**Figure 2.** Bar graphs of berry weight (W), total soluble solids (TSS), malic acid (MA), tartaric acid (TcA), potassium in must (K), and yeast assimilable nitrogen (YAN) (2015). Average values are displayed within bar graphs. Standard er‐ rors are shown as bars (±1 SE mean). CS.C and SY.C: control in Cabernet sauvignon and Syrah, respectively; CS.VpV

and SY.VpV: NAA sprayed 5 days pre‐ and postveraison in Cabernet sauvignon and Syrah, respectively.

high must sugar levels at harvest time.

366 Grape and Wine Biotechnology

**Figure 3.** Relationship between malic acid (MA), yeast assimilable nitrogen (YAN), and total soluble solids (TSS) levels in grapes at harvest time. TSS in Brix scale.

## **3. Effects of Mg2+ foliar fertilization on berry sugar content**

It must be recognized that grapevine nutrition remains an important part of managing a vineyard since it impacts on berry development and, finally, wine quality is derived to a large degree from berry composition. Some grape growers avoid any fertilizer for fear of oversti‐ mulating growth, whereas in other cases vineyard blocks might be fertilized when only specific areas of the block require fertilizer. Therefore, it is important that growers have a sound basis for determining the fertilizer needs of their vines [22]. Elsewhere, since the general relationship between vine nutritional status (in both nutrient macro‐ and microelements) and grape composition is obscure, further efforts are necessary to acquire greater knowledge in this topic. This is an important knowledge gap because these elements should necessarily influence grape juice quality and, therefore, the vinification process.

It is recognized that plants need K+ for the formation of sugars and starches, for protein synthesis, and for cell division. Additionally, K+ also neutralizes organic acids, regulates the activities of other mineral nutrients in plants, activates certain enzymes, and helps adjust water relationships (Hewitt, cited by [20]), but free potassium ions are released when the grape cell membranes are broken during grape processing, and form crystals with tartrate, which drop grape juice and wine acidity [11]. On the basis of the antagonistic interaction between levels of K+ and Mg2+ reported by several authors at the root‐soil interface [23, 24], another study was performed during 2014 vintage in the PDO area of Ribera del Duero, Spain. The impact of Mg2+ supply on berry chemistry attributes from this trial is shown below.

**Figure 4.** Bar graphs of berry weight (W), total soluble solids (TSS), real acidity (pH), malic acid (MA), tartaric acid (TcA), and yeast assimilable nitrogen (YAN) (2014). Average values are displayed within bar graphs. Standard errors are shown as bars (±1 SE mean). C: control; Mg0.5: foliar Mg at 0.5 kg/ha; Mg1.0: foliar Mg at 1.0 kg/ha.

The cultivar chosen, *Vitis vinifera* L. Tempranillo, is important in Spanish PDOs such as Rioja, Navarra, and Toro, and in many other countries [25]. The trial consisted on a randomized triplicate design with control and a foliar Mg2+ spray which was applied to cv. Tempranillo at two doses (0.5 kg/ha (Mg0.5) and 1.0 kg/ha (Mg1.0)) at veraison stage. Each replicate consisted of 10 treated vines (~150 bunches) in each replicate. The treatments were evaluated by data from cluster samples (10 clusters per replicate) at harvest time. One hundred berries from the clusters were removed and weighed (W). The berries were then crushed and several parame‐ ters (pH, TSS, MA, TcA, and YAN) were determined in the must. Means and standard errors of all evaluated parameters arranged by treatment are shown in **Figure 4**.

of K+

368 Grape and Wine Biotechnology

 and Mg2+ reported by several authors at the root‐soil interface [23, 24], another study was performed during 2014 vintage in the PDO area of Ribera del Duero, Spain. The impact of

**Figure 4.** Bar graphs of berry weight (W), total soluble solids (TSS), real acidity (pH), malic acid (MA), tartaric acid (TcA), and yeast assimilable nitrogen (YAN) (2014). Average values are displayed within bar graphs. Standard errors

The cultivar chosen, *Vitis vinifera* L. Tempranillo, is important in Spanish PDOs such as Rioja, Navarra, and Toro, and in many other countries [25]. The trial consisted on a randomized triplicate design with control and a foliar Mg2+ spray which was applied to cv. Tempranillo at two doses (0.5 kg/ha (Mg0.5) and 1.0 kg/ha (Mg1.0)) at veraison stage. Each replicate consisted

are shown as bars (±1 SE mean). C: control; Mg0.5: foliar Mg at 0.5 kg/ha; Mg1.0: foliar Mg at 1.0 kg/ha.

Mg2+ supply on berry chemistry attributes from this trial is shown below.

Several *F*‐tests were carried out to study the effect of foliar Mg on W, TSS, pH, MA, TcA, and YAN. According to the ANOVA, foliar Mg treatments had no significant effects on any of the must quality parameters evaluated. Although none of TSS differences were statistically significant, as can be seen from **Figure 3** only Mg1.0 treatment showed a lower TSS level than control subplots, whereas Mg0.5 showed a greater TSS level than other treatments. Interest‐ ingly, although there are no significant differences with the control subplots in terms of grape weight (W) an increasing trend could be observed in Mg treated subplots (Control (2.25 g ± 0.29 95% CI) < Mg0.5 (2.30 g ± 0.31 95% CI) < Mg1.0 (2.31 g ± 0.22 95% CI)). In a similar way, leaf and shoot removal also affected YAN levels in such way that both Mg treatments may be associated with an increase in this key quality parameter. However, with a small sample size (*n* = 9 and 1 year), caution must be applied in both cases (W and YAN), because it is important to bear in mind the possible bias in the response to a foliar Mg treatment. Thus, if we consider our results collectively, they do not allow us to draw clear conclusions about the impact of foliar Mg treatments on harvest parameters, and therefore on TSS.

## **4. Effects of leaf removal and lateral shoot removal on berry sugar content**

One of the most important and commonly applied summer canopy management operations in viticulture is the removal of leaves [26] and shoots in the fruit zone. Both practices are performed on grapevines to increase air circulation, light exposure, penetration of fungicide sprays, as well as decrease disease incidence. In general, exposing fruit to the sun will increase fruit temperature along with the enzymatic activities therein. Consequently, when compared to shaded fruit, exposed fruit will normally contain higher soluble solids [27]. Nevertheless, it should be noted that these actions on the vine canopy microclimate, which basically depends on the amount and distribution of leaf area in space and its interaction with above‐ground climate [28], will have different effects on harvest quality according to the time and the shoot position when they were carried out. Most canopy microclimate components are of different values than those around the canopy, due to attenuation by the canopy. The degree of shading within grapevine canopies can be altered by three principal means: by varying the shoot number, the vine vigor, and/or the training system employed [28]. At the same time, a number of viticultural practices in wine grape improvement programs have been a topic of discussion in the scientific community in order to improve grape quality at harvest: optimum balance in vine pruning, shoot thinning, leaf and lateral shoot removal, early cluster thinning, late cluster thinning, shoot positioning, and tipping or irrigation scheduling.

On the basis of the above, a research was performed during 2014 vintage in the PDO area of Ribera del Duero, Spain. The cultivar chosen was *Vitis vinifera* L. Tempranillo. The trial consisted on a randomized triplicate design with control and two leaf removal treatments both at veraison stage. One of the treatments was a leaf and lateral shoots removal between the clusters positioned at bottom and top in the productive shoots (LRbt), whereas the other one was a leaf and lateral shoots removal below the clusters positioned at bottom in the productive shoots (LRbl). Each replicate consisted of 10 treated vines and 100 berries from the clusters were weighed (W) and then crushed to evaluate several must quality parameters at harvest time (pH, TSS, MA, TcA, and YAN). Means and standard errors of must parameters arranged by treatment are shown in **Table 1**.

LRbt: leaf and lateral shoots removal between the clusters positioned at bottom and top in the productive shoots; LRbl: leaf and lateral shoots removal below the clusters positioned at bottom in the productive shoots.

**Table 1.** Means and standard errors (SE) of berry weight (W), total soluble solids (TSS), real acidity (pH), malic acid (MA), tartaric acid (TcA), and yeast assimilable nitrogen (YAN) (2014).

Several *F*‐tests were carried out to study the effect of management practices on W, TSS, pH, MA, TcA, and YAN. According to the ANOVA these viticultural practices had no significant effects on any of the must quality parameters evaluated. However, fruit composition has been shown to be affected by microclimate manipulation. While it is true that leaf and shoot removal in both treatments (LRbt and LRbl) had no significant effect on W, both treatments increased consistently this parameter. In an opposite direction both treatments decreased consistently the real acidity (pH). On the other hand, only LRbt treatment showed a lower TSS level than Control (Control (23.6 °Brix ± 2.39 95% CI) > LRbt (23.1 °Brix ± 1.29 95% CI)). In contrast to earlier findings [28, 29] levels of tartaric acid (TcA) are increased by shade (C > LRbl > LRbt). The latter showed that this decrease in the concentrations of tartaric acid in the shaded berries was due to an increase in berry size. Additionally, in contrast to Petrie et al. [30], levels of pH decreased by shade (C > LRbl > LRbt). It is difficult to explain these controversies with previous studies, but it might be related with the time of carrying out both viticultural practices. In this regard, because when veraison begins to occur green growth slows to a stop (while the vine directs energy toward the grape clusters) [31], the choice of another phenological stage for making these viticultural practices might have a different impact on harvest parameters. The lack of scientific evidence and controversies have been reported by several authors. For instance, whereas Bledsoe et al. [32] found that yield and yield components were not signifi‐ cantly affected by the timing of leaf removal in Sauvignon Blanc grapes, Hunter and Visser [33] found that 33% defoliation prior to berries reaching pea size reduced berry size and yield, but had no effect when applied at veraison. Thus, understanding the impact of the timing of leaf and shoot removal on vines is crucial for vineyard managers and winemakers.

## **5. Biotechnological approaches to reduce the alcohol content in wine**

consisted on a randomized triplicate design with control and two leaf removal treatments both at veraison stage. One of the treatments was a leaf and lateral shoots removal between the clusters positioned at bottom and top in the productive shoots (LRbt), whereas the other one was a leaf and lateral shoots removal below the clusters positioned at bottom in the productive shoots (LRbl). Each replicate consisted of 10 treated vines and 100 berries from the clusters were weighed (W) and then crushed to evaluate several must quality parameters at harvest time (pH, TSS, MA, TcA, and YAN). Means and standard errors of must parameters arranged

**Mean SE Mean SE Mean SE**

**Must parameter Control LRbt LRbl**

leaf and lateral shoots removal below the clusters positioned at bottom in the productive shoots.

(MA), tartaric acid (TcA), and yeast assimilable nitrogen (YAN) (2014).

**W (g)** 2.25 0.15 2.50 0.25 2.57 0.06 **TSS (Brix)** 23.6 1.22 23.1 0.66 23.6 0.81 **pH** 3.49 0.04 3.46 0.05 3.48 0.06 **MA (g/l)** 2.05 0.13 1.93 0.18 2.29 0.08 **TcA (g/l)** 3.06 0.52 2.92 0.19 2.98 0.36 **YAN (mg/l)** 256 1.68 239 8.85 270 22.4

LRbt: leaf and lateral shoots removal between the clusters positioned at bottom and top in the productive shoots; LRbl:

**Table 1.** Means and standard errors (SE) of berry weight (W), total soluble solids (TSS), real acidity (pH), malic acid

Several *F*‐tests were carried out to study the effect of management practices on W, TSS, pH, MA, TcA, and YAN. According to the ANOVA these viticultural practices had no significant effects on any of the must quality parameters evaluated. However, fruit composition has been shown to be affected by microclimate manipulation. While it is true that leaf and shoot removal in both treatments (LRbt and LRbl) had no significant effect on W, both treatments increased consistently this parameter. In an opposite direction both treatments decreased consistently the real acidity (pH). On the other hand, only LRbt treatment showed a lower TSS level than Control (Control (23.6 °Brix ± 2.39 95% CI) > LRbt (23.1 °Brix ± 1.29 95% CI)). In contrast to earlier findings [28, 29] levels of tartaric acid (TcA) are increased by shade (C > LRbl > LRbt). The latter showed that this decrease in the concentrations of tartaric acid in the shaded berries was due to an increase in berry size. Additionally, in contrast to Petrie et al. [30], levels of pH decreased by shade (C > LRbl > LRbt). It is difficult to explain these controversies with previous studies, but it might be related with the time of carrying out both viticultural practices. In this regard, because when veraison begins to occur green growth slows to a stop (while the vine directs energy toward the grape clusters) [31], the choice of another phenological stage for making these viticultural practices might have a different impact on harvest parameters. The lack of scientific evidence and controversies have been reported by several authors. For instance, whereas Bledsoe et al. [32] found that yield and yield components were not signifi‐

by treatment are shown in **Table 1**.

370 Grape and Wine Biotechnology

Currently, several different technological strategies are available in order to reduce the ethanol content in final wines. Yeasts are the main microorganisms involved in the ethanol production from grapes and wine production, and accordingly, some of these strategies are based in a different management of wine yeasts, including the isolation of strains with a lower ability to produce ethanol.

Natural screening might be the first attempt to obtain lower ethanol‐producing strains. However, this approach is unlikely to succeed because different aspects of the biochemistry, physiology, and genetics of *Saccharomyces cerevisiae*. In fact, this microorganism has evolved by natural selection to boost the ethanol production, even when oxygen is available [1, 34]. On the other hand, an attractive option would be to develop engineered wine yeast with reduced ethanol yield. So far, one of the most promising strategies is to redirect the metabolic flux toward an increased production of glycerol instead ethanol [2, 35]. However, this strategy has shown some unwanted effects like an overproduction of undesirable compounds from an organoleptic point of view [1]. Indeed, glycerol and ethanol produced during alcoholic fermentation are important regulators of the cellular redox balance, and consequently any attempt to redirect carbon flux by gene manipulation would modify the concentration of a range of other metabolites in order to correct the redox imbalance [36]. Acetaldehyde, acetate, succinate, acetoin, diacetyl, and 2,3‐butanediol, among others, are some of the compounds to be avoided, since their presence at levels exceeding their sensorial threshold may be detri‐ mental to the final wine quality [1, 36]. Furthermore, the public attitude toward the use of genetically modified organisms (GMOs) in food products and the legal restrictions in their use suggest that novel yeast strains will have to be generated using non‐GM approaches.

