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. 2017 Mar 29;54(6):1555–1564. doi: 10.1007/s13197-017-2587-7

Screening of enzymatic activities within different enological non-Saccharomyces yeasts

Rocío Escribano 1, Lucía González-Arenzana 1, Patrocinio Garijo 1, Carmen Berlanas 1, Isabel López-Alfaro 1, Rosa López 1, Ana Rosa Gutiérrez 1,✉, Pilar Santamaría 1
PMCID: PMC5430188  PMID: 28559615

Abstract

Ninety-seven non-Saccharomyces wine yeast strains belonging to ten different genera and species (Candida spp. and Criptococcus spp.; Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri, Sporidiobolus salmonicolor, Torulaspora delbrueckii, Williopsis pratensis and Zygosaccharomyces bailii) were screened for 13 enzymes related to wine aroma, color and clarity. Understanding the yeasts’ influence in these wine characteristics provides a platform for selecting strains for their development as starter cultures and for the management of alcoholic fermentation. Most of the strains showed the presence of one or more enzymes of biotechnological interest. Our screening demonstrated several intraspecific differences within the yeast species investigated, indicating that strain selection is of great importance for their enological application, and also that some non-Saccharomyces that have not been thoroughly explored, may deserve further consideration. This research represents the first stage for selecting non-Saccharomyces strains to be used as a starter along with Saccharomyces cerevisiae to enhance some particular characteristics of wines.

Keywords: Enzymes, Winemaking, Non-Saccharomyces, Aroma, Color

Introduction

During the last decade, yeast species other than Saccharomyces cerevisiae have been proposed for winemaking as they could positively impact on wine quality by acting in mixed fermentation with S. cerevisiae. Traditionally, yeasts have been selected for their fermentative power and low acetic acid production, but nowadays new selection criteria are being used to obtain inocula that improve the technological properties and sensorial features of wines. Nowadays it is becoming ever more important to select yeasts that are good for each kind of wine, region and even microclimate (Suárez-Lepe and Morata 2012).

Knowledge about non-Saccharomyces yeasts has greatly increased over the last few years, which has led to some of them being used commercially as starters (T. delbrueckii, M. pulcherrima, P. kluyveri, L. thermotolerans) (Masneuf-Pomarede et al. 2015), while several other species are being subjected to studies to assess their effects on wine quality (Bely et al. 2013; Maturano et al. 2015). However, there are other species that remain unconsidered, although they have been repeatedly isolated from winemaking-related ecosystems. With a better understanding of the properties of yeasts, selection procedures could be adapted to obtain strains that could improve wine quality (e.g. enhancing positive aroma compounds and reducing those with negative impact, improving flavor and clarity, modulating color, etc.). Non-Saccharomyces wine yeasts could provide different desired characteristics (Jolly et al. 2014). High fermentation efficiency, high sulfite tolerance and killer activity for example, might not be needed with the new technology of wine production, (many consumers prefer more natural and healthier wines with less quantity of additives and low alcohol degree).

Enzymes play an important role in winemaking. They come from grapes, microorganisms and can also be added from commercial preparations. They help to address problems related to fining, filtration and increased aroma and color in wines (Merín and Morata de Ambrosini 2015; Belda et al. 2016b). In a selection process of microorganisms involved in the winemaking process, it is important to analyze the potential of strains for producing extracellular enzymes in order to change the composition of musts and for enhancing the sensory attributes of the wines (Van Resburg and Pretorius 2000). Maturano et al. (2012) determined the ability of different non-Saccharomyces yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases and xylanases) during fermentation, and observed that although non-Saccharomyces populations were not detected at the end of vinification, their secreted enzymes remained in the fermenting media. The secretion of each enzyme is not characteristic of a particular genus or species, but depends on the yeast strain analyzed (Ganga and Martínez 2004). It is therefore interesting to characterize every isolate so as to determine its potential as an enzyme producer. Selection within each genera or species could help the wine industry to develop the biotechnological use of non-conventional yeasts to improve the quality and differentiation of wines.

The aim of this study was the enzymatic characterization of yeast clones belonging to different non-Saccharomyces genera, as the first step in selecting wild strains with beneficial technological properties which can be potentially used as starters in the production of different wines.

Materials and methods

Yeast isolates

Ninety-seven non-Saccharomyces wine yeast strains belonging to 10 different genera taken from ICVV (Instituto de Ciencias de la Vid y el Vino) collections were used. These strains have been previously identified as being present in different ecosystems of the Rioja Qualified Designation of Origin (D.O.Ca. Rioja) wineries, such as on grapes, during fermentation, in wine, winery facilities and in the air (Ocón et al. 2010a, b, 2013). The wine yeast strains belonged to species used commercially as starters (Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri and Torulaspora delbrueckii), and to species that still remain unconsidered and that have been isolated in D.O.Ca. Rioja enological ecosystems (Candida spp. and Criptococcus spp. genera, Debaryomyces hansenii, Sporidiobolus salmonicolor, Williopsis pratensis and Zygosaccharomyces bailii). Criptococcus spp. and Sporidiobolus salmonicolor were isolated in the air of all the wineries where this medium was studied. However, Williopsis pratensis was detected only in the air of the bottling area of one winery during two consecutive years. Debaryomyces hansenii and Candida spp. were isolated in most of the studied wineries, the first one in winery air and facilities, and the second one in alcoholic fermentations and facilities. Finally, Zygosaccharomyces bailii has been detected in approximately 15% of vinifications studied by our research group during alcoholic fermentation. The study of enzymatic activities was carried out with the yeast grown for 48 h at 24 °C on a GYP medium (20 g/L dextrose, 5 g/L yeast extract, 5 g/L peptone and 20 g/L agar).

