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Article

Characterization of Nero Antico di Pretalucente Wine and Grape Fungal Microbiota: An Expression of Abruzzo Region Cultivar Heritage

by
Giorgia Perpetuini
1,
Alessio Pio Rossetti
1,
Lucia Giordano
2,
Marta Pulcini
1,
Beatrice Dufrusine
1,
Noemi Battistelli
3,
Camillo Zulli
3,
Giuseppe Arfelli
1,
Alberto Palliotti
2,*,
Enrico Dainese
1,* and
Rosanna Tofalo
1,*
1
Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, via R. Balzarini 1, 64100 Teramo, Italy
2
Department of Agriculture, Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy
3
Orsogna 1964 Winery, via Ortonese 29, Orsogna, 66036 Chieti, Italy
*
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(2), 150; https://doi.org/10.3390/fermentation9020150
Submission received: 1 December 2022 / Revised: 27 January 2023 / Accepted: 30 January 2023 / Published: 3 February 2023
(This article belongs to the Section Fermentation for Food and Beverages)
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Abstract

:
The aim of this study was to characterize the ampelographic and genetic profiles of Vitis vinifera L. cv. Nero Antico di Pretalucente and to describe the grape-borne fungal communities. The oenological characteristics and the aroma profile of wine obtained by spontaneous fermentation were also investigated. Microsatellite profiles and ampelographic traits indicated that this cultivar presented a unique profile, and therefore it can be considered a cultivar in its own right and autochthonous of Gessopalena village. Next-generation sequencing analysis revealed that Aureobasidium spp. was the main genus detected on grapes. At the species level, Aureobasidium pullulans was the main species, followed by Alternaria alternata. Wines were characterized by a final ethanol content of 12.75% (v/v), a pH of 3.4, a volatile acidity lower than 0.6 g/L, a content of glycerol of 8.56 g/L, and a concentration of polyphenols and anthocyanins of 977 GAE/L and 266 mg/L, respectively. The intensity and tonality of the wine as well as the active odor compounds found were described. The results obtained could improve the knowledge concerning the agronomic traits and the wine obtained from this ancient and autochthonous grapevine variety cultivated in a foothill area, in order to offer consumers a wine with unique traits.

1. Introduction

The domestication of the grapevine (Vitis vinifera L.) happened in the sixth millennium B.C.E. in the region comprising Eastern Anatolia, the South Caucasus, and Western Asia and was likely concurrent with the development of winemaking techniques based on wild grapes and other luscious fruits [1]. In recent years, developments in the wine market have resulted in a decline in wine consumption in European nations, despite a significant desire for wines that meet health standards and satisfy the senses. Market trends show that consumers are attracted to grapes and wines that are strictly related to the production area [2]. Minor Vitis vinifera L. grape varieties that are typical of a certain geographical area seem to be an interesting option to increase sales as they link the product to the territory of origin and differentiate the obtained wines from others, because they are not found anywhere else [2]. Furthermore, the exploitation of minor local varieties may be ecologically interesting and useful in increasing environmental biodiversity. Due to the changing marketing techniques of wine firms and the shifts in wine consumption patterns, some grape types that had been neglected by the wine industry for decades have reemerged in recent years, enabling their economic and social rehabilitation [2]. Local varieties are well adapted to the environment and allow for more sustainable viticulture. In addition, these varieties could have a selective advantage because, for example, they are able to cope with the climatic changes in their environment due to global warming [3]. The preservation of grapevine biodiversity and genetic variety is unquestionably one of the primary objectives of wine research [4].
The Italian National Catalogue of Grapevine Varieties lists approximately 610 Vitis vinifera L. cultivars used for winemaking in a country devoted to viticulture [5]. Occasionally, the prevalence of synonyms (multiple names for the same genotype) and homonyms (identical or very similar cultivar names but different genotypes) is one of the primary problems in contemporary viticulture. Simple sequence repeat (SSR) markers can be used to identify synonyms and homonyms and to analyze the parentage of locally or widely cultivated grapevine varieties in Italy. The characterization of genotypes, together with the study of morphological features, represents a useful tool to clarify synonyms and homonyms, detect the origin of species, and eventually describe new cultivars [6]. According to the regional vineyards register, the Abruzzo region currently has 33,319 hectares of vineyards, principally located in the province of Chieti (23,355 hectares), and in total represents 6% of the total Italian wine production [7]. The wine-growing area in the Abruzzo region is concentrated in the coastal or inland hilly areas (96%). In the mountainous areas of the Abruzzo Apennines, the vineyard area is around 4% [7] and represents a precious reservoir of biodiversity. The main grape varieties present in the Abruzzo region are Montepulciano, Trebbiano Abruzzese, Passerina, Montonico, and Pecorino, among others.
Vitis vinifera L. cv. Nero Antico di Pretalucente is one of these minor cultivars. It is a black grape variety located in ancient vineyards in the Gessopalena and Torricella Peligna villages (Chieti, Italy). The vineyards are at altitudes between 600 m and 900 m above sea level, and grapes are harvested at the end of October. They are called “uva nera antica” (ancient black grapes) by people inhabiting these areas. The word “Pretalucente” refers to the large chalk boulder on which the medieval core of the village of Gessopalena was built and which is characterized by the presence of large selenite crystals that reflect sunlight. Grapes were used by old farmers not only for winemaking but were also stored in houses, hanging from the ceiling, to be consumed during the Christmas holidays. The oenological potential of this cultivar is unknown, and no data are available concerning the fungal communities associated with grapes or how these could influence the characteristics of the wine obtained. Phenotypic and genotypic characteristics were evaluated through an ampelographic study of the Vitis vinifera L. cv. Nero Antico di Pretalucente shoots, mature leaves, bunches, berries, and seeds, while an international set of nine SSR markers was employed for its genetic characterization. Furthermore, the fungal communities of Vitis vinifera L. cv. Nero Antico di Pretalucente grapes were described using next-generation sequencing technology. The oenological characteristics and aroma profile of the wine produced through spontaneous fermentation were also investigated.

