Vinylphenolic pyranoanthocyanin pigments identified by HPLC/ESI‐MS in musts containing extra hydroxycinnamic acids fermented by selected yeast strains.
Colour is the first impression that the consumer receives from wine and it influences the taste. Colour gives an idea about wine quality, age, oxidation and structure, so it has an important repercussion on the consumer perception of wine. Yeasts promote the formation of stable pigments by the production and release of fermentative metabolites affecting the formation of vitisin A and B type pyranoanthocyanins. The hydrox- and ycinnamate decarboxylase activity showed by some yeast strains produces highly reactive vinylphenols stimulating the formation of vinylphenolic pyranoanthocyanins from grape anthocyanin precursors during fermentation. Some yeasts also influence the formation of polymeric pigments by unclear mechanisms that can include the production of linking molecules such as acetaldehyde. Grape anthocyanins adsorbed in yeast cell walls during fermentation are removed from wine after racking processes affecting final pigment content. Moreover, the intensive use of non‐Saccharomyces yeasts in current oenology makes it interesting to assess the effect of new species in the improvement of wine colour.
- polymeric pigments
In the past, the formation and evolution of wine colour was conditioned by the anthocyanin composition of the grape variety, the degree of extraction during winemaking and the physicochemical evolution of the pigments during tank, barrel and bottle ageing. In these last processes, the influence of grape proanthocyanins, other flavonoids and oxygen affects the formation of stable polymeric pigments .
Grape pigments are monomeric anthocyanins glycosylated in position 3. The colour of anthocyanins is strongly dependent on pH, SO2 levels and hydration. According to these factors, several anthocyanin derivatives can be found (Figure 1). Moreover, the solubility of anthocyanins is affected by the polarity of the medium. During fermentation, alcohol production reduces must polarity and the concentration of anthocyanins decreases.
Since 1990s the role of yeast in colour stability has been studied deeply. In fact, anthocyanin insolubilisation during fermentation is a consequence of yeast metabolism. However, that is not the only contribution of yeasts to wine colour, the production of derived pigments during fermentation using yeast metabolites as precursors or by means of yeast enzymatic activities are also major concerns that have been analysed in detail in the last decades [2, 3].
Pigment adsorption by yeast cell walls reduces the concentration of anthocyanins affecting wine colour, especially in low colour varieties. This property has been used traditionally to reduce pigment contents in the production of white sparkling wines from red varieties (
The formation of polymeric pigments has been considered as a way of colour stabilization during ageing. Grape anthocyanins condense with other flavonoids forming polymeric structures (
The strong effect of pH in anthocyanins colour intensity can cause slight modifications in wine colour by some yeast species able to either degrade or produce organic acids during fermentation.
Some of these processes with effect on colour can also be enhanced by the use of non‐
2. Formation of pyranoanthocyanins with yeast metabolites
Some yeast metabolites can react with grape anthocyanins during fermentation forming derived pigments with enhanced stability in oenological conditions and slightly different chromatic features. The formation of vitisin A, a pyranoanthocyanin pigment formed by chemical reaction between malvidin‐3‐
Vitisins A and B are stable pyranoanthocyanin in oenological conditions with stable colour intensity under variable pH . They are also more resistant to oxidative damage probably because of the higher number of resonant forms they have due to the double pyranose ring structure. Moreover, they are not sensitive to sulphur dioxide bleaching because position C4 is fully saturated being unable to react with the bisulphite ion (Figure 3).
Chromatic properties of vitisins are slightly different from grape anthocyanins. The maximum of absorption in visible spectra for malvidin is approximately 528 nm, but vitisin A shows a maximum of 515 nm and vitisin B of 495 nm (Figure 4). That means that vitisins are red‐orange pigments and consumers currently prefer red‐bluish colours in wines. However, the normal evolution in wine colour during ageing is from purple to red‐orange, and, in this situation, colour of vitisins can be better integrated in wine appearance, indeed, they can even improve it. The stability of vitisins that makes them more persistent during ageing must be also considered.
Vitisins are usually analysed by HPLC‐DAD separation and quantitation, using an external standard of malvidin‐3‐
Vitisins A and B can be also identified by mass spectrometry (MS) by the specific fragmentation patterns; in fact, MS facilitates the full identification of these pigments after LC separation. The
Concentration of vitisins in wines could range from traces to a few mg/l. The amount formed during fermentation can be improved by using selected
The production of pyruvate and acetaldehyde behaves differently in
Selected yeast strains of
Recently, we have observed that some non‐
Vitisins are formed by chemical condensation of malvidin with pyruvate and acetaldehyde so the addition of these precursors in wines enhances their formation. The addition of pyruvate is especially effective . However, pyruvate and acetaldehyde are not allowed as oenological additives. Moreover, the use of acetaldehyde has many other effects on reactions between phenolic compounds promoting the condensation between tannins and the precipitation of phenols and pigments.
