Non-Saccharomyces biodiversity in wine and the ‘microbial terroir’: a survey on Nero di Troia wine from the Apulian region, Italy

Abstract

The aim of this work was to study the biodiversity of cultivable non-Saccharomyces yeasts isolated from the autochthonous wine variety ‘Uva di Troia’ from the North-Apulian region during vintages 2012 and 2013. Grapes were collected in vineyards from four different geographical areas on which four different wines with the status of geographical indication are produced. Different restriction profiles of ITS–5.8S rDNA regions, corresponding to those of Candida boidinii, Candida zemplinina, Hanseniaspora guilliermondii, Issatchenkia terricola, Zygosaccharomyces bailii, Hanseniaspora uvarum, Zygoascus hellenicus and Hanseniaspora opuntiae, were observed. The most abundant genera were represented by Hanseniaspora guilliermondii and Candida zemplinina. Significant differences among locations and vintages were observed. This first report on non-Saccharomyces diversity during the early steps of spontaneous alcoholic fermentation of Nero di Troia wines provides the basis for an improved management of non-Saccharomyces in typical Apulian wines, which will be important for the development of the local wine industry and to achieve an enhanced standard of safety in the final production.

Introduction

Grape juice is a non-sterile mixture with several types of microorganisms belonging to the microbiota naturally present on grape berries. This microbial consortium is important for wine production. In particular, yeasts can ferment the substrate, promoting the transformation of grape sugars into ethanol, carbon dioxide and hundreds of other metabolites (alcoholic fermentation), while lactic acid bacteria (LAB) are responsible for the decarboxylation of l-malic acid into l-lactic acid and CO₂ (malolactic fermentation). Spontaneous fermentation is carried out through a sequence of different yeast species, according to their metabolic aptitudes and alcohol tolerance: non-Saccharomyces yeasts are the most abundant at the beginning of alcoholic fermentation (AF), replaced, after 3–4 days, by Saccharomyces cerevisiae (Pretorius 2000; Ciani et al. 2009). S. cerevisiae is considered the principal microorganism responsible for vinification, since it completes the fermentation of available sugars. However, during spontaneous AF several yeast genera, such as Hanseniaspora, Kloeckera, Candida, Pichia, Zygosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces and Metschnikowia, have been isolated (Fleet 2003, 2008; Jolly et al. 2014).

Non-Saccharomyces yeasts, generally considered spoilage yeasts, in some cases display physiological characteristics that lead them to be considered as potential starter cultures. Indeed, some strains may produce compounds that exert a positive influence on the quality of the wine (Fleet 2003) and/or may be used with a specific technological purpose (e.g., to decrease volatile acidity, decrease alcohol content) (Bely et al. 2008; Contreras et al. 2013). Moreover, several authors have recently proposed the direct application of non-Saccharomyces as biocontrol agents against molds or spoilage microorganisms, including spoilage lactic acid bacteria (LAB) or yeast belong to Brettanomyces bruxellensis species (Úbeda et al. 2014; Oro et al. 2014). For this reason, in order to improve the aroma and flavor of wine, several studies suggest the inclusion of non-Saccharomyces wine yeasts, together with Saccharomyces strains as part of mixed and multi-starter fermentations (Rojas et al. 2003; Romano et al. 2003a; Ciani et al. 2006; Jolly et al. 2006). From this point of view, the study of non-Saccharomyces microbial biodiversity represents a subject of increasing interest in wine regions. This attention is also attested by studies highlighting the importance of ‘virtuous’ microbial diversity during fermentation of artisanal/typical fermented foods and geographical indications (Capozzi and Spano 2011; Capozzi et al. 2012a, b). Additionally, in the grape/wine environment, recent studies investigating the microbial biogeography—the so-called “microbial terroir” (Gilbert et al. 2014)—have revealed a close relationship between production region, climate and microbial patterns (Bokulich et al. 2013). Such evidence sheds new light also on the selection and characterization of autochthonous microbes from regional wines and autochthonous grapevine varieties, such as microbial resources isolated from the Northern Apulian region (Capozzi et al. 2010, 2014; Lamontanara et al. 2014; Di Toro et al. 2014). It is crucial to stress that non-Saccharomyces yeast are a very heterogeneous group of microbes, representing not only a vast source of direct applications (Jolly et al. 2013), but also posing risks to wine quality. Among the potential risks due to the non-Saccharomyces presence in wine are the production of biogenic amines (Tristezza et al. 2013), generation of off-flavors (acetic acid, esters, acetaldehydes, H₂S) (Fleet 2003, 2008) and competition for the availability of nutrients during fermentation with S. cerevisiae (Taillandier et al. 2014).