Enzymatic characterization

Enzymes related to aroma

Sulfite reductase (SR) activity

The H2S-production potential of the yeasts was determined by plating them onto a solid juice indicator agar (250 mL grape juice, 15 mL succinate pH 5.1, 735 mL distilled water, 11 g/L bismuth citrate and 30 g/L agar) (Strauss et al. 2001). After 24–48 h of incubation at 30 °C, a low H2S-producing colony was identified by its white color whereas a high H2S producing colony was characterized by a black color.

Hydroxycinnamic acid decarboxylase (HCDC) activity

Decarboxylation of ferulic and p-coumaric acids by yeasts was determined following the protocol described by Viana et al. (2008). The detection of the activity was performed using YPD plates containing 0.01% (w/v) bromocresol purple (Sigma Aldrich) supplemented with 0.145% (w/v) p-coumaric acid (Sigma Aldrich). Aliquots (10 µL) of cell extracts prepared in a 10 mM phosphate buffer pH 7 (0.869 g/L K2HPO4, 0.519 g/L KH2PO4) from 24-h must cultures were laid on the surface of the plates and incubated for 24 h at 37 °C. Hydroxycinnamic acid decarboxylase activity can be detected by a color shift from yellow to purple as a result of a pH increase due to the decarboxylation of the hydroxycinnamic acid which leads to an alkalization of the sample environment.

API-ZYM

Seven enzymatic activities were measured using an API-ZYM test system (BioMérieux, France), according to the manufacturer’s instructions (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase and ß-glucosidase). API-ZYM is a semi-quantitative test system used for screening 19 different enzyme activities. Yeast cultures were previously grown on YPD agar (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract and 20 g/agar) at 30 °C for 24 h. Subsequently, the cultures were suspended in distilled water until suspensions reached 5–6 McFarland turbidity. Suspensions were inoculated in the microwells on the API-ZYM strip at a level of 65 µL for each cupule. The strips were incubated at 37 °C for 4–5 h. Later ZYM A and ZYM B reagents were added to each cupule. Enzyme activity was measured comparing the color produced with the APY-ZYM color reaction chart.

Enzymes related to color and fining

Pectinase activity

The yeast replica were plated onto a polygalacturonate agar medium (12.5 g/L polygalacturonic acid, 6.8 g/L potassium phosphate (pH 3.5), 6.7 g/L Yeast Nitrogen Base (YNB, Sigma), 10 g/L glucose and 20 g/L agar). The plates were incubated at 30 °C for five days. The colonies were rinsed off with distilled water before staining the plates with 0.1% ruthenium red. Colonies showing a purple halo were identified as positive activity.

Cellulase activity

Cellulase production was determined by plating the yeast replica onto YPD plates containing 0.4% de carboxymethilcellulose (CMC, Sigma). The plates were incubated for five days at 30 °C. The colonies were rinsed off with distilled water before staining with 0.03% congo red, followed by destaining with 1 M NaCl. Cellulase activity was determined as positive when a clear halo appeared around the colony.

Xylanase activity

Yeast replica were screened for hemicellulase activity by plating onto SC plates (6.7 g/L YNB, 20 g/L agar) containing 0.2% Remazol Brilliant Blue Xylan (RBB-Xylan, Sigma). The plates were incubated for five days at 30 °C. The colonies were washed off the plates with distilled water. Colonies showing activity were identified by a clear zone around the colony.

Glucanase activity

Production of β-glucanase activity was determined by replica plating the yeast onto YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 20 g/L agar) containing 0.2% Yeast Beta Glucan (Megazyme). The plates were incubated for five days at 30 °C. Colonies were rinsed off with distilled water before staining the plates with 0.03% congo red (Merck) for 15 min. Yeasts showing a clear halo around the colonies were considered as glucanase positive.

Results and discussion

Screening of enzymatic activities

Enzymes involved in off-flavors

The volatile sulfur compounds in wines come mainly from the metabolism of yeast and have a significant role in the flavor of wines associated with the reduction character (Mendes-Ferreira et al. 2001; Shinohara et al. 2000). This production has been related to the presence of sulfite reductase activity in yeasts. In this work, the SR activity was highly present in T. delbrueckii (100%) and M. pulcherrrima (94%) (Table 1). In two other yeasts (Cryptococcus spp. and D. hansenii) SR activity was present to a lesser extent (20 and 33% respectively). The six remaining species did not show this activity (Candida spp., L. thermotolerans P. kluyveri, S. salmonicolor, and W. pratensis). Comitini et al. (2011) found that 100% of T. delbrueckii and M. pulcherrima tested (nine and seven strains respectively) were SR positive. However, unlike in our study, they also found that 100% of L. thermotolerans and Candida spp. (five and thirteen strains respectively) were also positive for SR. These differences could be explained by the low number of strains analyzed in both studies, or as a strain-related characteristic (strains isolated in different zones).

Table 1.