Microorganisms, and particularly yeast, have a huge ability to adapt rapidly to different environmental conditions. This property has been used in recent years to modify the natural properties of yeasts by conducting adaptive laboratory evolution (ALE) experiments [2]. This approach mimics the natural evolution, by environmental or metabolic constraints, with the main purpose of obtaining improve yeast strains for several biotechnological applications and, of course, in winemaking processes [37–40]. A recent ALE study, by using KCl as osmotic and salt stress agent during 450 generations, achieved a wine with 0.6% (v/v) less ethanol in pilot scale fermentation when it was compared to the previous ancient strain. Besides that, the use of intrastrain hybrids by breeding techniques (a non‐GMO technique) has proven the reduction of the alcoholic strength to 1.3% (v/v) [2].

An alternative approach to modify the final alcohol content of wines is related to the perform‐ ance of modified fermentation procedures. Although *S. cerevisiae* is the main yeast species responsible for conducting the alcoholic fermentation, the contribution of a nonnegligible number of other yeast species associated to the initial stages of the fermentation and their contribution to final sensorial properties are well established [41–43]. These strains are naturally present in sound grapes and might be easily isolated from the grape must at initial fermentation stages.

There are significant differences in sugar metabolism between some of these species and *S. cerevisiae*. The non‐*Saccharomyces* strains actually allow an increased breakdown of sugars via respiratory pathways than through fermentation. An enhancement of this respiratory catab‐ olism has been suggested by several authors in order to reduce the amount of sugar conversion into ethanol [44, 45]. For this reason, mixed cultures between a non‐*Saccharomyces* strain and a *Saccharomyces cerevisiae* strain might be a good alternative to the previously mentioned approaches. Further, an additional advantage of this strategy would be to use autochthonous yeast strains in order to maintain the typicality of wines elaborated in this way [46].

**Table 2.** Yeast species isolated at different stages of the fermentation process and identified from spontaneous fermentations of natural "Tinta de Toro" grape juice obtained from vineyards of different ages.

Although high levels of ethanol content in final wines is a worldwide issue, as mentioned earlier, in Spain this problem is still more pronounce in the Denomination of Origin (DO) Toro (Toro, Zamora, Spain), whose wines easily reach and exceed 15–16° alcohol content. For this reason a biotechnological‐based approach was developed with the final aim to reduce their ethanol levels. During 2013 vintage, the population of indigenous strains associated to a winery belonged to DO Toro was characterized. Spontaneous fermentations were carried out on natural grape juice ("*Tinta de Toro*" grape variety), obtained from grapes of three different vineyards. Microfermentations were conducted at fixed temperature (21°C) and were daily monitored by measuring their weight loss until completion (constant weight). Yeast isolation was made from three different phases of the fermentation process: initial, medium, and final stage (final wine). Yeast strains were randomly selected for genetic typing. Yeast identification was carried out by RFLP analysis of the 5.8S‐ITS‐rRNA region amplified by using ITS1 and ITS4 primers [47], and confirmed by sequencing the D1‐D2 regions of 26S rDNA using the NL‐1 and NL‐4 primers [48]. The results are shown in **Table 2**.

responsible for conducting the alcoholic fermentation, the contribution of a nonnegligible number of other yeast species associated to the initial stages of the fermentation and their contribution to final sensorial properties are well established [41–43]. These strains are naturally present in sound grapes and might be easily isolated from the grape must at initial

There are significant differences in sugar metabolism between some of these species and *S. cerevisiae*. The non‐*Saccharomyces* strains actually allow an increased breakdown of sugars via respiratory pathways than through fermentation. An enhancement of this respiratory catab‐ olism has been suggested by several authors in order to reduce the amount of sugar conversion into ethanol [44, 45]. For this reason, mixed cultures between a non‐*Saccharomyces* strain and a *Saccharomyces cerevisiae* strain might be a good alternative to the previously mentioned approaches. Further, an additional advantage of this strategy would be to use autochthonous

yeast strains in order to maintain the typicality of wines elaborated in this way [46].

**Young vineyard Middle-age vineyard Old vineyard**

10 **2** *Lachancea*

*thermotolerans* **4** *Metschnikowia aff.*

*fructicola* **1** *Metschnikowia chrysoperlae* **3** *Metschnikowia pulcherrima*

10 **2** *Hanseniaspora uvarum* **5** *Lachancea thermotolerans* **3** *Saccharomyces cerevisiae*

10 **8** *Saccharomyces cerevisiae* **2** *Saccharomyces paradoxus*

**Table 2.** Yeast species isolated at different stages of the fermentation process and identified from spontaneous

Although high levels of ethanol content in final wines is a worldwide issue, as mentioned earlier, in Spain this problem is still more pronounce in the Denomination of Origin (DO) Toro (Toro, Zamora, Spain), whose wines easily reach and exceed 15–16° alcohol content. For this reason a biotechnological‐based approach was developed with the final aim to reduce their

fermentations of natural "Tinta de Toro" grape juice obtained from vineyards of different ages.

**Identification Yeast**

**analyzed**

**Identification**

10 **1** *Debaryomyces hansenii*

**3** *Lachancea thermotolerans* **2** *Metschnikowia aff.*

*fructicola* **2** *Metschnikowia pulcherrima* **1** *Kluyveromyces dobzhanskii*

10 **5** *Hanseniaspora uvarum* **1** *Lachancea thermotolerans*

10 **10** *Saccharomyces cerevisiae*

**1** *Saccharomyces cerevisiae* **3** *Saccharomyces bayanus*

**1** *Hanseniaspora uvarum*

**analyzed**

fermentation stages.

372 Grape and Wine Biotechnology

**Fermentation phase**

**Yeast analyzed**

*Initial* 10 **2** *Hanseniaspora*

*Medium* 10 **5** *Hanseniaspora*

*Final* 10 **10** *Saccharomyces*

**Muestra/microvinification**

*uvarum* **1** *Lachancea thermotolerans* **4** *Metschnikowia aff.*

*fructicola* **3** *Metschnikowia pulcherrima*

*uvarum* **1** *Lachancea thermotolerans* **4** *Saccharomyces cerevisiae*

*cerevisiae*

**Identification Yeast**

*S. cerevisiae* strain typing was performed by RFLP‐mtDNA analysis with *Alu*I restriction enzyme [49]. The RFLP profiles were compared using the InfoQuest FP software package (Bio‐ Rad, Hercules, CA, USA). Nine different *S. cerevisiae* strains were identified (**Figure 5**), resulting no matches with the commercial strains routinely used in the winery.

The predominant *S. cerevisiae* strain (**Sc-1**, 44% of *S. cerevisiae* total population) and two non‐ *Saccharomyces* strains, *Lachancea thermotolerans* and *Kluyveromyces dobzhanskii*, were selected to conduct experimental fermentation with mixed cultures. The final aim was to the decrease the ethanol content by increasing the respiratory catabolism of sugars by non‐*Saccharomyces* strains in the initial stages of the alcoholic fermentation. Two different methodologies were assayed for both non‐*Saccharomyces* strain: coinoculation with the *S. cerevisiae* strain; and sequential‐ inoculation, by adding first the non‐*Saccharomyces* strain and then (15 days later) the *S. cerevisiae* strain. Microvinifications were conducted in triplicate by inoculation with the selected strains. Microfermentations were carried out at 25°C and monitored by measuring the loss of weight, as described above. Once fermentations were completed, yeast cells were removed by centri‐ fugation and analysis. Samples for quantitative analysis were stored at −20°C until analyses were performed.

The analysis of final wines were performed by HPLC using an Agilent 1200 series (Agilent Technologies, Santa Clara, CA, USA) chromatograph equipped with a HyperREZ XP Carbo‐ hydrate H+ column (8 μm particle size, 300 × 7.7 mm) and a HyperREZ XP carbohydrate H+ Guard pre‐column (Thermo Scientific, Waltham, MA, USA), maintained at 50°C. Samples were filtered using 0.45 μm cellulose acetate filters (Costar, Washington, DC, USA) prior to analysis. A refraction index detector (RID) (positive polarity) at a flow rate of 0.8 ml/min with 4 mmol/ l H2SO4 as mobile phase (injection volume 25 μl) was used to detect glycerol and ethanol. One‐ way analysis of variance was carried out to determine the influence of the "yeast used" factor on ethanol and glycerol content. The results are shown in **Table 3**. Coinoculation methodology decreased the alcohol level in final wines from 0.55 to 0.62% (v/v) when a *L. thermotolerans* or *K. dobzhanskii* strains had been used, respectively. A two‐step (sequential) inoculation strategy achieved a higher reduction in ethanol values: up to 0.79% (v/v) reduction was obtained by using *K. dobzhanskii* strain, whereas a 0.82% (v/v) decrease was obtained by using the *L. thermotolerans* strain. Although some differences in the glycerol content were detected in the final wines, sensory analyses by a panel of expert tasters did not find significant differences in the wines thus elaborated.

**Figure 5.** Dendrogram (top) based on RFLP‐mtDNA analyses with the restriction endonuclease AluI (center) of all the *S. cerevisiae* strains (bottom) isolated from the three vineyards belonging to the winery (DO Toro, Zamora, Spain). The strain selected for the further oenological approach is highlighted in bold. Commercial strains have not been detected in neither case.

significantly different according to ANOVA results (*p* < 0.05).

**Table 3.** Ethanol and glycerol content of the final wines analyzed by HPLC.

Recently, a novel study addressed the same issue with a similar experimental design. In fact, Morales et al. [45] used a *Metschnikowia pulcherrima*/*S. cerevisiae* mixed cultures. The authors used an oxygen flux during the first stages of the fermentation. An alcohol reduction of 2.2% (v/v) was achieved with correct levels of volatile acidity. These data suggest that a higher reduction in the ethanol level could be obtained by increasing aeration at the beginning of the alcoholic fermentation, in order to increase the sugar consumption rate by non‐*Saccharomyces* strains (respiration).

Therefore, the implementation at the industrial level of strategies to lower the ethanol content of wine, owing to breakdown of sugars by non‐*Saccharomyces* yeasts, appears to be an inter‐ esting challenge. However, a further optimization is required in several aspects as yeast strains selection, inoculation protocols, aeration conditions, and other requirements.

## **6. Conclusion**

Taken together the findings showed in this chapter, it has become evident that there are several potential efficient practices to overcome high must sugar levels at harvest time. Most favorable results were obtained by using plant growth regulators (auxins) and yeast selection. The generalizability of these results could be subject to certain limitations. Thus, in the case of auxins the cultivar behaves as an important factor to be taken into consideration, whereas in the case of yeast selection, more research is required to determine the efficacy of implementa‐ tion at the industrial level.

## **Acknowledgements**

**Figure 5.** Dendrogram (top) based on RFLP‐mtDNA analyses with the restriction endonuclease AluI (center) of all the *S. cerevisiae* strains (bottom) isolated from the three vineyards belonging to the winery (DO Toro, Zamora, Spain). The strain selected for the further oenological approach is highlighted in bold. Commercial strains have not been detected

in neither case.

374 Grape and Wine Biotechnology

We are grateful to the R Core team for their hard work and nonprofit effort with the software.

## **Author details**

Miguel Ángel Olego1\*# , José Manuel Álvarez‐Pérez2# , Miguel Javier Quiroga2 , Rebeca Cobos1 , Mario Sánchez‐García2 , Jesús Esteban Medina2 , Sandra González‐García2 , Juan José Rubio Coque2 and José Enrique Garzón‐Jimeno2

\*Address all correspondence to<EMAIL_ADDRESS>
1 RGA Bio‐Investigación, SL, Laboratorio 29, Instituto de Recursos Naturales, Universidad de León, León, Spain

2 Instituto de Investigación de la Viña y el Vino (IIVV), Universidad de León, León, Spain

#These authors contributed equally to this work

## **References**

of putative stagespecific grapevine berry biomarkers and omics data integration into networks. Plant Physiology. 2010;154:1439–1459.

[10] Varela C, Dry PR, Kutyna DR, Francis IL, Henschke PA, Curtin CD, Chambers PJ. Strategies for reducing alcohol concentration in wine. Australian Journal of Grape and Wine Research. 2015;1:670–679.

**Author details**

376 Grape and Wine Biotechnology

León, León, Spain

**References**

Miguel Ángel Olego1\*#

Mario Sánchez‐García2

Juan José Rubio Coque2

, José Manuel Álvarez‐Pérez2#

and José Enrique Garzón‐Jimeno2

1 RGA Bio‐Investigación, SL, Laboratorio 29, Instituto de Recursos Naturales, Universidad de

2 Instituto de Investigación de la Viña y el Vino (IIVV), Universidad de León, León, Spain

[1] Kutyna DR, Varela C, Henschke PA, Chambers PJ, Stanley GA. Microbiological approaches to lowering ethanol concentration in wine. Trends in Food Science and

[2] Tilloy V, Ortiz‐Julien A, Dequin S. Reduction of ethanol yield and improvement of glycerol formation by adaptive evolution of the wine yeast *Saccharomyces cerevisiae* under hyperosmotic conditions. Applied and Environmental Microbiology.