The aim of this work was to study, for the first time, the biodiversity of cultivable non-Saccharomyces yeasts associated with grapes collected from North-Apulian region on the autochthonous vine variety ‘Uva di Troia’ (’Nero di Troia’ is the corresponding wine obtained with ‘Uva di Troia’ as unique variety) during the vintages ‘2012’ and ‘2013’. We collected grapes in vineyards from four different geographical areas (San Severo, Barletta, Lucera, Ascoli Satriano) that produce four different wines with the status of geographical indication (wine with appellation of origin) (‘San Severo Rosso DOC, ‘Rosso Barletta DOC’, ‘Cacc’e Mmitte DOC’, ‘Tavoliere delle Puglie DOC’). The common denominator of these wines is the autochthonous grape variety ‘Uva di Troia’, which can be used at different percentages for specific product formulations.

Materials and methods

Spontaneous fermentation and yeast isolation

Spontaneous alcoholic fermentations were performed by sampling Nero di Troia grape cultivars in the North Apulia area from four vineyards located in the geographical areas of San Severo, Barletta, Lucera, and Ascoli Satriano, during vintages 2012 and 2013. The fermentations were carried out using samples from 1 kg to 5 kg grape berries in 1-L tanks, then spontaneous AF was carried out in the laboratory at 25 °C temperature without further inoculation of starter culture and monitored for 1 month. Yeasts were sampled at the beginning of AF, which was determined on the basis of alcohol content (about 1 %). Decimal saline dilutions were plated on Wallerstein Laboratory (WL) nutrient agar (Oxoid, Basingstoke, UK) and Lysine medium (Oxoid), supplemented with 10 mg/L chloramphenicol to inhibit bacterial growth, according to Lopandic et al. (2008). About 25 green colonies, with different morphologies, were selected for isolation and identification from every fermentation stage and stored at −80 °C in YPD medium supplemented with glycerol (30 % v/v). All assays were conducted in duplicate.

Molecular characterization of non-Saccharomyces yeast

The isolates were identified by PCR-RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers (ITS), performed according to Esteve-Zarzoso et al. (1999), with some modifications. The amplification reactions were performed using a PCR reaction mix containing 0.5 μM of each primer (ITS1 and ITS4), 200 μM dNTP, buffer 10X, solution Q and 1.25 units of Qiagen Taq DNA Polymerase (Taq PCR Core, Qiagen, Hilden, Germany). PCR was performed in a thermocycler (I-Cycler, Bio-Rad, Richmond, CA), using the following program: initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturing at 94 °C for 1 min, annealing at 55.5 °C for 2 min and extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min; samples were then conserved at 4 °C. Products of amplification were verified on 2 % agarose gels run in 1X TBE buffer and stained with ethidium bromide. After electrophoresis, gels were visualized under UV light and photographed (Versa Doc, Bio-Rad). Sizes were estimated by comparison against a DNA length standard (50 bp ladder, Promega) with Quantity One Software (Bio-Rad). PCR products were then digested without further purification with the Fast Digest® restriction endonucleases HaeIII, HhaI (CfoI) and HinfI (Fermentas, M-Medical, Milan, Italy), although in some cases endonuclease DdeI was used.

Restriction analysis was performed following the manufacturer’s instructions, using a mix containing 10 μL (about 0.2 μg) PCR product, 2 μL 10X Fast Digest® Green buffer, 1 μL of endonuclease and 17 μL bi-distilled water. The mix was then incubated at 37 °C for 20 min using a thermo cycler (I-Cycler, Bio-Rad). The restriction fragments were separated on a 3 % agarose gel with 1X TBE buffer and stained with ethidium bromide. After electrophoresis, gels were visualized under UV light and photographed (Versa Doc, Bio-Rad). Sizes of the PCR products obtained were estimated by comparison against a DNA length standard (1 kb ladder, Promega). Two randomly selected PCR fragments for each restriction pattern obtained with RFLP-PCR were purified using the QIAquick PCR purification kit (Qiagen) and sent to Primm Biotech (Milano, Italy) for sequencing. Strains were identified by comparison with sequences available at the NCBI database (GenBank) using the standard nucleotide_nucleotide homology search Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST).

GenBank accession number

The 5.8 ITS sequences obtained were deposited with the GenBank data library under the accession numbers listed in Table 1.

Table 1 Identification of yeasts isolated from grape must collected from four wineries (Barletta, San Severo, Lucera, Ascoli Satriano) by internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) analysis and sequencing

Statistical data analysis

Data generated were analyzed by one-way ANOVA, Turkey test (P < 0.005). All statistical analyses were performed using Past, version 3.05 (Hammer et al. 2001).