Production of extracellular enzymes

Yeast Species N Enzymatic activities
SR HCDC Esterase Esterase-Lipase Lipase LeucineA ValineA CystineA ß-glucosidase Pectinase Cellulase Xylanase Glucanase
Candida spp. 12 0 0 83 100 0 100 75 0 25 50 0 0 0
Cryptococcus spp. 10 20 0 100 100 0 100 0 0 60 60 60 0 60
Debaryomyces hansenii 5 33 0 67 67 0 0 0 0 0 100 33 0 67
Lachancea thermotolerans 3 0 0 94 100 12 100 94 6 6 12 75 62 81
Metchnikowia pulcherrima 6 94 50 100 100 0 94 44 0 63 69 37 0 6
Pichia kluyveri 4 0 0 100 100 0 100 0 0 0 100 0 0 0
Sporodiobulus salmonicolor 7 0 0 100 100 33 100 100 0 0 67 0 0 0
Torulaspora delbrueckii 18 100 0 56 56 0 67 0 6 0 83 11 0 0
Williopsis pratensis 16 0 0 100 100 0 100 100 90 0 0 0 0 0
Zygosaccharomyces bailii 16 43 0 71 86 0 100 0 0 14 71 29 0 0
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Numbers indicate the percentage of isolates with positive activity into each species

N, number of isolates studied; SR, sulfite reductase; HCDC, hydroxycinnamic acid decarboxylase

Vinyl- and ethylphenols can produce phenolic off-flavors in wine (Suárez et al. 2007; Wedral et al. 2010). The ability of wine yeasts to decarboxylate ferulic and p-coumaric acids by HCDC activity is related to the production of phenolic off-flavors in winemaking. Ethylphenol producers are yeasts belonging to the genus Brettanomyces/Dekkera, while the synthesis of vinylphenols varied among yeast species. Hence, the importance of controlling phenolic off-flavor production by using wine yeast strains with low HCDC productivity. In our study, only 50% of M. pulcherrima strains were HCDC positive (Table 1), whereas the rest of the genera and species did not show this activity. Shinohara et al. (2000) observed that 78–83% of non-Saccharomyces yeasts studied (23 strains) produced phenolic off-flavors in wine fermentation. These yeasts belonged to the Rhodotorula, Candida, Cryptococcus, Pichia, Hansenula and Brettanomyces genera, whereas in the T. delbrueckii, Z. bailii and M. pulcherrima species the productivity was absent. Differences in Candida, Cryptococcus and M. pulcherrima were found in comparison to our work. Those differences could be explained, as in the SR activity, by the number of strains studied, or as a strain-related characteristic (strains coming from different locations). The fact that only 50% of M. pulcherrima strains studied showed production of HCDC suggested that this is not characteristic of a particular genus or species, but depends on the yeast strain, as Ganga and Martínez (2004) had already pointed out. Results found in this research were quite good for a selection process, because most of the strains studied were unable to release HCDC activity.

Enzymes related with aroma

All the enzymes shown in Table 1 are involved in the final aroma of wines: the group of esterases, esterases-lipases and lipases, the group of aminopeptidases (leucine arylamidase, valine arylamidase and cystine arylamidase), β-glucosidase and the group of carbohydrolases (pectinase, cellulase, xylanase, glucanase). Besides, HCDC and SR are also aroma-related enzymes, although with negative effects.

Lipases contribute to an increase in the concentrations of free fatty acids from grapes or microorganisms, and esterases catalyze the synthesis of esters that contribute to the secondary aroma of wines. Esterase and esterase-lipase activities were present in all the species studied (Table 1), even in some of them in all the strains. 100% of the strains of Cryptococcus spp. genera and W. pratensis, S. salmonicolor, P. kluyveri and M. pulcherrima species showed esterase activity, and in the remaining ones this activity was variable (from 56% in T. delbrueckii to 94% in L. thermotolerans). Something similar happened with esterase-lipase activity which was positive for 100% of Candida spp., Cryptococcus spp., W. pratensis, S. salmonicolor, P. kluyveri, M. pulcherrima and L. thermotolerans. In the three remaining species (Debaryomyces hansenii, Torulaspora delbrueckii and Zygosaccharomyces bailii) more than 50% of strains displayed this activity. Viana et al. (2008) also revealed the potential of some non-Saccharomyces to produce acetate esters, specifically for the Hanseniaspora and Pichia genera. However, lipase activity was scarcely detected and only 33% of S. salmonicolor strains and 12% of L. thermotolerans showed this activity. Charoenchai et al. (1997) revealed that several wine yeasts (Candida stellata, Candida pulcherrima, Candida krusei and T. delbrueckii) had the potential to show extracellular lipolytic activity, degrading lipids and releasing free fatty acids. The liberation of medium chain fatty acids, such as decanoic or octanoic acids, could inhibit the growth of Saccharomyces cerevisiae and malolactic bacteria, leading to stuck fermentations. In contrast, in the current study no lipase activity was found for Candida spp. studied, or T. delbrueckii, which would result positive by preventing fermentation depletions.