2. Materials and Methods

2.1. Sampling Site

The accession samples for the ampelographic, molecular, and oenological studies were collected in an 18-year-old vineyard located in Gessopalena, Chieti, Abruzzo Region, Central Italy (42°03′22″ N, 14°16′46″ E; 660 m a.s.l.) on a clayey and sandy soil type. The vines were ungrafted, planted with 3.5 × 1.5 m inter- and intra-row spacing, and trained to a classic or bilateral Guyot system. The vineyard was approximately 0.5 ha in size and hosted only the Nero Antico grape variety. The vineyard was managed as organic in accordance with Reg. EC 834/2007 [8], EC 889/2008, and EC1235/2008 [9]. In particular, pest management was achieved only through copper/sulfur-based products.

2.2. Ampelographic Descriptions

The morphological characterization was made during the 2019, 2020, and 2021 growing seasons. The young shoots, leaves, bunches, and berries were evaluated using 19 descriptors which were selected for their power of discrimination and objectivity, according to the International Organization of Vine and Wine descriptors [10]. The OIV descriptors evaluated were: OIV codes 001, 002, 004, 006, 007, 008, and 016 for shoots; 051 for young leaves; 065, 067, 068, 069, and 080 for mature leaves; 202, 204, and 208 for bunches; 223 and 225 for berries; and 241 for seeds (Table S1).

2.3. Yield and Grape and Berry Characteristics at Harvest

In all of the years of the trial, grapes from 50 vines were individually picked, and the crop weight and bunch number per vine were recorded. The average bunch weight was calculated, and berry fresh weight and number of berries per bunch were measured. Finally, the percentage of skin by weight was also assessed.

2.4. Plant Material Sampling

Plant material was collected in May 2021. Young leaves were collected from different rows using sterile scissors and immediately placed on dry ice for transport to the laboratory, where they were quickly immersed in liquid nitrogen and stored at −80 °C until analysis. Grape samples of Vitis vinifera L. cv. Nero Antico di Pretalucente were collected aseptically in September 2021. Three sampling points were identified at distal spatial points of different rows, where samples of grape berries were collected and processed independently (representing three biological replicates). Healthy and undamaged grapes (about 1 kg) were collected using sterile scissors and placed in sterile plastic bags, transported to the laboratory in refrigerated boxes, and processed within 12 h. To prevent cross-contamination, sampling tools were sterilized with 75% ethanol before sampling.