3. Hydroxycinnamate decarboxylase activity influence on the formation of vinylphenolic pyranoanthocyanins
Vinylphenolic pyranoanthocyanins (VPAs) are also stable pigments with similar properties to vitisins. They were discovered initially in pinotage (
Later, the formation of these pigments derived from malvidin and hydroxycinnamic acids (HCAs) (either caffeic, ferulic or
The use of HCDC+ strains of
The selection of HCDC+ and determination of the intensity of enzymatic activity can be tested by using a fermentative medium with hydroxycinnamic acids. The use of
HCAs are precursors of ethyl phenols (EPs) in wines, controversial off‐smells that highly degrade the wine quality. During barrel ageing, some spoilage yeasts are able to transform grape HCAs into ethyl phenols by means of two enzymatic steps. First, an HCDC activity transform HCAs into VPhs and later a vinylphenol reductase enzyme induce the reduction of VPhs in EPs. Sensory threshold of EPs is very low, about 400 ppb of 4‐ethylphenol can be perceptible in wines, although it also depends on wine polyphenolic structure. Higher concentrations can strongly depreciate the wine quality. Formation of VPAs by yeasts during fermentation is a natural way to block VPhs and, consequently, to reduce EP precursors of wines (Figure 9).
A reduction in the amount of HCAs, correlated with the amount of VPAs that were formed, has been observed when the effect of ferment with HCDC+
Other problem in the formation of ethylphenols in wines is that the levels of tartaric esters of HCAs (TE‐HCAs) are frequently higher than free HCAs. These esters can release free HCAs during storage and barrel ageing increasing the amount of ethylphenol precursors. The use of cinnamyl esterases enzymes (CEs) during maceration is a way to release free HCAs. If, at the same time, fermentation is done with HCDC+ yeasts, the HCAs can be used to form VPAs reducing the precursors of EPs. This is a natural enzymatic‐biologic‐chemical way to decrease the precursors of ethylphenols protecting wines against ulterior contaminations by
The simultaneous use of CEs and HCDC+ yeasts promotes the formation of stable pigments decreasing at the same time the amount of EPs precursors. We can observe the effect on scale fermentations when HCDC+ (
4. Formation of polymeric pigments
It is known that the wine colour evolves from red‐bluish to red‐orange during the ageing and this phenomenon is affected by oxygen levels and temperature (Figure 12). During barrel ageing, microoxygenation through the porous surface of wood promotes the browning of the wine pigments, and, at the same time, helps to modulate the aromatic profile and causes tannins smoothing. It is also known that long reductive ageing, as happens in vintage Porto wines, helps to keep red‐bluish pigments and to preserve initial colour. During ageing, the colour of wine, initially due to grape anthocyanins, is being substituted by polymeric pigments; these pigments could be responsible of 50% of the colour density after the first year .
Polymeric pigments are formed by more than one flavonoid unit, which means compounds with structure
Polymeric pigments show red‐orange colours and higher stability against both oxidative damage and SO2 bleaching, so they are really important in the colour of aged red wines. The analysis of these pigments can be done by LC‐MS, capillary electrophoresis and gel electrophoresis. When wine anthocyanins are separated by mass/charge ratio in gel electrophoresis, the monomeric grape anthocyanins run faster being easily separated in the gel front and polymeric pigments are delayed at the end, because all wine anthocyanins have a positive charge in the pyrilium ring, but the mass increase strongly in the oligomers depending on the number of flavonoid units. A red‐bluish or red colour in anthocyanin monomers and a red‐orange colour in oligomeric pigments can be observed (Figure 13).
Capillary electrophoresis (CE) has also been used to separate polymeric anthocyanins. This technique is quite similar to gel electrophoresis, however, it improves the resolution that is possible to get in traditional gel technique. Although LC separations are usually preferred for separation of monomeric anthocyanins, its performance is worst to identify and separate polymeric pigments. However, CE is good for charged compounds such as anthocyanins that can be easily separated according to the charge/mass ratio.
Formation of polymeric pigments has been traditionally considered as a natural chemical process produced during ageing and promoted in acidic media and under oxidative conditions of barrel ageing. However, recently, the role of yeast in the formation of polymeric pigments during must fermentation has been considered. Moreover, some polymeric pigments can be formed faster because of the connection between anthocyanin and catechins or procyanidins by acetaldehyde bridges. When musts supplemented with catechin and procyanidin B2 were fermented by several selected
We are also studying the role of non‐
5. Pigment adsorption in yeast cell walls
During fermentation, yeasts are able to adsorb the molecules in external cell wall surface. The adsorption of anthocyanins [24, 25], phenols [26, 27], aromatic compounds  and toxic molecules [29, 30], have been previously reported. In the exponential fermentation, yeast population range 108–109 cfu/ml, and considering the typical elliptic geometry and size of
Moreover, cell adsorption is a strain‐dependant phenomena being possible to select yeasts with lower anthocyanin adsorption than others . The adsorption of anthocyanins in cell walls is not yet well understood, but probably depends on cell wall surface structure and composition. It has been observed that the amount of each anthocyanin type molecule adsorbed on cell walls is affected by the polarity of the anthocyanin. Polarity of grape anthocyanins is affected by B ring substitution pattern (methoxylation makes anthocyanin more apolar, hydroxylation makes it more polar), and the type of acylation: none, acetylation, coumaroylation or caffeoylation in decreasing order of polarity. It has been observed that apolar anthocyanins can be more strongly adsorbed than polar ones.