The proteolytic enzymes of arylamidases or aminopeptidases (A) catalyze the hydrolysis of N-terminal amino acids from peptides. The increase in the amino acids content enhances the nutrient content in must and the production of aroma compounds in wine (Trinh et al. 2010). Some amino acids such as valine, leucine, isoleucine and phenylalanine (Santamaría et al. 2015), are known for being precursors of active aroma compounds produced by the yeasts. These enzymatic activities are also involved in the reduction of the proteic instability in wines (Dizy and Bisson 2000). In relation to these groups of enzymes it was found that D. hansenii did not show any activity, while W. pratensis was positive for the three activities in most of the strains (100% for leucine, 100% for valine and 90% for cystine). L. thermotolerans strains also produced the three activities but with lower percentages of positives (100, 94 and 6% respectively). In the remaining species’ strains, one or two activities could be found (One in Criptococcus spp., P. kluyveri and Z. bailii and two in Candida spp., M. pulcherrima, S. salmonicolor and T. delbrueckii). Other authors (Charoenchai et al. 1997; Strauss et al. 2001) found Kloeckera apiculata, M. pulcherrima, Pichia anomala, C. stellata and also D. hansenii strains with protease activity, and Ganga and Martínez (2004) showed that this activity was present in a greater percentage of isolates from M. pulcherrima (78,4%) and Candida spp. (56,7%). The methodologies employed to determine the proteolytic activity in the previous works differed from the present methods, which could explain the different results obtained in the current study. Belda et al. (2016a), detected absence of proteolytic activity in the majority of S. cerevisiae strains and highlighted the potential of Wickerhamomyces anomalus, M. pulcherrima and Kluyveromyces marxianus as proteolytic enzyme producers. Within the group of three enzymes included in proteolytic activity or aminopeptidases in this work, the most frequent one was leucine arylamidase, present in nine species with a high percentage (average of 96% from those nine species, with the minimum percentage found being 67% in T. delbrueckii), and the lowest cystine arylamidase, which was only detected in three species. The only species in which this last activity was detected to a great extent was W. pratensis.

Terpenes are a type of varietal compound that contributes to improve wine aroma. Most of them are bound to sugar molecules to form odorless compounds. β-glucosidase breaks them down to release free terpenes, thus enhancing the wine aroma. The β-glucosidase activity was absent in half of the studied species, and its presence was only significant in Cryptococcus spp. and M. pulcherrima (60% and 63% respectively). β-glucosidase activity was detected in other species, although in low ratios (25% in Candida spp., 14% in Z. bailii and 6% in L. thermotolerans). Mendes Ferreira et al. (2001) indicated that Kloeckera apiculata and M. pulcherrima were potential candidates for releasing terpenes due to their high β-glucosidase activity. Moreover, Rossouw and Bauer (2016) found isolates of non-conventional yeasts (Cryptococcus spp. and Candida spp.) producing high amounts of terpenes. In the current research, the strains of Cryptococcus spp., M. pulcherrima and Candida spp. were the ones that most displayed this enzymatic activity. Other studies have shown that species from the genera Debaryomyces, Kloeckera, Kluyveromyces, Metschnikowia, Dekkera, Hansenula, Hanseniaspora species and Zygosaccharomyces genera (Ganga and Martínez 2004) and even Pichia (Charoenchai et al. 1997) displayed this activity. However, Strauss et al. (2001) did not find β-glucosidase activity in 245 isolates of K. apiculata, D. hansenii and Candida spp. analyzed, and Fernández et al. (2000) indicated that the enzyme β-glucosidase was mainly linked to M. pulcherrima species. These latter results were in agreement with those described in this report.

The glycosidically bound terpenols are located mainly in the grape skins. So, enzymatic degradation of the cell wall by carbohydrolases (pectinase, cellulase, xylanase and glucanase) contributes to the release of those precursors from the berries to the must. It has been proposed that the cell wall degradation of the grapes allows many aromatic or potential aromatic compounds to form part of the must, resulting in a better aromatic profile (Pérez-González et al. 1993). Our results showed that the only non-Saccharomyces species where the four carbohydrolases activities were present was L. thermotolerans, and this species was the only one showing xylanase activity. On the other hand, within the W. pratensis no strain showed activities in this group. Interestingly, within Cryptococcus spp. and D. hansenii, both non-conventional yeasts in fermentation processes, a high percentage of the tested strains exhibited three activities which could be relevant in a selection process in order to improve the aromatic profile of wines.

Enzymes related to color and fining

Some of the enzymes, such as pectinase, cellulose, xylanase and glucanase, related to aroma were also directly involved in color and in fining. Indeed, the degradation of the structural polysaccharides by these carbohydrolases can result in higher juice extraction, improvement in fining and filterability during winemaking, increasing the extraction of substances that enhance color and aroma during maceration. The rise in the extraction of phenolic compounds led to the formation of more polymeric pigments in aged red wine, resulting in an increase in color intensity and stability. Louw et al. (2006) carried out vinifications with a recombinant Saccharomyces strain able to degrade glucan and xylan, which resulted in significant increases of free-run wine, significant differences in color intensity, color stability and volatile composition. In the screening of pectinase, cellulose, xylanase and glucanase activities that have been carried out in 10 different species, differences among them were found. Pectinase was the most widespread activity among the non-Saccharomyces yeasts studied in this work, with nine out of ten species having it, and in most cases, with high percentages (average of 68% from those nine species). Besides, this enzyme was the only one from this group of enzymatic activities present in Candida spp., S. salmonicolor and P. kluyveri. Some of these carbohydrolases have been detected in different genera (Kluyveromyces, Metschnikowia, Candida, Dekkera, Torulaspora, Zygosacharomyces, Pichia and Hanseniaspora) (Ganga and Martínez, 2004; Manzanares et al. 1999). Belda et al. (2015) demonstrated the impact of one M. pulcherrima strain producer of pectinolytic enzymes on wine traits: color, anthocyanin and polyphenol content of wines increased when that strain was used in mixed fermentations.