2.5. DNA Extraction and Microsatellite Analysis

Genomic DNA from 300 mg of frozen leaf tissue was extracted using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The yield and quality of nucleic acid extraction were determined using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA 02451). A DNA sample was analyzed at the nine international reference SSR loci for Vitis vinifera L. accessions: VVS2 [11], VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32 [12,13], VrZAG62, and VrZAG79 [14]. PCR amplification was performed in a Biometra Thermocycler T-Gradient ThermoBlock (Analytik Jena GmbH, Jena, Germany) in a 25 µL final volume using the PCR Core kit (Roche Diagnostics GmbH by Sigma-Aldrich, Mannheim, Germany) and adapting the method previously described by Panara et al. [15]. Briefly, the reaction mixture contained an adequate volume of PCR grade water, 100 µg of bovine serum albumin (Promega, Madison, WI, USA), a 2.5 mM final concentration of MgCl2, 2.5 µL of 10x PCR-reaction buffer without MgCl2, 200 µM of each dNTP final concentration, 0.625 U of Taq DNA Polymerase, 0.2 µM of each primer pair, and 150 ng of extracted DNA. All the forward primers were fluorescein-5′-labeled. PCR products were verified at 2% (w/v) agarose gel electrophoresis. The length of amplified SSR loci was assessed via capillary electrophoresis at BMR Genomics srl (Padua, Italy). The resulting genetic profile was compared with the SSR profile accessions already available in the following databases: European Vitis Database (www.eu-vitis.de, accessed on 21 November 2022), Italian Vitis Database (www.vitisdb.it, accessed on 21 November 2022), Greek Vitis Database (www.biology.uoc.gr/gvd, accessed on 21 November 2022), Swiss Vitis Microsatellite Database (www1.unine.ch/svmd/, accessed on 21 November 2022), and Vitis International Variety Catalogue (www.vivc.de, accessed on 21 November 2022).

2.6. Next-Generation Sequencing Analysis

Grape berries were processed using a PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. Total genomic DNA was measured, and its purity at A260 nm/A280 nm was evaluated. Using the ITS3 (5′-GCATCGATGAAGAACGCAGC) and ITS4 (5′-TCCTCCGCTTATTGATAGATAC-3′) primers, the fungal community was amplified. In a final volume of 25 µL, PCR mixtures contained 1x reaction buffer (5x Go Taq DNA Polymerase, Milan, Promega), 1 µL of each primer (10 µM), and 2 µL of DNA template. Initial denaturation at 95 °C for 3 min was followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min, with a final elongation step at 72 °C for 5 min. At BMR Genomics srl, DNA sequencing and taxonomy analysis were performed.

2.7. Laboratory Scale Wine Fermentations

The grapes were crushed, and the grape mash (must and skins) was placed in sterilized 500 mL glass bottles with sterile Müller valves. Each of the 400 mL glass bottles included must and sterilized paraffin oil in order to allow the carbon dioxide evolved during the fermentation process to escape from the fermenting juice, to ensure semi-anaerobic conditions and to prevent external contamination, according to the procedure reported by Englezos et al. [16]. The spontaneous fermentations, without SO2 addition, were conducted at 25 °C under static conditions. The solids (cap) were not handled due to the small volume employed. The fermentation kinetic was evaluated daily by monitoring the loss of weight due to CO2 emission. Once a steady weight was achieved, the fermentation was deemed complete. Samples were aseptically collected with sterile serological pipettes at different stages of the alcoholic fermentation (T0—after crushing; T7—after 7 days of spontaneous fermentation; and at the end of alcoholic fermentation) for yeast cell count and physical and chemical analysis. Three biological and three technical replicates were conducted.

2.8. Viable Yeasts Count

Fifty milliliters of must were suspended in 450 milliliters of physiological solutions (NaCl 0.85% w/v) and diluted serially. To detect total yeasts and non-Saccharomyces yeasts, cell suspensions were plated on YPD agar (Yeast Extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L, Agar 18 g/L) and Lysine medium (LM, Oxoid, Milan, Italy). YPD is a medium supporting the growth of all yeasts, while LM allowed the enumeration of non-Saccharomyces, since S. cerevisiae is not able to use lysine for its growth. Plate counts were performed in triplicate at different times (T0—after crushing, T7—after 7 days of spontaneous fermentation, and at the end of alcoholic fermentation). Plates were incubated for 48 h at 30 °C.

2.9. Main Physical–Chemical Parameters

FOSS WineScan™ FT120 rapid scanning Fourier Transform Infrared Spectroscopy with FOSS WineScan software version 2.2.1 was used to analyze the primary physico-chemical parameters. Previously, the equipment was calibrated using wine samples tested according to established OIV protocols. The pH was determined using a pH meter. The total polyphenol content was evaluated using the Folin–Ciocalteu colorimetric technique, calibrated against gallic acid standards, and expressed as mg gallic acid equivalents per liter (GAE/L). Briefly, the samples were diluted, and then Folin–Ciocalteu reagent and sodium carbonate were added. The solution was reacted at 40 °C for 30 min. A spectrophotometer (Jenway 6305 UV/vis, Jenway, Essex, UK) was used to determine the absorbance of the solution at 760 nm. Each analysis was conducted in duplicate.