[3] Matthews MA. Terroir and Other Myths of Winegrowing. Oakland, USA: University

[4] Jackson RS. Wine Science: Principles and Applications. 4th ed. London, UK: Elsevier;

[5] Piazzolla F, Pati S, Amodio ML, Colelli G. Effect of harvest time on table grape quality during on‐vine storage. Journal of the Science of Food and Agriculture. 2016;96:131–

[6] Creasy GL, Creasy LL. Grapes. Wallingford, UK: The Centre for Agriculture and

[7] Agasse A, Vignault C, Kappel C, Conde C, Gerós H, Delrot, S. Sugar transport and sugar sensing in grape. In: Roubelakis‐Angelakis KA, editor. Grapevine Molecular Physiology and Biotechnology. 2nd ed. Dordrecht, The Netherlands: Springer; 2009.

[8] Hufnagel JC, Hofmann T. Quantitative reconstruction of the nonvolatile sensometabo‐ lome of a red wine. Journal of Agriculture and Food Chemistry. 2008b;56:9190–9199. [9] Zamboni A, Di Carli M, Guzzo F, Stocchero M, Zenoni S, Ferrarini A, Tononi P, Toffali K, Desiderio A, Lilley KS, Pe ME, Benvenuto E, Delledonne M, Pezzotti M. Identification

, Jesús Esteban Medina2

\*Address all correspondence to<EMAIL_ADDRESS>
#These authors contributed equally to this work

Technology. 2010;1:293–302.

of California Press; 2015. 328 p.

Bioscience International; 2009. 312 p.

2014;80:2623–2632.

2014. 978 p.

pp. 105–139.

139.

, Miguel Javier Quiroga2

,

, Sandra González‐García2

, Rebeca Cobos1

,

[36] Schmidtke LM, Blackman JW, Agboola SO. Production technologies for reduced alcoholic wines. Journal of Food Science. 2012;77:25–41.

[22] Poling B, Spayd S. The North Carolina Winegrape Grower's Guide. Raleigh, USA:

[23] Ohno T, Grunes DL. Potassium magnesium interactions affecting nutrient uptake by

[24] Huang JW, Grunes DL, Welch RM. Magnesium, nitrogen form, and root temperature effects on grass tetany potential of wheat storage. Soil Science Society of America

[26] Poni S, Casalin L, Bernizzon F, Civardi S, Intrieri C. Effects of early defoliation on shoot photosynthesis, yield components, and grape composition. American Journal of

[27] Reynolds AG. Viticultural and vineyard management practices and their effects on grape and wine quality. In: Reynolds AG, editor. Managing Wine Quality. Volume 1: Viticulture and Wine Quality. Cambridge, UK: Woodhead Publishing Limited; 2010.

[28] Smart, RE. Principles of grapevine canopy microclimate manipulation with implica‐ tions for yield and quality. A review. American Journal of Enology and Viticulture.

[29] Crippen, D, Morrison J. The effects of Sun exposure on the compositional development of Cabernet Sauvignon berries. American Journal of Enology and Viticulture.

[30] Petrie PR, Trought MCT, Howell G, Buchan G. The effect of leaf removal and canopy height on whole‐vine gas exchange and fruit development of *Vitis vinifera* L. Sauvignon

[31] Stanley M. Creating World Class Red Wine. West Chester, USA: Ascension Press; 2014.

[32] Bledsoe A, Kliewer W, Marois J. Effects of timing and severity of leaf removal on yield and fruit composition of Sauvignon Blanc. American Journal of Enology and Viticul‐

[33] Hunter J, Visser J. The effect of defoliation on growth characteristics of *Vitis vinifera* Cabernet Sauvignon II. Reproductive growth. South African Journal of Enology and

[34] Piškur J, Rozpedowska E, Polakova S, Merico A, Compagno C. How did Saccharomyces

[35] Varela C, Kutyna DR, Solomon MR, Black CA, Borneman A, Henschke PA, et al. Evaluation of gene modification strategies for the development of low‐alcohol‐wine

evolve to become a good brewer?. Trends in Genetics. 2006;22:183–186.

yeasts. Applied and Environmental Microbiology. 2012;78:6068–6077.

wheat forage. Soil Science Society of America Journal. 1985;49:685–690.

Publications Office, North Carolina University; 2015. 196 p.

[25] Walker L. It's time for Tempranillo. Wines & Vines. 2006;May:77–80.

Journal. 1990;82:581–587.

pp. 365–444.

378 Grape and Wine Biotechnology

1985;36(3):230–239.

1986;37(4):235–242.

ture. 1988;39:49–54.

Viticulture. 1990;11:26–32.

150 p.

Enology and Viticulture. 2006;57:397–407.

Blanc. Functional Plant Biology. 2003;30:711–717.

#### **Innovations in the Use of Bentonite in Oenology: Interactions with Grape and Wine Proteins, Colloids, Polyphenols and Aroma Compounds Innovations in the Use of Bentonite in Oenology: Interactions with Grape and Wine Proteins, Colloids, Polyphenols and Aroma Compounds**

Milena Lambri, Donato Colangelo, Roberta Dordoni, Fabrizio Torchio and Dante Marco De Faveri Milena Lambri, Donato Colangelo, Roberta Dordoni, Fabrizio Torchio and Dante Marco De Faveri Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64753

#### **Abstract**

[48] O'Donnell K. Fusarium and its near relatives. In: Reynolds DR, Taylor JW, editors. The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systemat‐

[49] Querol A, Barrio E, Ramón D. A comparative study of different methods of yeast strain

characterization. Systematic and Applied Microbiology. 1992;15:439–446.

ics. Wallingfork, UK: CAB International; 1993. pp. 225–233.

380 Grape and Wine Biotechnology

Bentonite is used in oenology to improve the limpidity and the stability of wine and to predict the formation of deposits in the bottle. The exchangeable cations in its lamellar structures strongly influence some properties, such as the specific surface, the exchange capacity and the adsorption behavior. The unintended use of bentonite for juice settling and/or for wine fining produces jeopardized effects on colloidal and protein stability, the aroma compounds and sensory profiles. The interactions with haze-forming proteins, other colloids, as well as aroma compounds and phenols would have been to discover as the modulation of wine colloids by an adjuvant severely affects the wine resilience and the sensory profile. This chapter reviews several studies that focus on the impact of commercial bentonite samples used for both juice clarification and wine fining on the colloids, proteins, phenols and aroma compounds of white and red wines. Some parameters of practical value, such as the wine heat stability, the concentrations of total and haze-forming proteins and the content of the most relevant aromas, have been assessed to track the effects of bentonite and to achieve findings that are applicable to the field of oenology.

**Keywords:** bentonite, colloids, proteins, polyphenols, aroma, wine

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Bentonite**

#### **1.1. Chemical structure**

Bentonite is a phyllosilicate of the class of dioctahedral smectites [1] with a general composition of (M+y・nH2O) (Al3+2-yMg2+y)Si4+4O10(OH)2.

The structures of phyllosilicates are all based on tetrahedral T and octahedral O sheets arranged in either a 1:1 (kaolinite, dickite and nacrite) or 2:1 (smectites, vermiculite, mica and chlorite) ratio to form an anisotropic TO or TOT layer, respectively (**Figure 1**).

**Figure 1.** Structure of a phyllosilicate of the class of dioctahedral smectites [1] as bentonite is.

The layers are stacked on top of each other to form what is called a 'particle'. In turn, an assembly of particles is known as an 'aggregate'. Thus, a particle is formed by stacked layers that are separated from each other by an interlayer space. For a single layer, each tetrahedron consists of a cation (Si4+, Al3+ and Fe3+ are the most common) coordinated to four oxygen atoms and linked to an adjacent tetrahedra by sharing three basal oxygen atoms to form a twodimensional 'hexagonal' pattern. However, each octahedron consists of a cation (Al3+, Fe3+, Mg2+, or Fe2+) coordinated by six oxygen atoms and linked to a neighbouring octahedra by sharing edges. In the 2:1 structures, such as smectites, one octahedral sheet is sandwiched between two tetrahedral sheets. The surface properties of clay minerals depend on many factors, such as their chemical composition, nature of the surface atoms, extent and type of charge, and type of exchangeable cations. There are two types of surfaces with different properties, the planar surface and the edge surface, which is affected by pH. In a 2:1 clay, such as bentonite, the oxygen atoms of the Si tetrahedron represent the atoms of the basal surface. The charge on the oxygen atoms depends on the difference in electronegativity between oxygen and the other atoms involved in the bond. Thus, the ionicity of the bonds follows the order H–O < Si–O < Al–O < Mg–O < Li–O.

Thus, in smectites, the planar surfaces are negatively charged, with the magnitude of the layer charge ranging from 0.2 to 0.6 per half-unit cell. The type of metals that are bonded to the oxygen atoms governs the hydrophobicity of the surface. In the absence of isomorphous substitutions, Si–O bonds prevail in the tetrahedra and Al–O bonds prevail in the octahedral sheets, resulting in a hydrophobic surface. When isomorphous substitutions occur, that is substitution of trivalent cations for Si4+ in tetrahedral sites and of divalent cations for Al3+ in octahedral sites, hydrophilicity is introduced and exchangeable cations are present [1].

Using the electronegativity equalization method (EEM) [2], beidellite and montmorillonite were shown to be the most electronegative and reactive 2:1 clays. The negative charges that result from isomorphous substitutions are balanced by the presence of exchangeable cations in the interlayer space. Na+ , Ca2+, Mg2+ and K+ are the most common ions. The mechanisms involved in the cation hydration for smectites are mainly electrostatic interactions that minimize water–water repulsion [3].

The presence of clustered or 'nanoconfined' water molecules around the exchangeable cations and the water bound to the clay through hydrogen bonds, charge dipole attractions and van der Waals interactions expands the interlayer space. This expansion or swelling depends on the cations present, the ionic strength of the medium and other factors. During hydration, water molecules orient their negative dipole towards the cations, thus weakening the electrostatic interactions with the charged layer and leading to increased separation between the two successive layers. As a result, the cations on the layer surface can be exchanged with the cations in the external solution and, based on the persistence or absence of significant interactions between two successive layers, this separation is called delamination or exfoliation, respectively [4]. In the first case, the smectite increases its volume to maintain its structure, whereas in the second case, isolated layers from the stacks of layers detach from the structure, becoming independently mobile.

Based on their swelling properties, sodium bentonite is different from calcium bentonite because the former is a high-swelling type of clay, while the latter is a low-swelling type. In fact, when sodium bentonite takes up water, exfoliation is more frequent than in the case of calcium bentonite. This difference is due to the type of ion that is intercalated on the surface of the layers. The amount of Na+ needed for the compensation of the negative charge on the layers is double that needed for Ca2+ because the charge of Na+ is half that of Ca2+. In addition, based on the hydrated ionic radii of Na+ and Ca2+, the number of water molecules intercalated in the interlayer space during wetting in the presence of Na+ is higher than that in the presence of Ca2+.

The other type of surface, the edge surface, is characterized by the presence of hydroxyl groups called terminal OH groups. These hydroxyl groups carry a charge that is dependent on the type of metal ion to which they are bonded in the structure and on the pH of the solution. The charging arises from the adsorption or dissociation of protons. At low pH values, the excess protons in the aqueous medium create positive edge charges, whereas negative charges are produced by the dissociation of silica and alumina groups at high pH values [5, 6].

#### **1.2. Extraction sites**

**1. Bentonite**

382 Grape and Wine Biotechnology

**1.1. Chemical structure**

of (M+y・nH2O) (Al3+2-yMg2+y)Si4+4O10(OH)2.

order H–O < Si–O < Al–O < Mg–O < Li–O.

Bentonite is a phyllosilicate of the class of dioctahedral smectites [1] with a general composition

The structures of phyllosilicates are all based on tetrahedral T and octahedral O sheets arranged in either a 1:1 (kaolinite, dickite and nacrite) or 2:1 (smectites, vermiculite, mica and chlorite)

The layers are stacked on top of each other to form what is called a 'particle'. In turn, an assembly of particles is known as an 'aggregate'. Thus, a particle is formed by stacked layers that are separated from each other by an interlayer space. For a single layer, each tetrahedron consists of a cation (Si4+, Al3+ and Fe3+ are the most common) coordinated to four oxygen atoms and linked to an adjacent tetrahedra by sharing three basal oxygen atoms to form a twodimensional 'hexagonal' pattern. However, each octahedron consists of a cation (Al3+, Fe3+, Mg2+, or Fe2+) coordinated by six oxygen atoms and linked to a neighbouring octahedra by sharing edges. In the 2:1 structures, such as smectites, one octahedral sheet is sandwiched between two tetrahedral sheets. The surface properties of clay minerals depend on many factors, such as their chemical composition, nature of the surface atoms, extent and type of charge, and type of exchangeable cations. There are two types of surfaces with different properties, the planar surface and the edge surface, which is affected by pH. In a 2:1 clay, such as bentonite, the oxygen atoms of the Si tetrahedron represent the atoms of the basal surface. The charge on the oxygen atoms depends on the difference in electronegativity between oxygen and the other atoms involved in the bond. Thus, the ionicity of the bonds follows the

Thus, in smectites, the planar surfaces are negatively charged, with the magnitude of the layer charge ranging from 0.2 to 0.6 per half-unit cell. The type of metals that are bonded to the oxygen atoms governs the hydrophobicity of the surface. In the absence of isomorphous

ratio to form an anisotropic TO or TOT layer, respectively (**Figure 1**).