On another note, some metabolites produced by yeasts during fermentation may condense with grape anthocyanins to produce highly stable adducts such as vitisines A and B. So, the yeast strains used in vinification have a substantial influence on the formation of stable pigments and, therefore proper yeast selection is important to ensure the stability of the wine coloring matter (Morata et al. 2016). Yeasts with HCDC activity can also be used to decarboxylate hydroxycinnamic acids and form vinylphenols, that condense with grape anthocyanins to produce vinylphenolic pyroanthocyanin adducts, molecules that show great color stability. Benito et al. (2011) showed that during mixed fermentations using non-Saccharomyces with high HCDC activity, the content of stable pigments increased without sensorial modifications. Besides this, the content of p-coumaric acid was also reduced, and the formation of 4-ethylphenol (off-flavor) caused by massive Dekkera/Brettanomyces contamination in the wines was also minimized (Benito et al. 2009). Taking into account this theory, non-Saccharomyces yeasts having HCDC activity could increase the color stability and avoid the accumulation of vinylphenols in wines. In our study, strains with HCDC activity were only found for M. pulcherrima. So, displaying HCDC activity could be positive if the purpose of the yeast selection were to improve the red wine color due to the accumulation of vinylphenol adducts, and negative if the objective were to improve the aroma of the wine because of the risk of ethylphenol synthesis by Dekkera/Brettanomyces. Something similar happens with β-glucosidase activity, which is a desired enzymatic activity for aroma improvement in wines, but its presence could be harmful for color in red wines due to the degradation of anthocyanins (Suárez-Lepe and Morata, 2015). Manzanares et al. (2000) indicated that some non-Saccharomyces included in Dekkera, Rhodotorula and Schizosaccharomyces genera were not able to produce β-glucosidase. In all the species studied in this research, there were strains that did not show this activity (meaning that β-glucosidase activity is a strain-dependent trait), which could be chosen for inocula in red vinifications. In fact, its presence was significant only in Cryptococcus spp. and M. pulcherrima (60 and 63% respectively).

Proteolytic enzymes hydrolyze proteins improving fining of musts and wines and could reduce the proteic instability in wines (Dizy and Bisson 2000). As previously indicated, D. hansenii did not show any of the three aminopeptidases assayed in this study, and in W. pratensis and L. thermotolerans we found strains with the three activities. The most frequent proteolytic enzyme detected was leucine arylamidase which was present in nine species in a high percentage (>67%).

Enzymatic profiles and preselection within species

All the strains studied showed several enzymes of biotechnological interest, but only in L. thermotolerans were all the enzymatic activities with positive influence in wine quality found. However, it was quite complicated for all of the activities to converge in the same strain. So, because the secretion of each enzyme depends on the yeast strain analyzed (Ganga and Martínez 2004), the strains have been grouped using their enzymatic profiles (Tables 2, 3) in order to choose the best strains within each species according to their final use (white or red wines), or the characteristic we want to modulate (aroma or color).

Table 2.

Specific enzymatic profiles related to wine aroma

Species/profiles Enzymatic activities N %
Esterase Esterase-Lipase Lipase LeucineA ValineA CystineA ß-glucosidase HCDC SR
Candida spp.
CA 1 + + − + + − − − − 5 42
CA 2 + + − + − − − − − 3 25
CA3 − + − + + − − − − 1 8
CA 4 − + − + + − + − − 1 8
CA 5 + + − + + − + − − 2 17
Cryptococcus spp.
CCA 1 + + − + − − + − − 2 40
CCA 2 + + − + − − + − + 1 20
CCA 3 + + − + − − − − − 2 40
Debaryomyces hansenii
DA 1 − − − − − − − − − 1 33
DA 2 + + − − − − − − − 1 33
DA 3 + + − − − − − − + 1 33
Lachancea thermotolerans
LA 1 + + − + + − − − − 11 70
LA 2 + + − + − − − − − 1 6
LA 3 − + − + + − − − − 1 6
LA 4 + + + + + − − − − 1 6
LA 5 + + + + + − + − − 1 6
LA 6 + + − + + + − − − 1 6
Metchnikowia pulcherrima
MA 1 + + − + − − − + + 5 32
MA 2 + + − + − − + + + 2 12
MA 3 + + − − + − − − − 1 6
MA 4 + + − + + − + − + 5 32
MA 5 + + − + − − + − + 2 12
MA 6 + + − + + − + + + 1 6
Pichia kluyveri
PKA 1 + + − + − − − − − 4 100
Sporodiobulus salmonicolor
SPA 1 + + − + + − − − − 4 67
SPA 2 + + + + + − − − − 2 33
Torulaspora delbrueckii
TA 1 + + − − − − − − + 1 6
TA 2 − − − − − − − − + 5 28
TA 3 + + − + − − − − + 8 44
TA 4 − − − + − − − − + 3 16
TA 5 + + − + − + − − + 1 6
Williopsis pratensis
WA 1 + + − + + + − − − 9 90
WA 2 + + − + + − − − − 1 10
Zygosaccharomyces bailii
ZA 1 + + − + − − − − + 1 14
ZA 2 + − − + − − − − + 1 14
ZA 3 + + − + − − + − + 1 14
ZA 4 − + − + − − − − − 2 29
ZA 5 + + − + − − − − − 2 29
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Presence (+) or absence (−) of each activity; SR, sulfite eductase activity; HCDC, hydroxycinnamic acid decarboxylase activity; N, number of isolates showing each profile; %, percentage of each profile within the species

Table 3.