2.10. Volatile Profile

Volatile organic compounds (VOCs) were measured by solid-phase microextraction coupled with gas-chromatography (GC-MS-SPME) utilizing a Clarus SQ8S chromatography/mass (GC-MS) spectrometer (Perkin Elmer, Boston, MA, USA) as described previously [17]. By comparing the retention duration of pure chemicals (Sigma-Aldrich, Milan, Italy) analyzed under identical conditions, volatile substances were found. The internal standard was 2-methylhexanol. A provisional identification was determined by comparing MS fragmentation patterns to those in the National Institute of Standards and Technology database (NIST version 2005). All analyses were conducted in duplicate.

2.11. Odor Activity Values (OAVs)

The odor activity value (OAV) was determined to assess the contribution of a chemical ingredient to the wine’s scent. It was determined by dividing the concentration of an individual compound by the perceptual threshold (OTV) stated in the literature.

2.12. Statistical Analysis

The program Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to analyze data and create graphs. The results were reported as the mean value ± standard deviation.

3. Results and Discussion

3.1. Ampelographic Descriptions

Over the decades, a program of rediscovery and recovery of minor grapevine varieties has been launched in Abruzzo Region (central Italy) in order to meet consumers’ demand for products strictly linked to the territory of origin and with unique sensorial traits. Vitis vinifera L. cv. Nero Antico di Pretalucente can be considered a minor grapevine variety located in ancient vineyards in the Gessopalena and Torricella Peligna villages. The ampelographic characteristics assessed in Vitis vinifera L. cv. Nero Antico di Pretalucente, reported in Table S1, constitute a unique profile as emerges from the morphological characteristics of the shoot, leaf, cluster and berry shape. The bunches are medium-large and loose, and the berries are medium in size, with thick skin.

3.2. Yield, Vine Phenology, and Grape and Berry Characteristics

Pooling the data across trial seasons 2019, 2020, and 2021 showed that Vitis vinifera L. cv. Nero Antico di Pretalucente is a grapevine variety characterized by a late budburst and medium fertility of the buds, with 1.4 bunches produced for each bud maintained in the vine with winter pruning (Table 1). Under the conditions examined, the yield per vine was 6.7 kg attributable above all to the high average bunch weight. The flowering is on average, during the first ten days of June, the veraison is late in that is about half way through September, and the ripening is quite late. In all years, the harvest was, in fact, carried out during the third week of October (Table 2). Interestingly, there was a high percentage of skin, at about 16% of fresh weight. In other black grape varieties, this percentage usually ranges from 10% to 12%, which gives a greater resistance against filamentous fungi and harmful insects.

3.3. Analysis of SSR Regions

SSR markers have been widely used for the identification of synonyms and homonyms, as well as the analysis of the parentage of grapevine varieties. The Italian Vitis Database (http://www.vitisdb.it, accessed on 21 November 2022) and the Registro Nazionale delle Varietà di Vite collect ampelographic and ampelometric descriptions, as well as biochemical and microsatellite profiles of approximately 800 grapevine varieties cultivated on the Italian Peninsula. This information is used to establish the parentage and relatedness of grape varieties. In this study, nine SSR loci were studied to assess the genetic profile of Vitis vinifera L. cv. Nero Antico di Pretalucente (Table 3). These loci were chosen due to their combination of polymorphism, reproducibility, and their codominant nature and have been added to the OIV register. It is assumed that two different plants having the same profile for the 9 SSR loci represent the same grape genotype; two related varieties in the first degree share at least one allele per marker [18]. The allele sizes expressed in base pairs for the nine SSR loci are reported in Table 3. The loci VVS2, VVMD7, VrZAG79, VVMD25, VVMD28, and VVMD32 were heterozygous, while VVMD5, VVMD27, and VrZAG62 were homozygous. The Italian Vitis Database revealed a unique SSR profile, suggesting that Vitis vinifera L. cv. Nero Antico di Pretalucente could be considered a new cultivar. Moreover, the frequency of homozygous loci could be due to natural events of self-fertilization linked to its geographic isolation. This event has also been previously described for Tannat, an ancient “cépage” planted in the Madiran region of France [19].