The selection of yeasts with low anthocyanin adsorption helps to keep more anthocyanins in solution, what means wines with higher amount of anthocyanins. Of course, this will be especially interesting for those grape varieties in which production of anthocyanins is low (Pinot noir, Grenache) or in regions where the synthesis of anthocyanins is inhibited by unsuitable climatic conditions. Although global adsorption in
To evaluate the ability to adsorb anthocyanins by yeast, two kinds of procedures have been used. The first one, that is fast and easy to apply, is the plating in agar medium enriched in grape anthocyanins. The medium is a YEPD‐agar but supplemented with a high concentration of anthocyanins extracted from grape skins [18, 31]. When yeasts colonies grow and develop in plate surface adsorb anthocyanins from the surrounding medium, and this adsorption is proportional to the affinity of their cell walls to link anthocyanins. So, more pigmented colonies are coming from strains with strong anthocyanin adsorption (Figure 14). This technique allows to perform a fast screening for selecting either yeast strains or species with low anthocyanin adsorption.
The second procedure to evaluate anthocyanins adsorption by yeasts, that is more precise but more difficult and tedious to apply, is the recovery of anthocyanins adsorbed from cell walls and the characterization and quantitation of them by LC‐DAD or LC‐ESI/MS [24, 25]. The procedure requires separating all the lees from fermentation and it is possible to do it when red wines are fermented without skins maceration. If winemaking is done with skin maceration, what is the usual industrial process, which is very difficult to separate the yeast lees? But, it is easy to produce a red must with enough colour and tannins to make fermentation in absence of skins by using accelerated maceration, for example: heating and pressing the grapes, freezing or using ultrasounds. Thus, the lees can be separated from the wine by centrifugation at the end of fermentation.
Later, the lees can be washed with water or water‐ethanol (88/12, v/v) solutions to remove anthocyanins that are not strongly adsorbed in cell wall but only in suspension in the surrounding medium among cells. It is not easy to evaluate what is the degree of extraction in this preliminary clean up and maybe some of the removed anthocyanins can be partially/weakly retained on cell walls. The following step, that is particularly delicate, is the separation of anthocyanins from cell walls. Yeast cell wall is a thick layer that externally covers the cell, and is involved in relationship function and giving resistance to osmotic pressure. It is formed by globular mannoproteins sustained by a net of fibrillar polysaccharides mainly formed by β‐glucans and chitin [32, 33]. Anthocyanins might be forming links by polar interactions with these cell wall components, but the nature of this process is not yet well described.
Separation of anthocyanins from cell walls must be done by using solvents. The use of formic acid‐methanol mixtures has been described . Several washings are necessary to remove most of the adsorbed compounds and some anthocyanins can still remain linked after extraction. The detachment process can be facilitated by applying energy in form of shaking, ultrasounds or temperature. After each washing, supernatant is recovered by centrifugation at 3000 ×
6. Biological ageing: ageing on lees
Yeasts are used not only to ferment musts but also in the ageing process of wines. Many traditional wines as Sherry‐flor wines, natural sparkling wines and barrel fermented and aged Chardonnays improve their quality after a long period together with the yeast lees produced during fermentation. Along the biological ageing, many cell metabolites and structural components are released into the wine improving sensory quality. Also, the ageing on lees (AOL) technique can be used during barrel maturation of red wines . AOL has a reductive role because yeast lees are oxygen consumers and, moreover, some cell wall constituents such as glutathione (GSH) are antioxidant compounds with a protective effect on aromatic compounds and anthocyanins.
The simultaneous use of barrel ageing and AOL reduce the oxidative degree partially preserving anthocyanins from oxidative degradations . Yeast selection is also a tool to get better strains to improve the wine quality and to protect pigments during AOL . The use of non‐
7. Future trends
Probably, the future of red winemaking will be the separation of maceration and fermentation by means of fast macerations (minutes‐hours) using new technologies such as high hydrostatic pressures , pulsed electric fields , irradiation  and ultrasounds, among others, to ensure enough amounts of anthocyanins and tannins in the must. In this situation, the fermentation will be produced in absence of skins and seeds and at low temperature to preserve sensory quality. The use of selected strains of
This work was funded by the Spanish Ministry of Economía y Competitividad, Project AGL2013‐40503‐R.