Specific enzymatic profiles related to color and fining

Species/profiles Enzymatic activities N %
Pectinase Cellulase Xilanase Glucanase LeucineA ValineA CystineA HCDC
Candida spp.
CC 1 + − − − + + − − 3 25
CC 2 + − − − + − − − 3 25
CC 3 − − − − + + − − 6 50
Cryptococcus spp.
CCC 1 − + − + + − − − 2 40
CCC 2 + + − + + − − − 1 20
CCC 3 ++ − − − + − − − 2 40
Debaryomyces hansenii
DC 1 + − − + − − − − 1 33
DC 2 + + − + − − − − 1 33
DC 3 + − − − − − − − 1 33
Lachancea thermotolerans
LC 1 − + + + + + − − 9 56
LC 2 − + − + + + − − 2 13
LC 3 − − − + + + − − 1 6
LC4 − − − − + + − − 2 13
LC 5 + − − − + + − − 1 6
LC6 + + + + + + + − 1 6
Metchnikowia pulcherrima
MC 1 + − − − + − − + 5 31
MC 2 + + − − − − − − 2 13
MC 3 + − − − + + − − 2 13
MC4 + + − − + − − + 1 6
MC 5 − + − − + + − − 2 13
MC6 − − − + + + − + 1 6
Pichia kluyveri
PKC 1 + − − − + − − − 4 100
Sporodiobulus salmonicolor
SPC 1 + − − − + + − − 4 67
SPC 2 − − − − + + − − 2 33
Torulaspora delbrueckii
TC 1 + − − − − − − − 5 28
TC 2 − − − − − − − − 1 6
TC 3 − − − − + − − − 2 11
TC4 + + − − + − − − 2 11
TC 5 + − − − + − − − 7 38
TC6 + − − − + − + − 1 6
Williopsis pratensis
WC 1 − − − − + + + − 9 90
WC 2 − − − − + − − 1 10
Zygosaccharomyces bailii
ZC 1 + + − − + − − − 2 29
ZC 2 + − − − + − − − 3 42
ZC 3 − − − − + − − − 2 29
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Presence (+) or absence (−) of each activity; SR, sulfite reductase activity; HCDC hydroxycinnamic acid decarboxylase activity; N, number of isolates showing each profile; %, percentage of each profile within the species

In Table 2 the strains were gathered according to each species taking into account the enzymatic activities that could be involved in the aroma of finished wines. So, we found different enzymatic profiles in each species and each of them was made up of a different number of strains. Only one profile was described in P. kluyveri, two different profiles in W. pratensis and S. salmonicolor, three in Cryptococcus spp. and D. hansenii, five in Candida spp., Z. bailii and T. delbrueckii and six in M. pulcherrima and L. thermotolerans. Surprisingly, one D. hansenii profile (DA1) did not show any enzymatic activity and the TA2 profile in T. delbrueckii only showed the SR activity. The main differences in profiles within each species were mainly due to esterase, lipase and β glucosidase activity. Taking into account that information, for the next stage in the selection process based on aroma characteristics, we would choose the strains included in the following profiles, because they had all three enzymatic activities: CA5 of Candida spp., CCA1 of Cryptococcus spp., LA5 of L. thermotolerans, MA4 of M. pulcherrima, ZA3 of Z. bailii, and DA2 of D. hansenii, PKA1 of P. kluyveri, SPA2 of S. salmonicolor, TA5 of T. delbrueckii, WA1 of W. pratensis, and ZA5 of Z. bailii, as having two of the activities.

In Table 3 the strains were grouped according to the enzymatic activities involved in the color and clarity of the finished wines. The number of different enzymatic profiles found within each species were one in P. kluyveri, two in W. pratensis and S. salmonicolor, three in Candida spp., Cryptococcus spp., D. hansenii, and Z. bailii, six in T. delbrueckii and L. thermotolerans and finally nine in M. pulcherrima. In almost every species there were profiles which did not show any carbohydrolase activity: CC3 in Candida spp., SPC2 in S. salmonicolor, TC2 and TC3 in T. delbrueckii, MC8 and MC9 in M. pulcherrima and LC4 in L. thermotolerans. No strain within the W. pratensis species showed any of these activities. However nine out of the ten strains of this species showed the three aminopeptidase activities involved in proteolysis. In this last group of enzymes related to natural wine fining, there were also strains which did not exhibit any activity: all the D. hansenii strains, TC1 and TC2 in T. delbrueckii and MC2 in M. pulcherrima. The most surprising result was found in the profile LC6 of L. thermotolerans, as it was the only strain that exhibited all the activities studied included in this group; the four carbohydrolases and the three aminopeptidases. So, this strain could be an excellent candidate for improving color and limpidity in red wines. What is more, this strain could even increase acidity level in wines due to the ability of L. thermotolerans species to produce lactic acid during alcoholic fermentation (Gobbi et al. 2013).

Taking into account the overall results, the most suitable strains within each species to continue in a selection process based on color and limpidity characteristics would be: CC1 in Candida spp., CCC2 in Cryptococcus spp., DC2 in D. hansenii, LC6 in L. thermotolerans, MC4 and MC7 in M. pulcherrima, PKC1 in P. kluyveri, SPC1 in S. salmonicolor, TC4 in T. delbrueckii, WC1 in W. pratensis and ZC1 in Z. bailii,. However, to exploit the benefits of non-Saccharomyces yeasts in wine production it is necessary to know the implications of using these yeasts for winemaking practices. Further work is now required to examine both the production and the activity of the observed enzymes and the development of the selected strains under vinification conditions, and to investigate what impact such activities have on the sensory properties of wine.

Conclusion

This study has revealed the potential for non-Saccharomyces yeasts to produce extracellular enzymes of enological significance. The screening showed a wide intraspecific difference within the yeast species investigated, indicating that strain selection is of great importance, as not all strains within a species would necessarily show the same desirable or undesirable characteristics. Besides, these data confirmed the need to carry out a selection process according to the different kind of wines the yeasts are destined to produce, or the different wine characteristics they have to improve. However, the potential of these enzymes to modify grape and wine composition and the impact of these enzymatic activities on the aroma and color profile of finished wines needs to be studied further.