**Figure 1.** Structure of a phyllosilicate of the class of dioctahedral smectites [1] as bentonite is.

In 2009, the worldwide production of bentonite amounted to 9.66 Mt. Approximately 90% of the world production is concentrated in 13 countries, with 55% of the total production in Greece, the USA and the Commonwealth of Independent States. The different properties of bentonite reflect its different origins (47 countries contribute to the total production). Bentonite deposits were formed by diagenetic or hydrothermal alteration of volcanic glass and by authogenesis of smectite-rich materials in alkaline continental basins [7]. Dioctahedral smectites first become Fe3+-rich and then subsequently become Al3+-rich (montmorillonite) minerals formed in the absence of Mg2+, and the most important deposits are located in Wyoming, Montana, Arizona (USA) [8], India and Europe (Czech Republic, Denmark, Germany and Greece). Regarding the contents of Na+ and Ca2+, which are important for the swelling and rheological properties, there is high variability among the clays from different deposits in the same country. Generally, American bentonites are Na+ rich, while the European bentonites are Ca rich, with few exceptions.

#### **1.3. Industrial activation treatments**

To modify the properties of bentonite, industrial activation processes are performed on bentonites that do not present the optimal characteristics for the use for which they are designed. There are two types of modification processes, mineral activation and organic modification.

In the mineral activation process, wet mud is treated at 80°C in the presence of Na2CO3. During the process, Ca2+ ions are precipitated as CaCO3. Consequently, the Na/Ca ratio of the clay is modified by substituting the interlayer and surface Ca2+ ions for Na+ ions. The treatment improves the swelling ability of the slurries and enhances the adsorption of wine proteins [9]. Significant changes in the rheological characteristics (viscosity), pH, cationic exchange capacity (CEC), and structure of bentonite could also be produced after alkaline magnesium activation [10]. Another treatment that is used to improve the adsorbent performance of bentonite is the acid activation treatment with HCl and H2SO4, in which the main task is to increase the specific surface area (SSA) and the porosity by degrading the structure of the clay due to the leaching of Al3+, Fe2+ and Mg2+. In this case, the exchangeable cations are replaced by H+ . Organic modification is widely performed to improve the ability of the clay to remove heavy metals and organic compounds from water [11]; it reduces the total number of interlayer ions (Na, Ca, Mg) by replacing them with organic cations. Two categories of modified bentonite are produced, organoclays [12] and oxide pillared clays [13]. The main modifications induced by these processes are to the surface properties of the clays, which transition from hydrophilic to hydrophobic surfaces. This improves the ability of the modified bentonite to remove organic contaminants, such as phenols, from water [14].

## **2. Bentonite applications**

#### **2.1. Uses in non-food fields**

Based on its properties and its relatively low cost of production, bentonite use has attracted interest from researchers in several fields, particularly those in the industrial and environmental fields [15]. Due to its viscosity, thixotropy, plastering ability and plasticity, bentonite is used to grout cracks in rocks, for soil injections, as a thickener in paints and as an additive in ceramics [16]. To reduce gas release from landfill decomposition and to impede water percolation across the landfill, clay liners have been used as substitutes for the compacted soil components normally used [17]. In the context of disposal programmes, bentonite is projected to be used in engineered barriers for high-level waste and spent nuclear fuels [18]. A significant amount of the global bentonite production is destined to iron ore pelletizing, foundry moulding and oil-well drilling processes [19]. Bentonite is also used in animal feed, as a litter adsorbent and as an oil and grease adsorbent. Its importance in animal feed is derived from its ability to adsorb mycotoxins, such as aflatoxins [20]. Bentonite plays an important role in the development of health products [21], such as cosmetics and pharmaceuticals [22]. Moreover, it has been shown to be useful in different medical treatments [23]. Bentonite is also applied to the production of sunscreen lotions, as it acts as physical barriers against UV radiation [24] as well as a thickener in paste masks, eyeliners, nail lacquers, shampoos and toothpastes [25]. Other, less important applications include the production of additives for cement and mortar [26] and the cleaning of wastewaters. Recently, bentonites have been used for enzyme immobilization [27] and as bactericidal materials [28].

#### **2.2. Uses in syrup and juice production**

Greece, the USA and the Commonwealth of Independent States. The different properties of bentonite reflect its different origins (47 countries contribute to the total production). Bentonite deposits were formed by diagenetic or hydrothermal alteration of volcanic glass and by authogenesis of smectite-rich materials in alkaline continental basins [7]. Dioctahedral smectites first become Fe3+-rich and then subsequently become Al3+-rich (montmorillonite) minerals formed in the absence of Mg2+, and the most important deposits are located in Wyoming, Montana, Arizona (USA) [8], India and Europe (Czech Republic, Denmark,

swelling and rheological properties, there is high variability among the clays from different

To modify the properties of bentonite, industrial activation processes are performed on bentonites that do not present the optimal characteristics for the use for which they are designed. There are two types of modification processes, mineral activation and organic

In the mineral activation process, wet mud is treated at 80°C in the presence of Na2CO3. During the process, Ca2+ ions are precipitated as CaCO3. Consequently, the Na/Ca ratio of the clay is

improves the swelling ability of the slurries and enhances the adsorption of wine proteins [9]. Significant changes in the rheological characteristics (viscosity), pH, cationic exchange capacity (CEC), and structure of bentonite could also be produced after alkaline magnesium activation [10]. Another treatment that is used to improve the adsorbent performance of bentonite is the acid activation treatment with HCl and H2SO4, in which the main task is to increase the specific surface area (SSA) and the porosity by degrading the structure of the clay due to the leaching

modification is widely performed to improve the ability of the clay to remove heavy metals and organic compounds from water [11]; it reduces the total number of interlayer ions (Na, Ca, Mg) by replacing them with organic cations. Two categories of modified bentonite are produced, organoclays [12] and oxide pillared clays [13]. The main modifications induced by these processes are to the surface properties of the clays, which transition from hydrophilic to hydrophobic surfaces. This improves the ability of the modified bentonite to remove organic

Based on its properties and its relatively low cost of production, bentonite use has attracted interest from researchers in several fields, particularly those in the industrial and environmental fields [15]. Due to its viscosity, thixotropy, plastering ability and plasticity, bentonite is

of Al3+, Fe2+ and Mg2+. In this case, the exchangeable cations are replaced by H+

and Ca2+, which are important for the

rich, while the European

ions. The treatment

. Organic

Germany and Greece). Regarding the contents of Na+

bentonites are Ca rich, with few exceptions.

contaminants, such as phenols, from water [14].

**2. Bentonite applications**

**2.1. Uses in non-food fields**

**1.3. Industrial activation treatments**

modification.

384 Grape and Wine Biotechnology

deposits in the same country. Generally, American bentonites are Na+

modified by substituting the interlayer and surface Ca2+ ions for Na+

The application of bentonite's characteristics has been investigated in different juice and syrup production processes. Fruit juices are naturally cloudy due to the presence of polysaccharides (pectin, cellulose, hemicellulose, lignin and starch), proteins, tannins and metals [29]. Other molecules, such as flavonoids, polyphenols and organic acids, contribute to the dark colour of sugarcane and other juices. Because these components are present in varying amounts, clarification is one of the key steps in juice processing. The commonly used clarification methods include the use of enzymes, ultrafiltration, gelatine, bentonite, PVPP [30], lime, active carbon [31] and ion exchange resins [32]. Bentonite is cheap, does not pose environmental threats and represents one of the most suitable alternatives for the other methods. In particular, activated Na–Ca bentonite is the type of clay used in beverage technology [32]. It is used as the main clarifying agent or in preclarification steps [33]. In the clarifying step for concentrated must production, the basic treatment is based on the addition of bentonite and gelatine for various times of contact (usually 24 or 48 h). In fruit juice production, bentonite can be added to reduce the brown pigments resulting from enzymatic reactions: positive results concerning the browning index and the haze potential have also been attained for banana juice [34]. Finally, bentonite has been evaluated for use as a moisture-regulating adjuvant in food technology [35].

#### **3. Bentonite in oenology**

The addition of bentonite to musts and/or wines is aimed at reducing the protein contents, thus reducing the haze potential of the wine (fining). Fining is defined as the process of addition of substances that induce the precipitation of particles in suspension by promoting their sedimentation.

In oenology, bentonite could also be applied to the must to improve the precipitation of the suspended solids [36]. Clarification results in the variable reductions in the protein contents in the juice, but this aspect is not always directly related to the haze potential of the final wines because other wine components, such as phenolic compounds and non-proteinaceous colloids, and the storage temperature significantly affect the colloidal stability of the final wine [37].

Bentonite is mainly used on white wines, whereas in red wines, the aim of fining is softening, that is the removal of some of the tannins and polyphenols to improve the astringency of the product, and it is performed by applying fining agents other than bentonite (gelatine, albumin, isinglass, skim milk, casein and potassium caseinate) [38].

The main advantages of bentonite are its low cost, effectiveness and availability. Moreover, it is easily removed via sedimentation. Alternative treatments and adjuvants have been sought in oenology because of the drawbacks of bentonite, such as its direct and indirect costs. Indeed, the separation of the lees requires specific plants and is accompanied by the retention of variable quantities of wine (3–10%). The disposal of bentonite represents a limitation exacerbated by the fact that bentonite is not reusable [39], due to the lack of processes that promote the desorption of the adsorbed proteins.

Alternative treatments and adjuvants have been sought in oenology because of the drawbacks of bentonite, such as its direct and indirect costs. Indeed, the separation of the lees requires specific plants and is accompanied by the retention of variable quantities of wine (3–10%). Another concern regards the environmental impact of bentonite wastes, a problem that is exacerbated by the fact that bentonite is not reusable [39]: the enrichment of metals and important alterations of pH have been observed above certain levels of addition of bentonite in poor soils [40]. Even though the enrichment of organic matter by spent bentonite could be promising for poor soils and for applications in new vineyards, the disposal of big volumes bentonite should be, thus, carefully studied.

#### **3.1. International regulations on oenological use of bentonite**

According to the EU regulations, there is no upper limit for the addition of bentonite during the winemaking process. On the other hand, The International Oenological Codex [41] establishes the properties of the bentonites that are useful in oenology. The following three classes of bentonite are useful: calcium bentonite, sodium bentonite and activated bentonite, all of which are capable of swelling to different extents and must be dried at 80–90°C after grinding and before their commercialization.

According to the Resolution Oeno 11/2003 [42], the quality control of each bentonite should be based on 5 different test trials prior its application as an adjuvant. Mould contamination is not accepted, and as a result, no odour should be perceived. For the release of cations, the extraction trial should be performed in a tartaric acid solution (5 g/L, pH = 3). This recommendation seems to be insufficient to characterize the safety of a bentonite because the release of cations in wine has been shown to be more intense than extraction with tartaric acid [43].

The metals that are covered by the Resolution Oeno 11/2003 are Pb, Hg, As, Fe, Al, Ca, Mg and Na, with appropriate concentration limits for each metal. However, other metals can be part of the elemental composition of a bentonite, which include significant amounts of Li, Be, Sc, V, Co, Ni, Ga, Ge, As, Sr, Y, Zr, Nb, Mo, Cd, Sn, Sb, Ba, W, Tl and Bi [44]. The enrichment of cations from bentonite in wine partially results from the cation exchange mechanisms on which the adsorption of proteins is based [45] but also from the physicochemical conditions of the medium (pH, ethanol content and presence of other cations). Because the concentration of each cation in wine is important in the context of food safety and for the technological stability [46] and the release of metals from bentonite has been shown to be increased over the last few years [44], the control of the release phenomena has gained importance [45]. Other parameters that are controlled are the presence of large particles, the de‐acidification test, and the protein adsorption test [42].

#### **3.2. Physicochemical characteristics of oenological bentonite**

In oenology, bentonite could also be applied to the must to improve the precipitation of the suspended solids [36]. Clarification results in the variable reductions in the protein contents in the juice, but this aspect is not always directly related to the haze potential of the final wines because other wine components, such as phenolic compounds and non-proteinaceous colloids, and the storage temperature significantly affect the colloidal stability of the final wine [37].

Bentonite is mainly used on white wines, whereas in red wines, the aim of fining is softening, that is the removal of some of the tannins and polyphenols to improve the astringency of the product, and it is performed by applying fining agents other than bentonite (gelatine, albumin,

The main advantages of bentonite are its low cost, effectiveness and availability. Moreover, it is easily removed via sedimentation. Alternative treatments and adjuvants have been sought in oenology because of the drawbacks of bentonite, such as its direct and indirect costs. Indeed, the separation of the lees requires specific plants and is accompanied by the retention of variable quantities of wine (3–10%). The disposal of bentonite represents a limitation exacerbated by the fact that bentonite is not reusable [39], due to the lack of processes that promote

Alternative treatments and adjuvants have been sought in oenology because of the drawbacks of bentonite, such as its direct and indirect costs. Indeed, the separation of the lees requires specific plants and is accompanied by the retention of variable quantities of wine (3–10%). Another concern regards the environmental impact of bentonite wastes, a problem that is exacerbated by the fact that bentonite is not reusable [39]: the enrichment of metals and important alterations of pH have been observed above certain levels of addition of bentonite in poor soils [40]. Even though the enrichment of organic matter by spent bentonite could be promising for poor soils and for applications in new vineyards, the disposal of big volumes

According to the EU regulations, there is no upper limit for the addition of bentonite during the winemaking process. On the other hand, The International Oenological Codex [41] establishes the properties of the bentonites that are useful in oenology. The following three classes of bentonite are useful: calcium bentonite, sodium bentonite and activated bentonite, all of which are capable of swelling to different extents and must be dried at 80–90°C after

According to the Resolution Oeno 11/2003 [42], the quality control of each bentonite should be based on 5 different test trials prior its application as an adjuvant. Mould contamination is not accepted, and as a result, no odour should be perceived. For the release of cations, the extraction trial should be performed in a tartaric acid solution (5 g/L, pH = 3). This recommendation seems to be insufficient to characterize the safety of a bentonite because the release of cations in wine has been shown to be more intense than extraction with tartaric acid

isinglass, skim milk, casein and potassium caseinate) [38].

the desorption of the adsorbed proteins.

386 Grape and Wine Biotechnology

bentonite should be, thus, carefully studied.

grinding and before their commercialization.

[43].