3.4. Viable Yeast Count

The plate count was used to monitor the ratio between total yeast and non-Saccharomyces. The total yeast count after crushing (T0) was approximately 8 Log CFU/g, while non-Saccharomyces yeasts reached 7.3 log CFU/g, indicating that the majority of yeasts present did not belong to the Saccharomyces genus (Figure 1). This result is consistent with prior findings that non-Saccharomyces yeasts are the most prevalent on grapes [20]. A lower concentration is generally found in grapes. However, Guerzoni and Marchetti [21] and Sabate et al. [22] found a population ranging from 102 to 107 CFU/g of grape. This issue could be related to the occurrence of some apparently healthy berries that are not completely intact, which could result in an increase in the total number of copiotrophic species. The total yeasts reached 8.7 log CFU/mL after seven days of fermentation, but the quantity of non-Saccharomyces decreased by approximately one log (6.9 log CFU/mL). At the end of fermentation, non-Saccharomyces yeasts were almost not detected, but the total yeast concentration was 7.7 log CFU/mL.

3.5. Fungal Taxonomy Diversity

Grape berries host several microbes which are involved in the must fermentation, shaping a wine’s characteristics [23]. Therefore, it is important to identify the grape-borne microorganisms, especially in poorly studied cultivars, such as Vitis vinifera L. cv. Nero Antico di Pretalucente, in order to improve our knowledge concerning the “microbial terroir”. The fungal genera detected are reported in Figure 2A. Aureobasidium spp. (31.2%) was the main genus detected, followed by Metschnikowia spp. (19.1%), Hanseniaspora spp. (14.8%), Botrytis spp. (12.4%), Cladosporium spp. (8.7%), Pichia spp. (3.5%), Alternaria spp. (2.9%), Epicoccum spp. (2.9%), Vishniacozyma spp. (3.9%), and Candida spp. (0.6%).
At the species level, A. pullulans was the main species (34.2%), followed by A. alternata (25.9%), H. uvarum (16.8%), C. cladosporoides (14.9%), V. victoriae (4.8%), and E. nigrum (3.4%) (Figure 2B). For the other genera detected, it was not possible to assign the corresponding species. NGS analyses are principally targeted on highly conserved regions. It is not easy to obtain correct taxonomic assignments at lower taxonomic levels [24]. In fact, this method is more reliable for genus-level identification, or even higher taxonomic levels [25]. Moreover, the correspondence of OTUs with species can be unreliable because some species have genes that are 97% similar, which will result in merged OTUs containing multiple species [26]. DNA may not be recovered from all genotypes; in fact, less abundant ones could not be detected as in the case of S. cerevisiae on grapes [26]. In fact, it is found in low concentration on grapes [27].
Aureobasidium pullulans is a yeast-like fungus that can live as an endophyte or an epiphyte. It has been isolated from plant surfaces, soil, and grape berries [28,29]. According to prior studies, it is one of the most prevalent yeast species isolated from grape berries at all maturity stages and other vine tissues from both diseased and healthy vines [27]. The high relative abundance identified in this investigation is consistent with these findings. Its presence on Vitis vinifera L. cv. Nero Antico di Pretalucente may be of interest due to its biocontrol potential against significant grape diseases such as gray mold and bunch rot caused by the Aspergillus species [28]. In addition, it can breakdown and eliminate ochratoxin A, eliminating wine contamination [30]. Alternaria species are the predominant component of wine grape mycobiota in a variety of wine-producing countries across the world [31], mostly due to their unique lifestyle, which produces highly melanized hyphae capable of resisting and growing under extreme UV radiation. It is capable of producing chemicals that are harmful to grapes [31].
Cladosporium cladosporioides is known as the causal agent of Cladosporium rot in vineyards and can develop as a symbiont with many plants [32].
The presence of H. uvarum, E. nigrum, and V. victoriae is notable because of their biocontrol properties. According to Barata et al. [27], H. uvarum is regarded a copiotrophic group with higher nutritional requirements, necessitating a high availability of nutrients, as is the case with harvest-ready grape berries. It is a slightly fermentative yeast with anti-Alternaria spp. action [33]. In addition, Liu et al. [33] demonstrated the organism’s biological control activity against B. cinerea on Kyoho wine grapes.
Recently, V. victoriae was identified in biodynamic Montepulciano grapes [29]. It demonstrates biological control activity against the bunch rot pathogen B. cinerea, and Penicillium expansum, the causative agent of table grape postharvest rot [34].
Epicoccum nigrum is connected with various crops, such as grapevine [35]. It exhibits biological control activity against grape fungal diseases, including B. cinerea and Plasmopara viticola, respectively, the causative agents of grape grey mold and downy mildew [35]. The co-existence of E. nigrum and A. pullulans in the same grapevine tissues has been reported [35], suggesting that they coexist in the same host. Their co-occurrence might positively influence the grape microbial community and wine characteristics. For example, they can inhibit or retard the establishment of pathogens, e.g., B. cinerea, and at the same time reduce ochratoxin accumulation, inhibiting Aspergillus spp.