Results

Yeast species identification

A total of 200 purified colonies isolated from grape juice from the Uva di Troia variety during AF was subjected to PCR-RFLP analysis of the 5.8SITS rDNA region. Samples were collected from four different vineyards located in the north Apulia region (Fig. 1) during two consecutive vintages: 2012 and 2013. The yeast species identified and the isolation frequencies obtained during the spontaneous fermentations are shown in Table 1. A wide variety of non-Saccharomyces yeast was found. PCR products varying in length from 450 to 880 bp were digested with HhaI (CfoI), HaeIII, HinfI and DdeI enzymes, and the molecular mass of the restriction products obtained was compared with those described previously in the literature (Esteve-Zarzoso et al. 1999; Pham et al. 2011). In general, we observed eight different restriction profiles of the ITS–5.8S rDNA region, corresponding to Candida boidinii, Candida zemplinina, Hanseniaspora guilliermondii, Issatchenkia terricola, Zygosaccharomyces bailii, Hanseniaspora uvarum, Zygoascus hellenicus and Hanseniaspora opuntiae (Table 1). Two randomly selected strains for each pattern obtained were sequenced to confirm species assignation, performed by comparison with sequences available at the NCBI database (GenBank) using the standard nucleotide_nucleotide homology search Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST) (corresponding gene accession numbers are reported in Table 1).

Fig. 1

Schematic representation of the geographical vineyard sampling location

Several yeast species, e.g., C. boidinii, C. zemplinina, H. guilliermondii and I. terricola, represented a common denominator of all the vineyards studied. Other species were isolated from one vineyard only (for example, Zygoascus hellenicus from San Severo and H. opuntiae from Lucera) (Table 1). The predominance of non-Saccharomyces yeasts during the first step of the fermentation was observed for all the grape juice analyzed. Figure 2 shows the frequencies of strains identified from the four different vineyards, respectively, during the vintages 2012 and 2013, while Fig. 3 reports the frequencies of yeast isolated during vintages 2012 and 2013 in Nero di Troia vineyards. Among the non-Saccharomyces yeasts characterized in this study, the most abundant genera were Hanseniaspora (about 58 %, H. guilliermondii 53 %, H. uvarum 4 % and H. opuntiae 1 %) and Candida (about 32 %, C. zemplinina 19 % and C. boidinii 16 %) (Fig. 3). Analysis of non-Saccharomyces diversity in the four different areas revealed a great variability, showing, in several cases, statistically significant differences among locations (Fig. 2) and vintages (Fig. 3). The presence of H. guilliermondii is different in San Severo (46 and 49 %, respectively, for vintage 2012 and 2013) and Ascoli Satriano (59 and 60 %, respectively, for vintage 2012 and 2013) vineyards, while in Barletta and Lucera vineyards we found a similar frequency of H. guilliermondii (about 50 %). H. uvarum ecotypes were isolated only from San Severo and Lucera. In addition, H. uvarum was detected at higher frequency during vintage 2013 than 2012, (from 2 % to 16 %) (Fig. 2). C. boidinii shows a higher presence in Barletta and Ascoli Satriano vineyards, with respect to Lucera and San Severo. Considering the genus Candida, significant differences in C. boidinii selected ecotypes were found between the two vintages studied, with a frequency decrease from 21 % for the vintage 2012 to 11 % for the vintage 2013 (Fig. 3). During the vintage 2012, C. zemplinina exhibited the highest presence in San Severo vineyards (25 %), with lower frequencies in Barletta, Lucera and Ascoli Satriano. In contrast, during vintage 2013, the frequency of the same non-Saccharomyces species analyzed was higher in the Barletta area (23 %) and lower (about 20 %) in the other areas studied. Comparing the frequency of strains analyzed for C. boidinii, C. zemplinina, H. guilliermondii and H. uvarum, they also showed significant differences between the two vintages. Considering minor yeast genera isolated, I. terricola isolates were found to be lower in Barletta, Lucera and the Ascoli Satriano area (about 4 %) and higher in the San Severo area (about 10 %), although no significant differences were observed in the frequency of any of the I. terricola strains analyzed during vintage 2012 and 2013 (Fig. 3). Zygosaccharomyces bailii and Zygoascus hellenicus species were isolated only from specific vineyards: San Severo and Lucera for Zygosaccharomyces bailii and San Severo for Zygoascus hellenicus, respectively. However, even in this case, no significant differences in frequency were observed (Fig. 3).