**3.1. International regulations on oenological use of bentonite**

To optimize the performances of the fining of wines, the choice of the best bentonite is based on the characterization of its structural properties. The parameters that are considered are as follows:

The SCD is an expression of the substitution of ions into the bentonite structure, which was described in Section 1.1. Because the adsorption of proteins on bentonite is mainly driven by cation exchange [48], the higher the negative charge of the clay particles, the higher the removal of proteins, when all other parameters are equal. However, these data alone are not sufficient to describe the complete performance of a bentonite in wines. Although a correlation between the net charge of the bentonite and the removal of proteins in white wines was observed [49], other factors affect the ability of charged bentonite to remove positively charged colloids [50].

The SSA is an indirect measure of the medium‐sized bentonite particles and depends on the grinding process. The ratio between the SCD and the SSA results in the most significant parameter, the CDSU, and does not take into account the dose of the bentonite added to the wine and is only related to the surface of the clay. As shown for the SCD, the CDSU could sort the bentonites in the order of their charges.

Calcium bentonite is usually more acidic than an activated bentonite, which ranges from pH 6.5 to 8.5 and from pH 8.5 to 10.0, respectively, whereas natural sodium bentonite shows a quite wide range of pH values (4.5–10.0) [42]. The natural pH of a bentonite is strongly associated with its rheological properties, as the distribution of charges on the surface and along the edges of the of the platelets results in the organization of the platelets into a structure that confers viscosity to the medium. Currently, there is no global consensus on the type of structure formed, as some researchers [51] support the edge-to-face attraction mode, whereas other authors suggest the formation of a gel produced by long-range electrostatic double-layer repulsion both at the edges and at the faces [52]. The ionic strength and the pH should be considered when ascertaining the predominant type of interaction [53], particularly when the pH is lower than 4.5 [54], as in wine. Below pH 7, the edges of the platelets should be positively charged, allowing edge-to-face attractions, but very acidic pH values also increase the ionic strength of the medium [55].

Therefore, below pH 4.0, the acidic conditions could be so extreme that Al3+ and Mg2+ are dissolved and the clay structure is subsequently decomposed. In the same range of pH values, the high ionic strength of the medium could result in the compression of the double layers, thus reducing the edge-to-face attraction intensities [56]. The remaining card-house structure is thought to be an important property for the removal of proteins [45]. The capacity of the clay to maintain a residual three-dimensional structure is affected by the pH of the wine and by the natural pH of the bentonite.

## **4. Bentonite and wine colloids**

#### **4.1. Must and wine colloids**

The colloidal phenomena are articulated in two steps, which include the formation of small, invisible colloidal particles and their subsequent aggregation and formation of large particles that are able to diffuse light and to precipitate when the their size exceeds a certain limit.

In oenology [38], two groups of colloids may be distinguished.

In addition, polysaccharides could also function as protective colloids by coating the associated colloids and preventing their precipitation. The coating action that is typical of protecting colloids is exerted over a range of concentrations, below which a destabilizing effect takes over. The protective colloids are important in stabilizing different colloidal phenomena in wine, such as the precipitation of ferric phosphate and tartrate.

Colloids are also important for the quality properties required in specific products, such as the foamability of sparkling wines resulting from the presence of hydrophilic and hydrophobic domains in proteins that act as surfactants for the bubble film [57]. Further evidence was provided [58] in Prosecco wine, as high molecular weight glycocompounds showed a high capacity to foam.

Although there is some controversy about the degree of glycosylation of wine proteins [59], a variable fraction of the colloids from red wines, ranging from 4.3 to 5.2%, is composed of arabinogalactans [60], which have a protein content of <10%. These molecules are considered polysaccharides and are not enlisted in the class of wine proteins.

## **4.2. Must and wine proteins**

Calcium bentonite is usually more acidic than an activated bentonite, which ranges from pH 6.5 to 8.5 and from pH 8.5 to 10.0, respectively, whereas natural sodium bentonite shows a quite wide range of pH values (4.5–10.0) [42]. The natural pH of a bentonite is strongly associated with its rheological properties, as the distribution of charges on the surface and along the edges of the of the platelets results in the organization of the platelets into a structure that confers viscosity to the medium. Currently, there is no global consensus on the type of structure formed, as some researchers [51] support the edge-to-face attraction mode, whereas other authors suggest the formation of a gel produced by long-range electrostatic double-layer repulsion both at the edges and at the faces [52]. The ionic strength and the pH should be considered when ascertaining the predominant type of interaction [53], particularly when the pH is lower than 4.5 [54], as in wine. Below pH 7, the edges of the platelets should be positively charged, allowing edge-to-face attractions, but very acidic pH values also increase the ionic

Therefore, below pH 4.0, the acidic conditions could be so extreme that Al3+ and Mg2+ are dissolved and the clay structure is subsequently decomposed. In the same range of pH values, the high ionic strength of the medium could result in the compression of the double layers, thus reducing the edge-to-face attraction intensities [56]. The remaining card-house structure is thought to be an important property for the removal of proteins [45]. The capacity of the clay to maintain a residual three-dimensional structure is affected by the pH of the wine and by

The colloidal phenomena are articulated in two steps, which include the formation of small, invisible colloidal particles and their subsequent aggregation and formation of large particles that are able to diffuse light and to precipitate when the their size exceeds a certain limit.

**•** Associated colloids are charged particles composed of small molecules that are bound by weak bonds (Van der Waals, hydrogen bonds, hydrophobic interactions, etc.). This type of colloid is naturally present in wine as condensed phenols and colloidal colouring matter.

**•** Macromolecular colloids, such as polysaccharides and proteins, have high molecular masses

In addition, polysaccharides could also function as protective colloids by coating the associated colloids and preventing their precipitation. The coating action that is typical of protecting colloids is exerted over a range of concentrations, below which a destabilizing effect takes over. The protective colloids are important in stabilizing different colloidal phenomena in wine, such

strength of the medium [55].

388 Grape and Wine Biotechnology

the natural pH of the bentonite.

**4.1. Must and wine colloids**

and can form covalent bonds.

as the precipitation of ferric phosphate and tartrate.

**4. Bentonite and wine colloids**

In oenology [38], two groups of colloids may be distinguished.

Among the colloids that are present in musts and wines, proteins are the most important because they have a central role in haze formation.

Previous analyses have identified the most heat-unstable proteins in white wines, such as grape class IV chitinases, β-glucanases and a fraction of thaumatin-like (TL) proteins [61, 62]. These proteins are able to persist throughout the winemaking process because they are resistant to proteolysis and are stable at an acidic pH [63]. Conversely, yeast- and grape-derived highmolecular mass glycoproteins have been found to exert a stabilizing effect in white wines and are considered 'haze-protective factors' [39]. In recent years, several reports have attempted to characterize the factors affecting protein instability and haze formation in white wines. Because wines with different haze potentials typically contain very similar protein fractions [49], one or more non-proteinaceous wine components (sulphate anions, ionic strength, phenolic compounds, organic acids and pH values) are thought to have an impact on haze formation [39].

#### **4.3. Interactions of bentonite with grape and wine proteins**

The factors that can affect the capacity of a bentonite to bind proteins are its physical properties, the temperature, the pH and the ethanol content of the medium.

High temperature results in higher adsorption [48, 50], perhaps due to the conformational changes that occur in the proteins at low temperatures.

pH has a greater importance in the mechanisms of protein adsorption because it influences the charges of the proteins and, thus, the efficacy of the cation exchange onto bentonite [45, 49, 50].

The ethanol content of the wine influences the adsorption capacity of bentonite because ethanol molecules could displace water from the platelets and induce considerable swelling. In turn, increased swelling should increase the number of exchangeable cations, resulting in an increased adsorption capacity. Ethanol concentrations >10% in wine model solutions result in a considerable increase in the adsorption capacity [48]. However, the type of protein considered could influence this result because the separation of the clay platelets induced by ethanol molecules is finite. Under these conditions, the adsorption could only be attributed to those proteins that are small enough to enter the clay structure [64].

Various factors must be considered when the physical properties of bentonite are examined. CEC, SI and SSA are indeed directly correlated to the removal of proteins [50]. From a practical point of view, the choice of the correct contact time is important for effective fining operations. The general approach is to use a long period of contact time between the wine and bentonite, which is on the order of days [38]. In literature, the optimal contact time is reported to be shorter [48, 50]: a plateau trend is observed after a few minutes of contact between bentonite and wine, indicating that no further adsorption of proteins occurs.

The total protein content in wine is a useful, but it is not the definitive information that determines the risk of haze formation in a wine. Indeed, the types of proteins and their relative concentrations are more important than the total protein content [49].

An optimal performance by bentonite comprises the removal of those fractions that are heat unstable and responsible for the colloidal casse. Chitinases, β-glucanases and some TL protein isoforms are greatly affected by bentonite, but the more stable invertases require a very large amount of bentonite for removal [61, 65]. Extensive investigations [45, 49] on the effect of different bentonite labels on the removal of specific proteins in Chardonnay, Sauvignon and Erbaluce wines showed that bentonite could selectively remove specific protein bands. As a result, the resistance of the wine to haze formation after bentonite fining resulted in a distributed low haze up to 50°C and high cloudiness in the range of 60–80°C. When the distribution of the proteins was investigated with SDS-PAGE, a substantial but not total reduction in the concentration of the TL proteins was observed for all the fined wines. Small peptides were almost completely removed by all the labels tested, but some bentonites were able to remove some of the invertase bands.

The amount of protein removed by bentonite changes was affected by both the bentonite characteristics and the wine pH [45] over a typical oenological range (3.00–3.60). Low molecular mass proteins are efficiently removed by different Na-bentonites, regardless of the pH, but fewer high and medium molecular mass proteins are removed to different extents, based on the wine pH. Reductions in the amount of the vacuolar invertase (GIN1) and VVTL1 fractions of the TL-proteins are induced by bentonites with natural pH values of <10 and are affected, to a lesser extent, by the negative effect of acidic pH on the cardhouse-like structure. Moreover, at higher pH values, the poor removal of glycoproteins (YGP1 and Hmp1) contributed to the increased thermal stability of the wine. As a matter of fact, the modifications in the protein profile of a white wine in the pH range of 3.00–3.60 resulted in increased quantities of glycoproteins from grape and yeasts [66]. Minor pHdriven conformational changes are sufficient to weaken the interactions between the glycoproteins and the other wine macromolecules, especially tannins, resulting in increased release of hydrophobic glycoproteins. As a result, the temperature at which wine begins to show turbidity is higher.

#### **4.4. Interactions of bentonite with polysaccharides and tannins**

It has been shown that bentonite is capable of removing large phenolic compounds, such as anthocyanin, and phenolic compounds that are bound to proteins [67]. Consequently, bentonite may be responsible for modifying wine colour and astringency. The results of the fining of red wines [68] have shown that the high SCD of the bentonite could reduce the tannin levels to some extent, which is accompanied by a relatively high reduction in the anthocyanin levels. It is likely that the absorbance of positively charged anthocyanin induces a significant change in the properties of the bentonite surface such that tannins could be adsorbed via hydrogen bonds and π–π stacking. On the other hand, no effect of bentonites on the removal of tannins in red wines was observed [69], regardless of the molecular weight of the molecules, although the specific physical properties of the bentonite used were not reported in the study.

## **5. Bentonite and wine aroma compounds**

#### **5.1. Varietal odour-active compounds**

molecules is finite. Under these conditions, the adsorption could only be attributed to those

Various factors must be considered when the physical properties of bentonite are examined. CEC, SI and SSA are indeed directly correlated to the removal of proteins [50]. From a practical point of view, the choice of the correct contact time is important for effective fining operations. The general approach is to use a long period of contact time between the wine and bentonite, which is on the order of days [38]. In literature, the optimal contact time is reported to be shorter [48, 50]: a plateau trend is observed after a few minutes of contact between bentonite and wine, indicating that no further adsorption of proteins oc-

The total protein content in wine is a useful, but it is not the definitive information that determines the risk of haze formation in a wine. Indeed, the types of proteins and their relative

An optimal performance by bentonite comprises the removal of those fractions that are heat unstable and responsible for the colloidal casse. Chitinases, β-glucanases and some TL protein isoforms are greatly affected by bentonite, but the more stable invertases require a very large amount of bentonite for removal [61, 65]. Extensive investigations [45, 49] on the effect of different bentonite labels on the removal of specific proteins in Chardonnay, Sauvignon and Erbaluce wines showed that bentonite could selectively remove specific protein bands. As a result, the resistance of the wine to haze formation after bentonite fining resulted in a distributed low haze up to 50°C and high cloudiness in the range of 60–80°C. When the distribution of the proteins was investigated with SDS-PAGE, a substantial but not total reduction in the concentration of the TL proteins was observed for all the fined wines. Small peptides were almost completely removed by all the labels tested, but some bentonites were able to remove

The amount of protein removed by bentonite changes was affected by both the bentonite characteristics and the wine pH [45] over a typical oenological range (3.00–3.60). Low molecular mass proteins are efficiently removed by different Na-bentonites, regardless of the pH, but fewer high and medium molecular mass proteins are removed to different extents, based on the wine pH. Reductions in the amount of the vacuolar invertase (GIN1) and VVTL1 fractions of the TL-proteins are induced by bentonites with natural pH values of <10 and are affected, to a lesser extent, by the negative effect of acidic pH on the cardhouse-like structure. Moreover, at higher pH values, the poor removal of glycoproteins (YGP1 and Hmp1) contributed to the increased thermal stability of the wine. As a matter of fact, the modifications in the protein profile of a white wine in the pH range of 3.00–3.60 resulted in increased quantities of glycoproteins from grape and yeasts [66]. Minor pHdriven conformational changes are sufficient to weaken the interactions between the glycoproteins and the other wine macromolecules, especially tannins, resulting in increased release of hydrophobic glycoproteins. As a result, the temperature at which wine begins to

proteins that are small enough to enter the clay structure [64].

concentrations are more important than the total protein content [49].

curs.