3.6. Main Physico-Chemical Parameters of Wines

Vitis vinifera L. cv. Nero Antico di Pretalucente fermentation was spontaneously carried out in the laboratory, and the kinetics are reported in Figure 3. The fermentation started slowly, but in general, a typical fermentation kinetic, without stops in fermentation or too-long fermentation periods, was observed. Generally, spontaneous fermentation presents a longer initial phase and lasts longer than inoculated fermentations, which could positively impact the production of aroma compounds, especially in neutral grape varieties such as Trebbiano Abruzzese [22,36,37]. Despite these differences in the fermentation rate and lag phase, spontaneous fermentations, often, evolve correctly. For instance, Rodríguez et al. [37] revealed that the kinetic of inoculated fermentation was comparable to those presented by spontaneous fermentations carried out at laboratory scale. Uzkuç et al. [38] obtained that spontaneous fermentations took 4 and 7 days longer in Karalahna and Cabernet Sauvignon wines compared to inoculated fermentations. However, the fermentation process completed after 20 days without sluggish or stuck fermentations. Similar results have also been obtained by other authors [37,38,39].
The oenological parameters are reported in Table 4. The obtained wines had a final ethanol content of 12.75% (v/v), and the residual sugars were 0.71 g/L. The majority of sugars were consumed after 7 days (194.66 g/L), suggesting the development of fermentative strains. Autochthonous non-Saccharomyces yeasts trigger the alcoholic fermentation. Uzuk et al. [38] highlighted that spontaneously fermented Karalahna and Cabernet Sauvignon wines showed a higher ethanol content that wines resulting from inoculated fermentations. The great biodiversity of non-Saccharomyces yeasts occurring during the early stages of fermentation is related to several factors influencing the grape microbiota, e.g., vineyard localization, climatic conditions, cultivar, agronomic practices, stage of ripening, health of the grapes, harvesting procedures and the specific weather conditions in each vintage year [40]. An indigenous S. cerevisiae population is able to dominate the fermentation process, and the increasing ethanol content due to its development inhibits most of the non-Saccharomyces species due to their lower ethanol resistance [40]. The wines showed a pH of 3.4, and a volatile acidity lower than 0.6 g/L. These values are similar to those obtained in inoculated fermentations, suggesting the correct outcome of the fermentation process. A higher concentration of this last parameter is considered undesirable because it increasingly gives an acetic taste to the wine. The content of glycerol reached 8.56 g/L at the end of fermentation, suggesting the presence of glycerol-producing strains. However, the levels of glycerol depend upon many factors. In this study, the content of glycerol could be related to the presence of non-Saccharomyces and to the fermenting temperature (25 °C) [41]. In fact, glycerol is generally more abundant at temperatures of about 20–25 °C. Ciani and Comitini [42] demonstrated that increasing the fermentation temperature from 16 to 20 °C favored the accumulation of glycerol. This compound positively influences wine quality. In fact, when it is present in concentrations above the threshold taste level of 5.2 g/L in wine, it contributes to sweetness. In addition, it has been linked to mouth-feel sensations by imparting “fullness” (also known as “viscosity” or “weight”) to wine. Glycerol is also believed to improve the overall balance between alcoholic strength, acidity, astringency, and sweetness, so imparting a degree of roundness and smoothness to the tongue [43]. The obtained data revealed that spontaneous malolactic fermentation occurred. In fact, the malic acid was almost completely converted into lactic acid (Table 4). Wines were characterized by the content of polyphenols and anthocyanins similar to those obtained for other red wines [44]. The intensity and tonality reveal a light red color with purple nuances. The low intensity and tonality values obtained can be attributed to the fact that the fermentation trials were performed in a low volume (500 mL) and, therefore, the cap was not managed.

