able to release cell wall polysaccharides and mannoproteins faster and in higher concentrations than others (Figure 2, Loira et al, 2013). The selection of specific strains with faster autolysis can facilitate the process and also reduce the lees contact time.
The AoL starts at the end of fermentation with the death of the yeasts and the autolysis processes. Autolysis is the depolymerization of the cell wall envelopes (cell wall and membrane) by the own enzymes of the yeasts (Figure 2). Autolysis is a slow process taking more than 7 months to produce a significant release of cell wall components such as polysaccharides and mannoproteins. For that the recommended ageing on lees time in bottle for most of high quality natural sparkling wines is 9-15 months (Figure 2). Also we have observed that autolysis is a yeast strain-dependent process. Some yeast strains are able to release cell wall polysaccharides and mannoproteins faster and in higher concentrations than others (Figure 2, Loira et al, 2013). The selection of specific strains with faster autolysis can facilitate the process and also reduce the lees contact time.
However, autolysis process can be strongly speeded if enough mechanical energy is applied. The use of emerging non-thermal technologies such as High-pressure technologies (HHP and specially UHPH), pulsed technologies (PEFs) or ultrasounds (USs) can facilitate cell lysis and cell wall depolymerisation (Morata et al, 2019, Morata et al, 2020). We have explored the use of USs as a cheap, continuous, and efficient technology to facilitate yeast autolysis.
USs are sonic waves ranging 20-100 kHz of frequency that are able to produce intense shaking, cavitation and high localised temperatures and pressure, the global effect is achieved in short times (minutes) and with intense mechanical consequences. Cells can be depolymerised in minutes starting soon the release of cell components and facilitating faster and more efficient AoL (del Fresno et al, 2018, del Fresno et al, 2019).
Methods
These works have been developed in several experiments along more than 5 years and using several techniques, detailed methodologies can be found in the referenced articles Loira et al, 2013, del Fresno et al, 2018 and del Fresno et al, 2019. Briefly: Polysaccharides have been measured by LC-RID, oxygen by an oximeter, color evolution spectrophotometrically, anthocyanins by LC-DAD-ESI/MS.
The sonotrode and the US device used, the measures in the barrels, and the fermentation processes to produce the pure biomases of the single strains used in the AoL trials are showed in Figure 3.
Ultrasounds in AoL
USs are high intensity sound waves that by cavitation, local heating produce the disruption of the cells and the extraction and release of proteins, polysaccharides, and other cell components. When USs are used directly in wines under AOL the thermal side effects lead to undesired oxidations and affect wine quality even when the release of cell wall polysaccharides is accelerated. The application of USs to yeast biomasses exogenically produced help to overwhelm this inconvenience (Figure 3).
We have been using ultrasound as a disruptive technique to produce an intense and fast cell fragmentation to accelerate yeast lysis and to speed AoL processes. When USs are applied to yeast biomasses the typical turgent ellipsoidal shape of the cells (Figure 4a) is degraded and cells shows plasmolysis (Figure 4b), and also many cells are separated from their cell walls that can be observed as fragments in the autolysis media (Figure 4b).
When polysaccharides contents in the media are analyzed by LC-RID, it can be observed that the amount released is higher than in controls from the beginning and can be increased >50% in a few days (Figure 5a). Colloidal turbidity from lees due to the presence of cell fragments also has lower falling times after shaking what improves the clearness of the solutions as happens when wines are aged on lees during long time after the bâtonnage treatments (Figure 5b). This fast settling can be considered as an indicator of the intense evolution of the autolysis and ageing on lees.
Oxidation
By using US, autolysis processes can be extremely accelerated, reaching high contents of polysaccharides in a few months. Additionally, the sonicated lees produce a decreasing in the oxidation potential, protecting wine color and fruitiness during long periods. We have measured O2 contents <2 mg/L in AoL with sonicated lees when the controls showed contents >5 mg/L (del Fresno et al, 2019).
Ultrasounds and non-Saccharomyces yeasts in AoL
The use of USs to process the lees of non-Saccharomyces yeasts can also help to produce even faster autolysis processes and the release of even higher concentrations of polysaccharides in wines (Kulkarni et al, 2015, Figure 6). Some yeast species as Schizosaccharomyces pombe and Saccharomycodes ludwigii have thicker cell walls with higher contents of polysaccharides that can be easily depolymerized by US increasing even more the contents in wines.
Non-Saccharomyces associated to US treatments is an innovative biotechnology to speed AoL improving red wine quality. Appropriate species/strain selection can achieve truly effective AoL in very short periods of time (Figure 6).
Sensory assessment
The use of USs and AoL produces full bodied wines with well-integrated tanicity and good roundness. The aroma is complex with a good preservation of varietal aroma and fruitiness even in the long term.
Conclusions
USs and AoL is a successful combination for high quality red wines because of the reduction of ageing times but also reaching the same advantages than in the conventional ageing on lees. The wines show a good palatability and roundness but also a complex aromatic profile. Additionally, US can be applied to pure biomasses of selected Saccharomyces or non-Saccharomyces strains increasing the possibilities of this technology.
del Fresno, J.M.; Loira, I.; Morata, A.; González, C.; Suárez-Lepe, J.A.; Cuerda, R. Application of Ultrasound to Improve Lees Ageing Processes in Red Wines. Food Chem. 2018, 261, 157–163.
del Fresno, J.M.; Morata, A.; Escott, C.; Loira, I.; Cuerda, R.; Suárez-Lepe, J.A. Sonication of Yeast Biomasses to Improve the Ageing on Lees Technique in Red Wines. Molecules 2019, 24, 635. https://doi.org/10.3390/molecules24030635
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, 1255–1262.
Loira, I.; Vejarano, R.; Morata, A.; Ricardo-da-Silva, J.; Laureano, O.; González, M.; Suárez-Lepe, J. Effect of Saccharomyces Strains on the Quality of Red Wines Aged on Lees. Food Chem. 2013, 139, 1044–1051.
Morata, A.; Escott, C.; Bañuelos, M.A.; Loira, I.; del Fresno, J.M.; González, C.; Suárez-Lepe, J.A. Contribution of Non-Saccharomyces Yeasts to Wine Freshness. A Review. Biomolecules 2020, 10, 34. https://doi.org/10.3390/biom10010034
Morata, A.; Escott, C.; Loira, I.; Del Fresno, J.M.; González, C.; Suárez-Lepe, J.A. Influence of Saccharomyces and non-Saccharomyces Yeasts in the Formation of Pyranoanthocyanins and Polymeric Pigments during Red Wine Making. Molecules 2019, 24, 4490. https://doi.org/10.3390/molecules24244490
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