390 Grape and Wine Biotechnology

some of the invertase bands.

show turbidity is higher.

Grape aroma has an important contribution to wine flavour and characteristics, particularly in the aromatic cultivars, and it evolves during berry development according to the cultivar. Terpenes, which represent the main family of compounds and are present both as free and as glycosylated terpenoids, are mainly present in the grape skin and are present in high amounts in Muscat grape varieties [70]. Variable concentrations of norisoprenoids, that is C-13 norisoprenoids, such as β-damascenone, can improve the fruity note of wines, even at low concentrations [71], and are responsible for the aroma characteristics of Cabernet Sauvignon [72] and other non-floral grapes [73]. In rare cases, methoxypyrazines and sulphur compounds with thiol functional groups have been identified among the aroma compounds of wines. Methoxypyrazines are often associated with 'green' or 'herbaceous' aromas in Cabernet Sauvignon, Sauvignon Blanc, Cabernet Franc and Merlot Noir, among other varieties [74].

#### **5.2. Fermentative odour-active compounds**

The aroma complexity dramatically increases during alcoholic fermentation as a result of the synthesis of important volatile compounds and the release of some varietal aroma precursors [75]. The nature and amount of the synthesized volatile compounds depend on multiple factors, such as the nitrogen content of the must, the temperature of fermentation and the yeast strain. They consist of compounds with a wide range of polarity, solubility and volatility. The volatile compounds synthesized by wine yeasts include higher alcohols, medium- and long-chain volatile acids, acetate esters and ethyl esters and aldehydes, among others [76]. Aroma compounds interact with different macromolecules such as proteins or polysaccharides [77], so fining agents may fix substances that act as support for aromatic components [78].

## **5.3. Impact of bentonite on varietal odour-active compounds**

Protein stability and the presence of intense fine aromas are two important requirements for aromatic white wines. Bentonite treatment is typically performed on wine, but it can be applied to increase the rate of juice settling and to facilitate the precipitation of suspended solids [36].

The use of fining agents to clarify juice can positively or negatively affect the composition of the derived must. The must clarification prior to the onset of alcoholic fermentation improves the sensory characteristics of white wines [79]. The removal of grape solids from must enhances the production of ethyl esters and acetates and limits the release of fusel alcohols during alcoholic fermentation, which results in a global increase in wine aroma quality [36]. Nevertheless, a certain amount of colloids must be present because they confer structure and volume, contribute to the fixation of aromatic compounds and produce bubbles in sparkling wines as a result of their tensioactive properties [64]. Conversely, excessive turbidity in musts induces the presence of an herbaceous aroma in the resulting wines, which promotes an evolution towards reduction. Finally, excessive limpidity can cause a retardation or cessation of the fermentation process [79].

A report [37] studied the fates of proteins and terpenols during the processing of Chambave Muscat grapes. The experiments included bentonite addition to must only (100 g hl), to wine only (100 g hl) and double bentonite addition on must and wine in two vintages (2006 and 2007). The results of the experiments demonstrated that a reduced removal of free terpenols was observed in the samples from the double treatment (must + wine) compared with the wines that were only fined with bentonite after alcoholic fermentation. In general [80], bentonite alone has a small effect on the loss of terpenes, but it removed ethyl esters and fatty acids.

Our unpublished data attempted to optimize the clarification of Muscat Blanc must with Ca or Na bentonite or related mixes in a dose range of 10–100 g hL−1 through a central composite design (CCD). Response surface methodology (RSM) estimated the combined effect of the bentonite type and dose on the removal of aglycones and glycosylated aroma compounds. The RSM surface plots predicted the removal of the aglycones from aroma compounds by low doses of Na-bentonite and Ca-bentonite, regardless of the dose. Furthermore, the model estimated the removal of glycosylated aroma compounds, particularly by Ca-bentonite at 50 g hL−1.

#### **5.4. Impact of bentonite on fermentative odour-active compounds**

The effects of the clarification/stabilization treatments on the sensory qualities and aroma [36, 78] of wine have been studied, but the origin of these phenomena has rarely been explained.

The effect of the dosing time of bentonite on the aroma profile at both industrial and pilot scales has been evaluated [81]. The results seemed to indicate that the addition of bentonite not only affects the wine aroma by the adsorption of compounds but also by the production of these compounds during fermentation. Bentonite treatments at different stages of fermentation generally affected the production of volatile fermentative compounds. In addition to the possible loss of volatile compounds due to adsorption on bentonite, the effects on the production of fermentative compounds could be related to the variations in the nitrogen composition and other nutrients of musts and wines that are removed by the bentonite [82]. In a model solution, the presence of total and purified proteins (TL proteins and chitinase) and bentonite tended to increase the loss of esters with the longest carbon chains that is ethyl octanoate and ethyl decanoate [80], showing that hydrophobicity can be one of the driving forces involved in the interaction of aroma compounds with both bentonite and proteins.

As a matter of fact, it was demonstrated [83] that the effect of bentonite treatments on the aroma substances in white wine depended on the chemical nature and initial concentration of the volatile compounds and on the abundance and nature of proteins in the wine. In general, when low bentonite concentrations (20 g/hl) are used, the concentrations of most aromatic substances are not significantly affected. Most aroma compounds are removed as an indirect effect of deproteinization; some hydrophilic odour-active compounds undergo weak hydrogen binding with protein surfaces, whereas the more hydrophobic aromatic molecules can bind to interior protein sites with a stronger affinity for hydrophobic substances. Only a few odouractive compounds are directly adsorbed by the bentonite through an adsorption process, which is robustly fitted by the Freundlich equation, with a heterogeneous energy distribution of an infinite number of surface-active sites [84]. Bentonites with a lower SSA value and a greater CDSU value seemed to primarily interact with most of the odour-active compounds through physical mechanisms. In contrast, the clay with a large SSA value and a low CDSU value promoted stronger adsorptions that were probably driven by chemical interactions, particularly for the ethyl esters. For the fermentative odour-active compounds, the differences in the adsorption intensity and capacity mainly depended on the characteristics of the bentonite than on the properties of the substances. When yeast-derived material represents an important fraction of the wine macromolecules, colloids that favour aroma inclusion are held in suspension. In this situation, there may be fewer opportunities for the odour-active substances to be directly adsorbed onto the bentonite sheets [83].

## **6. Conclusions**

**5.3. Impact of bentonite on varietal odour-active compounds**

fermentation process [79].

392 Grape and Wine Biotechnology

acids.

50 g hL−1.

Protein stability and the presence of intense fine aromas are two important requirements for aromatic white wines. Bentonite treatment is typically performed on wine, but it can be applied to increase the rate of juice settling and to facilitate the precipitation of suspended solids [36]. The use of fining agents to clarify juice can positively or negatively affect the composition of the derived must. The must clarification prior to the onset of alcoholic fermentation improves the sensory characteristics of white wines [79]. The removal of grape solids from must enhances the production of ethyl esters and acetates and limits the release of fusel alcohols during alcoholic fermentation, which results in a global increase in wine aroma quality [36]. Nevertheless, a certain amount of colloids must be present because they confer structure and volume, contribute to the fixation of aromatic compounds and produce bubbles in sparkling wines as a result of their tensioactive properties [64]. Conversely, excessive turbidity in musts induces the presence of an herbaceous aroma in the resulting wines, which promotes an evolution towards reduction. Finally, excessive limpidity can cause a retardation or cessation of the

A report [37] studied the fates of proteins and terpenols during the processing of Chambave Muscat grapes. The experiments included bentonite addition to must only (100 g hl), to wine only (100 g hl) and double bentonite addition on must and wine in two vintages (2006 and 2007). The results of the experiments demonstrated that a reduced removal of free terpenols was observed in the samples from the double treatment (must + wine) compared with the wines that were only fined with bentonite after alcoholic fermentation. In general [80], bentonite alone has a small effect on the loss of terpenes, but it removed ethyl esters and fatty

Our unpublished data attempted to optimize the clarification of Muscat Blanc must with Ca or Na bentonite or related mixes in a dose range of 10–100 g hL−1 through a central composite design (CCD). Response surface methodology (RSM) estimated the combined effect of the bentonite type and dose on the removal of aglycones and glycosylated aroma compounds. The RSM surface plots predicted the removal of the aglycones from aroma compounds by low doses of Na-bentonite and Ca-bentonite, regardless of the dose. Furthermore, the model estimated the removal of glycosylated aroma compounds, particularly by Ca-bentonite at

The effects of the clarification/stabilization treatments on the sensory qualities and aroma [36, 78] of wine have been studied, but the origin of these phenomena has rarely been explained. The effect of the dosing time of bentonite on the aroma profile at both industrial and pilot scales has been evaluated [81]. The results seemed to indicate that the addition of bentonite not only affects the wine aroma by the adsorption of compounds but also by the production of these compounds during fermentation. Bentonite treatments at different stages of fermentation generally affected the production of volatile fermentative compounds. In addition to the possible loss of volatile compounds due to adsorption on bentonite, the effects

**5.4. Impact of bentonite on fermentative odour-active compounds**

Oenologists do not really know which parameters they must focus on to choose the bentonite that will obtain both the desired degree of limpidity and colloidal stability while avoiding undesirable side effects on the phenolic and aroma compounds.

This chapter detailed the structure, composition and non-food uses of bentonite and summarized the most recent scientific research that detected:

As wine haze formation is a product of different matrix parameters, including the wine pH and the concentration of different wine components, a detailed knowledge of the relationships between the adjuvant that is most commonly used to attain colloidal stability and the oenological matter is fundamental for process optimization and to increase wine resilience.

## **Author details**

Milena Lambri\* , Donato Colangelo, Roberta Dordoni, Fabrizio Torchio and Dante Marco De Faveri

\*Address all correspondence to<EMAIL_ADDRESS>
Institute of Enology and Agro-food Engineering, Università Cattolica del Sacro Cuore, Via Emilia Parmense, Piacenza, Italy

## **References**

Developments in Palygorskite- Sepiolite research. A new outlook on these nanomaterials. Developments in Clay Science. 2011;3:125–173.

[8] Grim RE, Güven N. Bentonites, Geology, Mineralogy, Properties and Uses, Development in Sedimentology. Vol. 24. Amsterdam: Elsevier; 1978.

**•** the interactions of bentonite with the free- and glycosylated-varietal aroma compounds; **•** the interactions of bentonite with fermentative aroma by indirect and direct removal.

As wine haze formation is a product of different matrix parameters, including the wine pH and the concentration of different wine components, a detailed knowledge of the relationships between the adjuvant that is most commonly used to attain colloidal stability and the oenological matter is fundamental for process optimization and to increase wine resilience.

, Donato Colangelo, Roberta Dordoni, Fabrizio Torchio and

Institute of Enology and Agro-food Engineering, Università Cattolica del Sacro Cuore, Via

[1] Bergaya B, Theng KG, Lagaly G, editors. Handbook of Clay Science, Developments in

[2] Mortier WJ. Electronegativity equalization and its applications. Structure and Bonding.

[3] Feller D, Glendening ED, Woon DE, Feyereisen MW. An extended basis-set ab-initio study of alkali-metal cation-water clusters. Journal of Chemical Physics. 1995;103:3526–

[4] Bergaya F, Jaber M, Lambert JF. Clays and clay minerals as layered nanofillers for (bio) polymers. In Averous L, Pollet E, editors. Environmental Silicates Nano-Biocomposites,

[5] Tournassat C, Ferrage E, Poinsignon C, Charlet L. The titration of clay minerals. II. Structure-based model and implications for clay reactivity. Journal of Colloid and

[6] Tournassat C, Greneche JM, Tisserant D, Charlet L. The titration of clay minerals. I. Discontinuous backtitration technique combined with CEC measurements. Journal of

[7] Galán R, Pozo, M. Palygorskite and sepiolite deposits in continental environments. Description, genetic patterns and sedimentary settings. In Galán E, Singer, A. editors.

Green Energy and Technology. London: Springer-Verlag; 2012. p. 41–75.

Clay Science. Vol. 1. Amsterdam: Elsevier; 2006. p. 1–1224.

**Author details**

394 Grape and Wine Biotechnology

Dante Marco De Faveri

Emilia Parmense, Piacenza, Italy

1987;66:125–143.

Interface Science. 2004;273:234–246.

Colloid and Interface Science. 2004;273:224–233.

3542.

\*Address all correspondence to<EMAIL_ADDRESS>
Milena Lambri\*

**References**

dietary aflatoxins on broilers reared to market weight. British Poultry Science. 2014;55:215–220.

[34] Lee WC, Yusol S, Hamid NSA, Baharin BS. Effects of fining treatment and storage temperature on the quality of clarified banana juice. LWT – Food Science and Technology. 2007;40:1755–1764.

dietary aflatoxins on broilers reared to market weight. British Poultry Science.

[21] Viseras C, Cerezo P, Sanchez R, Salcedo I, Aguzzi C. Current challenges in clay minerals

[22] Allo Wanda A, Haydn HM. Mineralogy, chemistry and potential applications of a white bentonite in San Juan province, Argentina. Applied Clay Science. 2004;25(3):237–243.

[23] Iliescu RI, Andronescu E, Voicu G, Ficai A, Covaliu CI. Hybrid materials based on montmorillonite and citostatic drugs: preparation and characterization. Applied Clay

[24] López-Galindo A, Viseras C. Pharmaceutical and cosmetic applications of clays. In Wypych F, Satyanarayana KG, editors. Clay Surfaces: Fundamentals and Applications.