Acknowledgements

This study has been undertaken with a Grant from the Instituto Nacional de Investigaciones Agrarias (INIA), Spain (Project RTA2013-0053-C03-03). We also would like to thank Victor Llop for his collaboration in laboratory analysis, and Ian Thomas for the English correction.

References

  1. Belda I, Conchillo LB, Ruiz J, Navascués E, Alonso A, Marquina D (2015) Rational selection of yeasts based on their pectinolytic activities and its incidence on technological and sensorial aspects of wine quality. In: VI European congress of microbiology-FEMS (Maastricht), pp 2420
  2. Belda I, Ruiz J, Alonso A, Marquina D, Navascués E, Santos A (2016a) Actividades enzimáticas de levaduras no Saccharomyces para su aplicación enológica. http://www.acenologia.com/cienciaytecnologia/actividades_enzimaticas_no_saccharomyces_cienc0715.htm, pp 1–8
  3. Belda I, Conchillo L, Ruiz J, Navascués E, Marquina D, Santos A. Selection and use of pectinolytic yeasts for improving clarification and phenolic extraction in winemaking. Int J Food Microbiol. 2016;223:1–8. doi: 10.1016/j.ijfoodmicro.2016.02.003. [DOI] [PubMed] [Google Scholar]
  4. Bely M, Renault P, de Silva T, Masneuf-Pomarede I, Albertin W, Moine V (2013) Non-conventional yeasts and alcohol level reduction. In: Alcohol level reduction in Wine. Vigne et Vin Publications Internationales, Bordeaux, France, pp 33–37
  5. Benito S, Palomero F, Morata A, Uthurry C, Suárez-Lepe JA. Minimization of ethylphenol precursors in red wines via the formation of pyroanthocyanins by selected yeasts. Int J Food Microbiol. 2009;132:145–152. doi: 10.1016/j.ijfoodmicro.2009.04.015. [DOI] [PubMed] [Google Scholar]
  6. Benito S, Morata A, Palomero F, González MC, Suárez-Lepe JA. Formation of vinylphenolic pyroanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies. Food Chem. 2011;124:15–23. doi: 10.1016/j.foodchem.2010.05.096. [DOI] [Google Scholar]
  7. Charoenchai C, Fleet G, Henschke P, Todd B. Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Aust J Grape Wine Res. 1997;3:2–8. doi: 10.1111/j.1755-0238.1997.tb00109.x. [DOI] [Google Scholar]
  8. Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol. 2011;5:873–882. doi: 10.1016/j.fm.2010.12.001. [DOI] [PubMed] [Google Scholar]
  9. Dizy M, Bisson L. Proteolytic activity of yeast strains during grape juice fermentation. Am J Enol Vitic. 2000;5:155–167. [Google Scholar]
  10. Fernández M, Úbeda JF, Briones AI. Typing of non-Saccharomyces yeasts with enzymatic activities of interest in wine-making. Int J Food Microbiol. 2000;59:29–36. doi: 10.1016/S0168-1605(00)00283-X. [DOI] [PubMed] [Google Scholar]
  11. Ganga MA, Martínez C. Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts. J Appl Microbiol. 2004;96:78–83. doi: 10.1046/j.1365-2672.2003.02080.x. [DOI] [PubMed] [Google Scholar]
  12. Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiol. 2013;33:271–281. doi: 10.1016/j.fm.2012.10.004. [DOI] [PubMed] [Google Scholar]
  13. Jolly N, Varela C, Pretorius IS. Not your ordinary yeasts: non-Saccharomyces yeast in wine production uncovered. FEMS Yeast Res. 2014;14(2):215–237. doi: 10.1111/1567-1364.12111. [DOI] [PubMed] [Google Scholar]
  14. Louw C, La Grange D, Pretorius IS, van Rensburg P. The effect of polisaccharide degrading wine yeast transformants on the efficiency of wine processing and wine flavour. J Biotechnol. 2006;125:447–461. doi: 10.1016/j.jbiotec.2006.03.029. [DOI] [PubMed] [Google Scholar]
  15. Manzanares P, Ramón D, Querol A. Screening of non-Saccharomyces wine yeasts for the production of β-d-xylosidase activity. Int J Food Microbiol. 1999;46:105–112. doi: 10.1016/S0168-1605(98)00186-X. [DOI] [PubMed] [Google Scholar]
  16. Manzanares P, Rojas V, Genovés S, Vallés S. A preliminary search for anthocyanin-β-d-glucosidase activity in non-Saccharomyces wine yeasts. Int J Food Microbiol. 2000;35:95–103. [Google Scholar]
  17. Masneuf-Pomarede I, Bely M, Marullo P, Albertin W. The genetics on non-conventional wine yeasts: current knowledge and future challenges. Front Microbiol. 2015;6(1553):1–15. doi: 10.3389/fmicb.2015.01563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maturano YP, Rodríguez Assaf LA, Toro ME, Nally MC, Vallejo M, Castellanos de Figueroa LI, Combina M, Vazquez F. Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation. Int J Food Microbiol. 2012;155:43–50. doi: 10.1016/j.ijfoodmicro.2012.01.015. [DOI] [PubMed] [Google Scholar]