3.7. Characterization of Aroma Compounds in Vitis vinifera L. cv. Nero Antico di Pretalucente Wine

The unique traits of a wine mainly rely on its volatile footprint. Therefore, the VOCs of spontaneously fermented Nero Antico di Pretalucente wine were determined (Table 5). A total of 45 compounds were detected, including: 13 higher alcohols, 18 esters, 6 organic acids, 3 ketones, 2 terpens, and 3 aldehydes. The most abundant compounds were esters (68 mg/L), and ethyl acetate (fruity, sweet, weedy, green), isoamyl acetate (sweet, banana, fruity), and diethyl succinate (fruity, cooked apple, ylang) were the main ones.
Several studies revealed that spontaneous fermentations are associated with greater wine body, unusual or odd aromas and flavors, and greater complexity [45]. The development of indigenous yeasts is associated with a higher amounts of esters in spontaneously fermented wines than inoculated wines [46,47]. The synthesis of esters may be a result of the high expression of acyltransferase and alcohol acetyltransferase during spontaneous fermentation, which is greater when the fermentation rate is slower [38]. For example, ethyl acetate, which is one of the most important volatile compounds in wines, is produced in greater quantities when the rate of spontaneous fermentation is lower and is primarily dependent on the metabolic activity of non-Saccharomyces yeasts, which produce more ethyl acetate than Saccharomyces strains [48]. The occurrence of the Pichia, Candida, and Metschnikowia genera detected in this study could be associated with ethyl acetate production. In fact, they have been described as producers of esters, especially ethyl acetate [49].
A final concentration of 65 mg/L of higher alcohols was reported. Below 300 mg/L, they contribute positively to wine complexity; over 400 mg/L, they have a negative effect on wine quality. These chemicals are secondary products of yeast metabolism and can be generated through either the anabolic pathway from glucose or the catabolic pathway from their respective amino acids [50]. Since the content of amino acids is dependent on the grape variety, all of these volatile chemicals are dependent on the grape variety [50]. 1-butanol-3-methyl (fusel, alcoholic, whiskey, fruity, banana) and phenylethyl alcohol (sweet, flowery fresh, bready, rose, honey) were the most abundant. These two compounds are often associated with the development of some non-Saccharomyces such as Hanseniaspora spp. or Pichia spp. Their production seems to be strain-dependent and related to the presence of S. cerevisiae [49]. However, it is important to underline that the amount of phenylethyl alcohol in wines is dependent on grape variety, ripeness, and winemaking processes [51], which suggests that Vitis vinifera L. cv. Nero Antico di Pretalucente grapes may contain its precursors. In addition, spontaneous fermentation could positively influence its buildup. Garde-Cerdan and Ancin-Azcilicueta [48] obtained a considerable yield of 2-phenyl ethanol from the spontaneously fermented musts of Parellada.
The effect of the fermentation process (spontaneous vs. inoculated) on higher alcohols is still a matter of debate. Varela et al. [45] stated that spontaneously fermented Chardonnay wines had higher concentrations of higher alcohols than inoculated wines. In a recent study, it is suggested that inoculated Cabernet Sauvignon wines had a greater concentration of higher alcohols [52]. In another study, no significant differences were found in the concentration of total higher alcohols between spontaneous and inoculated wines [48].
Terpenes add floral notes to wine and belong to the secondary plant constituents. Several studies reported that they could be used for grape characterization [49] since, even if microorganisms are able to synthetize terpenes, their concentration does not show significant changes during the fermentation [49]. Nerol showed OAV of >1 suggesting that it can be considered a terpen associated with the Nero Antico di Petralucente cultivar.
Organic acids were also detected, with octanoic acid being the main one. However, they were present in low amounts, and none of them reached the OAV. Yeast and bacteria produce this class of VOCs during fatty acid metabolism. They can contribute to the complexity of the wine bouquet even at sub-sensory threshold levels, but have a negative effect on the wine scent when present in excess of their thresholds [53].
Due to their low sensory threshold values, aldehydes play a crucial role in wine scent. They are the primary products of lipid oxidation and the degradation or autooxidation of unsaturated fatty acids via hydroperoxides, and they may be transformed to alcohols or acids during fermentation [53]. The content of aldehydes might be due to the occurrence of Hanseniaspora. In fact, the genus Hanseniaspora has been characterized as a high producer of aldehydes such as acetaldehyde, benzaldehyde, 4-ethylbenzaldehyde, and benzene acetaldehyde [49]. It is intriguing to emphasize nonanal’s presence; in fact, it is considered a potentially active odorant of wine even at a low concentration [54].
Three ketones have been identified: β-ionone, 2,3-butanedione, and 3-hexanone. Ketones are primarily produced through lipid oxidation, as well as citrate and glucose metabolism. Of relevance is the presence of β-ionone, which is characterized by a distinct aroma of violets. Like the monoterpenes, the norisoprenoids are varietal aroma compounds and occur predominantly as glycosidically bound precursors [55]. Consequently, their presence in Nero Antico di Pretalucente wines is directly tied to the aroma traits of this variety.
OAVs were computed in order to assess the contribution of observed volatile compounds to the olfactory perception of wines. Only compounds with OAVs of >1 contribute to wine fragrance. The detected VOCs have distinct odors that were described using a database (www.thegoodscentscompany.com, accessed on 21 November 2022). Fruity, sweet, and green were the most common scents, occurring in the vast majority of compounds with a descriptor. A total of 15 aroma compounds showed an OAV of >1 (Table 6). The highest values were for decanal, β-ionone, ethyl octanoate, and ethyl isovalerate, suggesting their contribution to the overall aroma of Nero Antico di Pretalucente wines. In particular, nerol and β-ionone could be considered important aromas of this variety.
The concentration and the type of volatile compounds are affected by indigenous microorganisms. Indigenous yeasts are better adapted to the micro-conditions of fermentation and the composition of the grape must, which is beneficial to producing wines with unique characteristics [56]. Previous studies highlighted that spontaneous fermentation improved the aroma complexity of some wines, e.g., Cabernet Sauvignon, Chardonnay and Vidal, proving the contribution of non-Saccharomyces to the formation of wine flavors [57].