[25] Murray HH. Bentonite applications. In Murray HH, editor. Developments in Clay

[26] Krøyer H, Lindgreen H, Jakobsen HJ, Skibsted J. Hydration of Portland cement in the presence of clay minerals studied by 29Si and 27Al MAS NMR spectroscopy. Advances

[27] Wicklein B, Darder M, Aranda P, Ruiz-Hitzky E. Bio-organoclays based on phospholipids as immobilization hosts for biological species. Langmuir. 2010;26:5217–5225. [28] Miyoshi H, Ohno H, Sakai K, Okamura N, Kourai H. Characterization and photochemical and antibacterial properties of highly stable silver nanoparticles prepared on montmorillonite clay in n-hexanol. Journal of Colloid and Interface Science.

[29] Vaillant F, Millan A, Dornier M, Decloux M, Reynes M. Strategy for economical optimisation of the clarification of pulpy fruit juices using crossflow microfiltration.

[30] Li ZX, Wang GM, Liang Q. Preparation and properties of the novel adsorbent agent for

[31] Laksameethanasan P, Somla N, Janprem S, Phochuen N. Clarification of sugarcane juice

[32] Jahed E, Khodaparast MHH, Khaneghah AM. Bentonite, temperature and pH effects on purification indexes of raw sugar beet juice to production of inverted liquid sugar.

[33] Novotny CJ. Ion exchange. In Chen JCP, editor. Cane Sugar Handbook. New York:

juices clarification. Advanced Materials Research. 2013;791–793:248–251.

for syrup production. Procedia Engineering. 2012;32:141–147.

for drug delivery. Applied Clay Science. 2010;48:291–295.

2014;55:215–220.

396 Grape and Wine Biotechnology

Science. 2011:52:62–68.

2010;345:433–441.

Amsterdam: Elsevier; 2004. p. 267–289.

in Cement Research. 2003;15:103–112.

Journal of Food Engineering. 2001;48:83–90.

Applied Clay Science. 2014;102:155–163.

Wiley; 1985. p. 591–598.

Science. Amsterdam: Elsevier; 2006. p. 111–130.

[59] Waters EJ, Wallace W, Tate ME Williams PJ. Isolation and partial characterization of a natural haze protective factor from wine. Journal of Agricultural and Food Chemistry. 1993;41:724–730.

[46] Catarino S, Curvelo-Gacia AS, Bruno de Sousa R. Contaminant elements in wine

[47] Ferrarini R, Celotti E, Zironi R. Importance of the surface electric charges of enological adjuvant, of particles, and of colloids in must and wine. French Review of Enology.

[48] Blade WH, Boulton R. Adsorption of protein by bentonite in a model wine solution.

[49] Lambri M, Dordoni R, Giribaldi M, Violetta MR, Giuffrida MG. Heat-unstable protein removal by different bentonite labels in white wines. LWT – Food Science and Tech-

[50] Xifang SUN, Chun LI, Zhansheng WU, Xiaolin XU, Ling REN, Hongsheng ZHAO. Adsorption of protein from model wine solution by different bentonites. Chinese

[51] Van Olphen H. Internal mutual flocculation in clay suspensions. Journal of Colloid

[52] Callaghan IC, Ottewill RH. Interparticle forces in montmorillonite gels. Faraday

[53] Rand B, Pekenć E, Goodwin JW, Smith RW. Investigation into the existence of edge face coagulated structures in Na-montmorillonite suspensions. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases. 1980;76:225–

[54] Khandal RK, Tadros TF. Application of viscoelastic measurements to the investigation of the swelling of sodium montmorillonite suspensions. Journal of Colloid and

[55] Heath D, Tadros TF. Influence of pH, electrolyte, and poly (vinyl alcohol) addition on the rheological characteristics of aqueous dispersions of sodium montmorillonite.

[56] Benna M, Kbir-Ariguib N, Magnin A, Bergaya F. Effect of pH on rheological properties of purified sodium bentonite suspensions. Journal of Colloid and Interface Science.

[57] Andrés-Lacueva C, Gallart M, López-Tamames E, Lamuela-Raventós RM. Influence of variety and aging on foaming properties of sparkling wine (Cava). Journal of Agricul-

[58] Vincenzi S, Crapisi A, Curioni A. Foamability of prosecco wine: cooperative effects of high molecular weight glycocompounds and wine PR-proteins. Food Hydrocolloids.

(review). Vine and Wine Science and Technique. 2008;23:3–19.

American Journal of Enology and Viticulture. 1988;39:193–199.

Journal of Chemical Engineering. 2007;15:632–638.

Discussions of the Chemical Society. 1974;57:110–118.

Journal of Colloid and Interface Science. 1983;93:307–319.

tural and Food Chemistry. 1996;44:3826–3829.

1996;36:5–10.

398 Grape and Wine Biotechnology

nology. 2012;46:460–467.

Science. 1964;19:313–322.

Interface Science. 1988;125:122–128.

1999;218:442–455.

2014;34:202–207.

235.

#### **The Trends and Prospects of Winemaking in Poland The Trends and Prospects of Winemaking in Poland**

[73] Pineau B, Barbe JC, Van Leeuwen C, Dubourdieu D. Which impact for β-damascenone on red wines aroma? Journal of Agricultural and Food Chemistry. 2007;55:4103–4108.

[74] Koch A, Doyle CL, Matthews MA, Williams LE, Ebeler SE. 2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype. Phytochemistry. 2010;71:2190–

[75] Swiegers JH, Bartowksy EJ, Henschke PA, Pretorius IS. Yeast and bacterial modulation of wine aroma and flavour. Australian Journal of Grape and Wine Research.

[76] Molina AM, Swiegers JH, Varela C, Pretorius IS, Agostin E. Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds.

[77] Guichard E. Flavour retention and release from protein solutions. Biotechnology

[78] Lubbers S, Voilley A, Charpentier C, Feuillat M. Mise en Evidences of interactions between wine macromolecules and aroma. French Review of Enology. 1993;144:12–18.

[79] Armada L, Falqué E. Repercussion of the clarification treatment agents before the alcoholic fermentation on volatile composition of white wines. European Food

[80] Vincenzi S, Panighel A, Gazzola D, Flamini R, Curioni A. Study of combined effect of proteins and bentonite fining on the wine aroma loss. Journal of Agricultural and Food

[81] Lira E, Salazar FN, Rodriguez-Bencomo JJ, Vincenzi S, Curioni A, Lopez F. Effect of using bentonite during fermentation on protein stabilisation and sensory properties of white wine. International Journal of Food Science and Technology. 2014;49:1070–1078.

[82] Lira E, Rodríguez-Bencomo JJ, Salazar FN, Orriols I, Fornos D, López F. Impact of bentonite additions during vinification on protein stability and volatile compounds of Albariño wines. Journal of Agricultural and Food Chemistry. 2015;63:3004–3011. [83] Lambri M, Dordoni R, Silva A, De Faveri DM. Effect of bentonite fining on odor active compounds in two different white wine styles. American Journal of Enology and

[84] Lambri M, Dordoni R, Silva A, De Faveri DM. Odor-active compound adsorption onto bentonite in a model white wine solution. Chemical Engineering Transactions (CET).

Applied Microbiology and Biotechnology. 2007;77:675–687.

2198.

400 Grape and Wine Biotechnology

2005;11:127–138.

Advances. 2006;24:226–229.

Chemistry. 2015;63:2314–2320.

Viticulture. 2010;61:225–233.

2013;32:1741–1746.

Research and Technology. 2007;225:553–558.

Alina Kunicka-Styczyńska, Agata Czyżowska, Katarzyna Rajkowska, Agnieszka Wilkowska and Piotr Dziugan Alina Kunicka-Styczyńska, Agata Czyżowska, Katarzyna Rajkowska, Agnieszka Wilkowska and Piotr Dziugan

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64976

#### **Abstract**

Viticulture and winery origins in Poland date to the tenth century, but their tradition has been reborn in the last ten years, resulting in a development of small vineyards producing excellent wines not only for the local market. Due to the cold climate, usually short summers with moderate and low temperatures, the grapes are characterized by lower sugar content and higher acidity compared to those grown in the south of Europe. According to the European Union regulations, Poland was classified as the coldest wine-growing region (A) and officially acknowledged as a wine-producing country. The grapevine cultivars adopted to the harsh climatic conditions give the Polish grape wines some unique sensory features. The most popular varieties of grapes for the production of red wine are Regent, Rondo, Pinot Noir, Maréchal Foch, Cabernet Cortis, Tryumf Alzacji, Cascade and Dornfelder. For white wine production, Solaris, Riesling, Seyval Blanc, Pinot Gris, Johanniter, Jutrzenka, Hibernal, Aurora, Bianka, Traminer, Jutrzenka and Siberia are mostly used in Poland. This chapter presents Polish grape winery with its specificity and prospects for the future. The traditional products of Polish fermentation industry, fruit wines and meads, are also mentioned.

**Keywords:** Polish wines, wine yeast, L-malic acid decomposition, Polish wine regions

## **1. Introduction**

However, Poland was not worldwide recognized as a wine-producing country, but the art of winemaking has been practiced there since the tenth century. Winery was introduced with Christianity and the first vineyards were cultivated by and wineries were established by Benedictine and Cistercian monks; however, wine was produced for religious purposes mainly.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The fruitful time for the Polish winery was the fourteenth century, during which many wineries were operating mainly in Silesia, Zielona Góra, Poznań, Toruń, Płock, Sandomierz, Lublin and Kraków.

Intensive development of wine making was in the age of enlightenment, when the viticulture and wine production were carried out in the Podole. Besides *Vitis vinifera*, hybrid varieties resistant to adverse climatic conditions were grown. After World War II, according to the authorities, two wine-growing regions were designated: the West (Zielona Góra region and Lower Silesia) and the Central (along the Pilica river). Vineyards planted in the communistic economy, however, have begun to bear losses, and in the 1960s, it was focused on the production of fruit wines. The tradition of viticulture and winery has been reborn in the last ten years, resulting in development of small vineyards producing excellent wines for the local market. Poland is located in the zone of the continental climate, where there are also wine regions such as Burgundy and the Loire Valley, Rioja, Piedmont and most of the vineyards of Austria, the Czech Republic, Slovakia or Romania. The climate in Poland is characterized by significant seasonal and daily fluctuations in temperature with the potential problems of frost and hailstorms during winters and springs. Due to the cold climate, usually short summers with moderate and low temperatures, the grapes are characterized by lower sugars content and higher acidity comparing to these grown in the south of Europe. A wine zone, most suitable for the cultivation of vines, is located on the latitude between 32°00′ and 52°00′ northern hemisphere and between 28°00′ and 42°00′ southern hemisphere [1].

Poland extends from the parallel 49°00′ N (south) to 54°50′ N (north), so about half of the country is situated in this area. At the latitudes between 49°00′ N and 52°00′ N, there are many regions in Europe known for their excellent wines, including some appellations in the region of Champagne (e.g. Reims) or German appellations the Rhine, mozelskie and Franconian. Krakow is located similarly [2]. Climate favourable for viticulture is characterized by an average annual temperature not less than 8°C, the average temperature of the hottest month not less than 17°C and the total active annual temperature 25°C. Some areas of Poland are also characterized by these conditions. The current climate changes are conducive to the development of Polish winemaking. The average annual temperature showed an upward trend (about 0.3°C per decade), transitional periods have been shortened, warm periods have been prolonged, the course of winters became milder, allowing the cultivation of early and very early varieties.

According to the European Union classification of climate for viticulture (Council Regulation (EC) No 479/2008), Poland was classified in the coldest wine-growing region (A) and officially acknowledged as a wine-producing country, altogether with, among others, Germany (except for Baden), the Czech Republic (except for Moravia), Belgium and the Great Britain.

Due to the thermal conditions, Polish territory was divided as follow: Region I—the west and southwest of the country, namely provinces of Lubusz, Lower Silesia, Opole, Silesian and southern parts of the provinces Wielkopolska and Lodz; Region II—threatened with greater extent of cold winters, covers the south and southeast of the country, i.e. the province Małopolska, Podkarpackie, świętokrzyskie and southern parts of the provinces of Lublin and Warsaw; and Region III—the other areas, where viticulture is impossible or very difficult [3]. Nowadays, the following winemaking regions can be distinguished in Poland; the largest and the best known vineyards are located in: (i) area of Zielona Góra (Lubuskie Province); (ii) Lower Silesia; (iii) vicinity of Kraków; (iv) area of Jasło and Krosno in the Podkarpackie Province; and (v) along the Vistula gorge (from Sandomierz to Puławy).