Fig. 2

a,b Percentage frequencies of non-Saccharomyces yeasts isolated from spontaneous fermentation of vine variety “Uva di Troia”, during vintages 2012 (a) and 2013 (b). Vineyard: Open bars Ascoli Satriano; black bars, San Severo; light grey bars, Lucera; dark grey bars, Barletta. Different letters above bars indicate statistical significance (one-way ANOVA, Turkey test P < 0.005)

Fig. 3

Percentage frequencies of non-Saccharomyces yeast during vintage 2012 (dark grey bars) and vintage 2013 (light grey bars). Different letters above bars indicate statistical significance (one-way ANOVA, Turkey test P < 0.005)

Discussion

The presence and variability of non-Saccharomyces on grapes, musts and wines are studied in order to determine their potential effects on the organoleptic qualities of the final products (González et al. 2006). For this reason, a greater understanding of non-Saccharomyces biodiversity in fermenting wines is an essential criterion for quality improvement programs in oenological production and, more specifically, in the sector of typical wine and oenological geographical indications. All samples analyzed in this study show the predominance of non-Saccharomyces yeasts in the first steps of a spontaneous AF (Ganga and Martínez 2003; Clavijo et al. 2010; Bezerra-Bussoli et al. 2013). In terms of yeast diversity, our results are similar to those found in other wine-producing areas. In fact, several studies have already reported a dominance of Candida and Hanseniaspora genera at the beginning of spontaneous AF in wine (Beltran et al. 2002; van Keulen et al. 2003; Combina et al. 2005; Romancino et al. 2008; Pramateftaki et al. 2012; Bezerra-Bussoli et al. 2013) although non-Saccharomyces yeast such as Lachancea, Wickerhamomyces and Torulaspora were sometimes reported as the main non-Saccharomyces dominant species (Cordero-Bueso et al. 2012). Furthermore, it is important to stress that, within Candida and Hanseniaspora genera, the species with the highest frequencies are usually different. For example, in this work, H. guilliermondii and C. zemplinina were found as dominant species, while Ocón et al. (2010), investigating the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation (D.O.Ca. Rioja, Spain), found C. stellata and H. uvarum in major proportions during fermentation. Our results can be correlated to specific pedoclimatic conditions. Indeed, both vintages analyzed in this study were characterized by high levels of precipitation.

Non-Saccharomyces yeast species play relevant roles in determining wine flavor and complexity. In particular, they can improve the chemical composition of wines due to several aromatic compounds, besides they are often isolated from wines with anomalous sensorial profiles and associated with the production of compounds toxic to human health (Tristezza et al. 2013). Several studies have proposed non-Saccharomyces as a tool to mimic natural biodiversity and to enhance the complexity and the particular characteristics of a wine, avoiding the risk of sluggish or stuck fermentation, as part of mixed/multi-strains starter cultures (Romano et al. 2003b; Ciani et al. 2006, 2009; Jolly et al. 2006, 2014; Ciani and Comitini 2011). The most represented species isolated in this study, in both vintages and in all locations, are H. guilliermondii and C. zemplinina, non-Saccharomyces yeasts of oenological interest. These findings lead us to suggest these two species as possible candidates for the design of mixed/multistrains autochthonous starter cultures for ‘San Severo Rosso DOC, ‘Rosso Barletta DOC’, ‘Cacc’e Mmitte DOC’, ‘Tavoliere delle Puglie DOC’, as well as for ‘Nero di Troia’ wines, with the final aim of achieving a product via a representation of autochthonous virtuous microbial diversity. Moreira et al. (2008) reported that wines inoculated with H. guilliermondii show higher levels of 2-phenylethyl acetate, 1-propanol and 3-(methylthio)propionic acid, highlighting that certain apiculate yeasts have the capacity to influence, in a positive way, the aromatic profile of wines. Moreover, C. zemplinina can be exploited advantageously in sweet wine production due to the lower amount of acetic acid produced and elevated concentration of glycerol (Sipiczki 2004; Rantsiou et al. 2012; Tofalo et al. 2012; Magyar et al. 2014). Whereas not present at higher concentrations, H. uvarum and C. boidii species are also representative of the non-Saccharomyces diversity observed. Concerning their oenological significance, with mixed fermentation, H. uvarum increased the content of isoamyl acetate in wines (Moreira et al. 2008), while C. boidinii is a species frequently isolated in cellar surfaces from spoiled wines (Saez et al. 2011).

With regard to non-Saccharomyces genera isolated at lower concentrations, it is crucial to highlight how they were often connected with spoilage and unwonted phenomena (Loureiro and Malfeito-Ferreira 2003; Pretorius 2000). Among yeasts with lower frequency, spoilage yeast, such as Zygosaccharomyces bailii, Zygoascus hellenicus, and I. terricola, were isolated. Zygosaccharomyces bailii is one of the main spoilage yeasts in the wine industry (Zuehlke et al. 2013), while several strains belonging to Zygoascus hellenicus and I. terricola have been characterized as producers of biogenic amines in wine (Tristezza et al. 2013).

In conclusion, this is the first report on yeast microbiota during the early steps of spontaneous AF from Apulian Nero di Troia wines. Our findings provide the basis for improved management of non-Saccharomyces in typical Apulian wines that will be important for the development of the local wine industry and to achieve an enhanced standard of safety in the final product.