  19. Maturano YP, Mestre MV, Esteve-Zarzoso B, Nally MC, Lerena MC, Vazquez F, Combina M. Yeast population dynamics during prefermentative cold soak of Cabernet sauvignon and Malbec wines. Int J Food Microbiol. 2015;199:23–32. doi: 10.1016/j.ijfoodmicro.2015.01.005. [DOI] [PubMed] [Google Scholar]
  20. Mendes-Ferreira A, Climaco MC, Medes-Faia A. The role of non-Saccharomyces species in releasing glycosydic bound fraction of grape aroma components: preliminary study. J Appl Microbiol. 2001;91:67–71. doi: 10.1046/j.1365-2672.2001.01348.x. [DOI] [PubMed] [Google Scholar]
  21. Merín MG, Morata de Ambrosini VI. Highly cold active pectinases under wine-like conditions from non-Saccharomyces yeasts for enzymatic production during winemaking. Lett Appl Microbiol. 2015;60:467–474. doi: 10.1111/lam.12390. [DOI] [PubMed] [Google Scholar]
  22. Morata A, Loira I, Heras JM, Callejo MJ, Tesfaye W, González C, Suárez-Lepe JA. Yeast influence on the formation of stable pigments in red winemaking. Food Chem. 2016;197:686–691. doi: 10.1016/j.foodchem.2015.11.026. [DOI] [PubMed] [Google Scholar]
  23. Ocón E, Gutiérrez AR, Garijo P, López I, López R, Santamaría P, Tenorio C. Quantitative and qualitative analysis of non-Saccharomyces in spontaneous alcoholic fermentations. Eur Food Res Tech. 2010;230(6):885–891. doi: 10.1007/s00217-010-1233-7. [DOI] [Google Scholar]
  24. Ocón E, Gutiérrez AR, Garijo P, López R, Santamaría P. Presence of non-Saccharomyces yeasts in cellar equipments and grape juice during harvest time. Food Microbiol. 2010;27:1023–1027. doi: 10.1016/j.fm.2010.06.012. [DOI] [PubMed] [Google Scholar]
  25. Ocón E, Garijo P, Sanz S, Olarte C, López R, Santamaría P, Gutiérrez AR. Screening of yeast mycoflora in winery air samples and their risk of wine contamination. Food Control. 2013;34:261–267. doi: 10.1016/j.foodcont.2013.04.044. [DOI] [Google Scholar]
  26. Pérez-González J, González R, Querol A, Sendra J, Ramón D. Construction of a recombinant wine yeast strain expressing β-(1, 4) endoglucanase and it use in microvinification processes. Appl Environ Microbiol. 1993;59:2801–2806. doi: 10.1128/aem.59.9.2801-2806.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rossouw D, Bauer FF. Exploring the phenotypic space of non-Saccharomyces wine yeast biodiversity. Food Microbiol. 2016;55:32–46. doi: 10.1016/j.fm.2015.11.017. [DOI] [PubMed] [Google Scholar]
  28. Santamaría P, López R, Portu J, González-Arenzana L, López-Alfaro I, Gutiérrez AR, Garde-Cerdán T (2015) Role of phenylalanine in Viticulture and Enology, In: Elliamson D (ed) Phenylalanine. Dietary, sources, functions and health effects. Nova Publisher, New York, pp 49–70
  29. Shinohara T, Kubodera S, Yanagida F. Distribution of phenolic yeasts and production of phenolic off flavors in wine fermentation. J Biosci Bioeng. 2000;90(1):90–97. doi: 10.1016/S1389-1723(00)80040-7. [DOI] [PubMed] [Google Scholar]
  30. Strauss M, Jolly N, Lambrechts M, van Rensburg P. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J Appl Microbiol. 2001;91:182–190. doi: 10.1046/j.1365-2672.2001.01379.x. [DOI] [PubMed] [Google Scholar]
  31. Suárez R, Suárez-Lepe JA, Morata A, Calderón F. The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera. A review. Food Chem. 2007;102:10–21. doi: 10.1016/j.foodchem.2006.03.030. [DOI] [Google Scholar]
  32. Suárez-Lepe JA, Morata A. New trends in yeast selection for winemaking. Trends Food Sci Technol. 2012;23:39–50. doi: 10.1016/j.tifs.2011.08.005. [DOI] [Google Scholar]
  33. Suárez-Lepe JA, Morata A (2015) Actividad β glicosidasa y degradación de antocianos. In: Madrid Vicente A (ed) Levaduras para vinificación en tinto. AMV ediciones, Madrid, pp 146–147
  34. Trinh TTT, Woon WY, Yu B, Curran P, Liu SQ. Effect of l-isoleucine and l-phenylalanine addition on aroma compound formation during Longan juice fermentation by a co-culture of Saccharomyces cerevisiae and Williopsis saturnus. S Afr J Enol Vitic. 2010;31(2):116–124. [Google Scholar]
  35. Van Resburg P, Pretorius IS. Enzymes in winemaking: harnessing natural catalysis for efficient biotransformations—a review. S Afr J Enol Vitic. 2000;21:52–73. [Google Scholar]
  36. Viana F, Gil JV, Genovés S, Vallés S, Manzanares P. Rational selection of non-Saccharomyces wine yeasts for mixed starter base on ester formation and enological traits. Food Microbiol. 2008;25:778–785. doi: 10.1016/j.fm.2008.04.015. [DOI] [PubMed] [Google Scholar]
  37. Wedral D, Shewfelt R, Frank J. The challenge of Brettanomyces in wine. LWT-Food Sci Technol. 2010;43:1474–1479. doi: 10.1016/j.lwt.2010.06.010. [DOI] [Google Scholar]

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