## **2. Wine yeasts**

The fruitful time for the Polish winery was the fourteenth century, during which many wineries were operating mainly in Silesia, Zielona Góra, Poznań, Toruń, Płock, Sandomierz, Lublin and

Intensive development of wine making was in the age of enlightenment, when the viticulture and wine production were carried out in the Podole. Besides *Vitis vinifera*, hybrid varieties resistant to adverse climatic conditions were grown. After World War II, according to the authorities, two wine-growing regions were designated: the West (Zielona Góra region and Lower Silesia) and the Central (along the Pilica river). Vineyards planted in the communistic economy, however, have begun to bear losses, and in the 1960s, it was focused on the production of fruit wines. The tradition of viticulture and winery has been reborn in the last ten years, resulting in development of small vineyards producing excellent wines for the local market. Poland is located in the zone of the continental climate, where there are also wine regions such as Burgundy and the Loire Valley, Rioja, Piedmont and most of the vineyards of Austria, the Czech Republic, Slovakia or Romania. The climate in Poland is characterized by significant seasonal and daily fluctuations in temperature with the potential problems of frost and hailstorms during winters and springs. Due to the cold climate, usually short summers with moderate and low temperatures, the grapes are characterized by lower sugars content and higher acidity comparing to these grown in the south of Europe. A wine zone, most suitable for the cultivation of vines, is located on the latitude between 32°00′ and 52°00′ northern

Poland extends from the parallel 49°00′ N (south) to 54°50′ N (north), so about half of the country is situated in this area. At the latitudes between 49°00′ N and 52°00′ N, there are many regions in Europe known for their excellent wines, including some appellations in the region of Champagne (e.g. Reims) or German appellations the Rhine, mozelskie and Franconian. Krakow is located similarly [2]. Climate favourable for viticulture is characterized by an average annual temperature not less than 8°C, the average temperature of the hottest month not less than 17°C and the total active annual temperature 25°C. Some areas of Poland are also characterized by these conditions. The current climate changes are conducive to the development of Polish winemaking. The average annual temperature showed an upward trend (about 0.3°C per decade), transitional periods have been shortened, warm periods have been prolonged, the course of winters became milder, allowing the cultivation of early and very early

According to the European Union classification of climate for viticulture (Council Regulation (EC) No 479/2008), Poland was classified in the coldest wine-growing region (A) and officially acknowledged as a wine-producing country, altogether with, among others, Germany (except

Due to the thermal conditions, Polish territory was divided as follow: Region I—the west and southwest of the country, namely provinces of Lubusz, Lower Silesia, Opole, Silesian and southern parts of the provinces Wielkopolska and Lodz; Region II—threatened with greater extent of cold winters, covers the south and southeast of the country, i.e. the province Małopolska, Podkarpackie, świętokrzyskie and southern parts of the provinces of Lublin and Warsaw; and Region III—the other areas, where viticulture is impossible or very difficult [3].

for Baden), the Czech Republic (except for Moravia), Belgium and the Great Britain.

hemisphere and between 28°00′ and 42°00′ southern hemisphere [1].

Kraków.

402 Grape and Wine Biotechnology

varieties.

Fermentation in winemaking can either be natural, conducted by native yeasts present on the fruit skins and in the winery, or by selected yeasts strains. The microflora of fruits vary according to a number of factors, among others, the fruit variety, temperature, climatic conditions, soil, viticultural practices and fungicides applied to vineyards [4]. In view of this variability, to obtain high-quality wines of defined aroma and flavour, the inoculation with pure cultures of selected commercial yeasts is recommended. In addition, starter culture fermentations offer the advantages of a more predictable and rapid process, giving wines with high consistency in quality. Criteria for the selection and development of yeasts for wine fermentation encompass: (i) vigorous and complete fermentation of fruit juice sugars, without excessive yeast growth; (ii) fermentation at low temperatures 10–12°C; (iii) production of ethanol in high concentrations and ethanol tolerance of yeasts; (iv) growth and fermentation in musts containing sulphur dioxide; (v) uniform dispersion and mixing throughout the fermenting juice; (vi) low foaming ability; (vii) effective sedimentation at the end of the fermentation; (viii) balanced array of flavour metabolites; and (ix) genetic stability [4–6].

Moreover, for areas with unfavourable climatic conditions including Poland, the high acidity of fruit wines caused inter alia by the excess of malic and tartaric acids in the raw material is specific. In terms of fruits used in the Polish winemaking industry, both organic acids account for up to 90% of all the acids present in grapes, and malic acid content reaches 94–98% of total acids present in apples and cherries [4, 7]. The high level of organic acids unfavourably influences the organoleptic properties and biochemical, physical and microbial stability of wine. One of the strategies for solving this problem is biological deacidification by wine yeasts with an extended ability to degrade L-malic acid. Demalication process is conducted simultaneously with fermentation. Malate decomposition varies greatly and may reach 48%, depending on the strain. However, industrial wine yeasts *Saccharomyces cerevisiae* and *Saccharomyces bayanus*, with a demalication activity reaching 68%, have also been selected and well-characterized [8, 9]. *Saccharomyces* sensu stricto strains are usually considered as less effective in malic acid metabolizing than *Schizosaccharomyces pombe* and *Zygosaccharomyces bailii*, which are additionally characterized by high resistance to acidity and sulphur dioxide [10, 11]. The rather low malate decomposition in *Saccharomyces* spp. is explained by the absence of an active L-malate transport system, the low substrate affinity of the malic enzyme and the mitochondrial location of two malic isoenzymes [12]. The alternative in reduction of the acidity of musts and wines may be the application of mixed cultures of yeasts, composed of species degrading L-malate and L-tartrate, such as *Schizosaccharomyces malidevorans, Kloeckera* spp., *Candida* spp. and *Hansenula* spp. [13]. However, inoculation of musts with yeasts other than *S. cerevisiae* may be associated with the possible interactions between populations and adverse organoleptic changes in the final product. To avoid these limitations and combine desired biochemical and technological properties of yeasts, interspecific hybrids between *S. cerevisiae* and *S. pombe*, and *S. cerevisiae* and *S. bayanus* were obtained using the protoplast fusion method [6, 9, 11]. The hybrids show an increase in degradation of L-malate compared to *S. pombe* and are able to decompose up to 77% of malic acid present in musts [14]. Moreover, hybrids ability to decompose L-malic acid seems to be linked to their high resistance to acidic stress [7, 14]. The application of natural yeast hybrids is fully acceptable by the industry in contrast to yeast engineered by molecular genetic methods.

Besides the search for new yeasts and modification of traditional wine-yeast strains, another important direction in Polish winery involves the immobilization of yeasts. The immobilization of yeast cells in winemaking practices offers many advantages: (i) prolonged activity and stability of the biocatalyst; (ii) high volumetric productivity; (iii) shorter fermentation time; (iv) elimination of non-productive cell growth phases; (v) increased substrate uptake and yield improvement; (vi) feasibility of continuous processing; (vii) increased tolerance to high substrate concentration and reduced end product inhibition; and (viii) reduction of risk of contamination by undesired microorganisms [15].

In winemaking, yeasts immobilization has been used in continuous and batch production of wine, in a secondary fermentation, and to improve the sensory quality of finished products [15, 16]. Bonin et al. [17] and Bonin and Wzorek [18] tested immobilization of *S. cerevisiae* and *S. bayanus* on foam glass for long-term continuous winemaking of high-sugar musts attaining substantial improvement in efficiency of the process.

Immobilization of yeasts other than *Saccharomyces* spp. may improve sensory quality of wine compared to that obtained with free cells. It was reported that immobilized *Kluyveromyces marxianus*, cultivated on typical fruit pomaces from Polish varieties of apples, cranberries and chokeberries left over from juice extraction, produced significant quantities of aromas δdecalactone and rose-like 2-phenyl ethyl alcohol [16].

Wines are considered to be the products rich in phenolic compounds. The phenolic wine constituents play an important role in the visual and gustative quality of red wines [19]. It is also known that wine yeasts are one of the factors decreasing the phenolic content of wines. The mechanism of this phenomenon is based on weak and reversible interactions mainly between anthocyanins and yeast walls by absorption. On the other hand, various yeast metabolites such as pyruvic acid and acetaldehyde were shown to react with different classes of phenolics, suggesting that it may be an important way of conversion into stable pigments during the maturation and ageing of wine [20]. Due to the impact of wine yeasts on the polyphenols content, studies are undertaken in order to select wine yeast strains that may advantageously modify phenolic profile and antioxidant properties of wine [21].

## **3. Grape winemaking in Poland**

Currently, there are approx. 500 vineyards in Poland, ranging from small ones (up to 10 acres) to large ones (4 hectares and more). The most popular varieties of grapes for the production of red wine are Regent, Rondo, Pinot Noir, Maréchal Foch, Cabernet Cortis, Tryumf Alzacji, Cascade, Dornfelder. For white wine production Solaris, Riesling, Seyval Blanc, Pinot Gris, Johanniter, Jutrzenka, Hibernal, Aurora, Bianka, Traminer, Jutrzenka, Siberia are mostly used [2, 22]. Distribution of the most popular varieties is unequal. In the four provinces with the largest number of vineyards, i.e. Podkarpackie, Małopolskie, Lublin and Świętokrzyskie, the hybrid varieties: Regent, Rondo, Seyval Blanc, Bianca, Johanniter and Solaris dominate. In the western provinces, due to the slightly higher temperatures, mostly grape vine (*V. vinifera*): Lubuskie—Zweigelt, Pinot Noir and Riesling and in Lower Silesia—Cabernet Sauvignon, Pinot Noir, Riesling, Chardonnay and Pinot Gris are grown [2].

organoleptic changes in the final product. To avoid these limitations and combine desired biochemical and technological properties of yeasts, interspecific hybrids between *S. cerevisiae* and *S. pombe*, and *S. cerevisiae* and *S. bayanus* were obtained using the protoplast fusion method [6, 9, 11]. The hybrids show an increase in degradation of L-malate compared to *S. pombe* and are able to decompose up to 77% of malic acid present in musts [14]. Moreover, hybrids ability to decompose L-malic acid seems to be linked to their high resistance to acidic stress [7, 14]. The application of natural yeast hybrids is fully acceptable by the industry in contrast to yeast

Besides the search for new yeasts and modification of traditional wine-yeast strains, another important direction in Polish winery involves the immobilization of yeasts. The immobilization of yeast cells in winemaking practices offers many advantages: (i) prolonged activity and stability of the biocatalyst; (ii) high volumetric productivity; (iii) shorter fermentation time; (iv) elimination of non-productive cell growth phases; (v) increased substrate uptake and yield improvement; (vi) feasibility of continuous processing; (vii) increased tolerance to high substrate concentration and reduced end product inhibition; and (viii) reduction of risk of

In winemaking, yeasts immobilization has been used in continuous and batch production of wine, in a secondary fermentation, and to improve the sensory quality of finished products [15, 16]. Bonin et al. [17] and Bonin and Wzorek [18] tested immobilization of *S. cerevisiae* and *S. bayanus* on foam glass for long-term continuous winemaking of high-sugar musts attaining

Immobilization of yeasts other than *Saccharomyces* spp. may improve sensory quality of wine compared to that obtained with free cells. It was reported that immobilized *Kluyveromyces marxianus*, cultivated on typical fruit pomaces from Polish varieties of apples, cranberries and chokeberries left over from juice extraction, produced significant quantities of aromas δ-

Wines are considered to be the products rich in phenolic compounds. The phenolic wine constituents play an important role in the visual and gustative quality of red wines [19]. It is also known that wine yeasts are one of the factors decreasing the phenolic content of wines. The mechanism of this phenomenon is based on weak and reversible interactions mainly between anthocyanins and yeast walls by absorption. On the other hand, various yeast metabolites such as pyruvic acid and acetaldehyde were shown to react with different classes of phenolics, suggesting that it may be an important way of conversion into stable pigments during the maturation and ageing of wine [20]. Due to the impact of wine yeasts on the polyphenols content, studies are undertaken in order to select wine yeast strains that may

Currently, there are approx. 500 vineyards in Poland, ranging from small ones (up to 10 acres) to large ones (4 hectares and more). The most popular varieties of grapes for the production

advantageously modify phenolic profile and antioxidant properties of wine [21].

engineered by molecular genetic methods.

404 Grape and Wine Biotechnology

contamination by undesired microorganisms [15].

substantial improvement in efficiency of the process.

decalactone and rose-like 2-phenyl ethyl alcohol [16].

**3. Grape winemaking in Poland**

As the climatic conditions in Poland are still too severe for *V. vinifera* vines, most of the planted vineyards are composed of hybrids between *V. vinifera* and native North America species (French hybrids), with higher resistance to cold and pests. For wine produced from such cultivars, both the taste and chemical compound content are different when compared to the ones made of *V. vinifera* varieties. The only Seyval Blanc, from older varieties, is suitable for cultivation in Poland [3] because of its high fertility, resistance to frost and diseases. The newest Bianca and Siberia varieties are comparable to Seyval Blanc in mould disease resistance. Muscat of Odessa and Hibernal varieties are the leaders among grapevines for white wine production. Among varieties grown for red wine the best are German Rondo and Ukrainian Wiszniowyj Rannij.

In Poland, the researches on the usefulness of multiple genotypes of vine grown in cool climate have been carried out for many years, with main objective to evaluate the yield, tolerance/ resistance to frost and susceptibility to disease-causing pathogens. The work conducted in 1987–1989 and 2005–2007 shown that plants assessed later, earlier began the growing season [23]. In 2012, technological maturity of grapes from a vineyard Srebrna Góra (Małopolska) near Krakow was examined [24]. It turned out that the fruit reached maturity about two weeks earlier than expected and technological parameters of must were similar to the parameters of musts obtained in a warmer climate.

The outstanding grapevine collection of the Research Institute of Pomology and Floriculture in Skierniewice, Central Poland, was established in 1992 and now comprises 234 cultivars. The investigations conducted in 2005–2009 [25] were focusing on the yielding, winter hardiness and susceptibility to fungal diseases (downy mildew, powdery mildew, grey mould and excoriose) of 25 selected cultivars: 14 white cultivars (e.g. Aurore, Bianca, Cayuga White, Reform, Refren, Seyval, Siberia, V 64035, V 71141), 9 red (e.g. Golubok, Cascade, Rondo, Marechal Foch, Regent, Leon Millot), and 2 rose (Delaware, Swenson Red). Berries ripened from the second half of August (Reform) until the second week of October (V 71141, Siberia). Vines of the hybrid V 64035 and cultivars Seyval and Cayuga White were the most productive. Vines of interspecific hybrids were less susceptible to frost damage and fungal diseases than cultivars of *V. vinifera* (Chasselas Dore, Ortega). Interspecific hybrids Seyval, Bianca, Siberia, Marechal Foch, Rondo and Regent were distinguished as the best yielding and the highest quality, suitable for commercial winemaking. Aurore, Delaware, Cascade and Golubok were relatively reliable in yielding, and their grapes may be used for the production of juice and home wines. The following cultivars of *V. vinifera* proved to be the most suitable for cultivation in Central Poland: Auxerrois, Pinot Gris, Pinot Noir, Riesling and Chasselas Blanc, classified as both wine and table cultivars [26].