4. Conclusions

The results obtained in this study contributed to the description and valorization of a local grape variety grown in a mountains area, where the interest for this cultivation is strongly improved, thanks also to several young producers, who are planting new vineyards of Vitis vinifera L. cv. Nero Antico di Pretalucente. This is a strength of the Italian viticultural–oenological system, which can offer many unique and unrepeatable products linked to a specific region on the global market. On the basis of SSR and ampelographic profiles, Vitis vinifera L. cv. Nero Antico di Pretalucente can be considered a new cultivar and can represent a reservoir of microbial biodiversity. Further studies are necessary to prepare a culture collection of wine microbes associated with this cultivar in order to preserve the microbial biodiversity and select strains useful to protect the characteristics of wines obtained from ancient cultivars, maintaining the link with the land of origin. This study represents a first step for the valorization of this variety which could also be used with success for the production of white and rosé wines, as well as sparkling wines, thanks to the high level of organic acids in the must at harvest. Vitis vinifera L. cv. Nero Antico di Pretalucente could help to showcase the regional terroir, and could expand the wine list of the Abruzzo Region together with other native vines already cultivated with success. Moreover, it could improve the development of these difficult mountain areas.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9020150/s1, Table S1: Descriptors proposed by the International Organisation of Vine and Wine (OIV, 2007) for the description of young shoots, shoots, young leaves, adult leaves, bunches and berries in one black-berried grapevine accession from the Abruzzo Region.

Author Contributions

Conceptualization, R.T., A.P. and E.D.; methodology, G.P., A.P.R., M.P., B.D., L.G., C.Z. and N.B.; formal analysis, G.P. and G.A.; data curation, G.P. and L.G.; writing—original draft preparation, R.T., E.D., G.P., A.P. and L.G.; writing—review and editing, R.T. and G.P.; supervision, R.T.; funding acquisition, R.T. All authors have read and agreed to the published version of the manuscript.

Funding

This reserach was funded by the Municipality of Gessopalena (Caratterizzazione ampelografica e valutazione viticola ed enologica di un vitigno minore abruzzese denominato Nero Antico di Petralucente). Marta Pulcini PhD position was funded by Fondo per lo Sviluppo e la Coesione (FSC).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors acknowledge the Orsogna winery for their kind collaboration.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00150 g001 550
Figure 1. Yeast cell counts detected during the vinification process.
Figure 1. Yeast cell counts detected during the vinification process.
Fermentation 09 00150 g001
Fermentation 09 00150 g002 550
Figure 2. Relative abundance of Vitis vinifera L. cv. Nero Antico di Pretalucente grape fungal community detected by NGS at harvest time. (A) Main genera detected; (B) main species detected.
Figure 2. Relative abundance of Vitis vinifera L. cv. Nero Antico di Pretalucente grape fungal community detected by NGS at harvest time. (A) Main genera detected; (B) main species detected.
Fermentation 09 00150 g002
Fermentation 09 00150 g003 550
Figure 3. Fermentation kinetic of Vitis vinifera L. cv. Nero Antico musts. Data are means ± standard deviations of two independent experiments, each carried out in triplicate.
Figure 3. Fermentation kinetic of Vitis vinifera L. cv. Nero Antico musts. Data are means ± standard deviations of two independent experiments, each carried out in triplicate.
Fermentation 09 00150 g003
Table
Table 1. Yield component at harvest (average values 2019–2021).
Table 1. Yield component at harvest (average values 2019–2021).
CharacteristicsNumber
Bud fertility 1.4 ± 0.2
Bunches per vine (n°)16 ± 3
Yield/vine (kg)6.7 ± 0.5
Bunch weight (g)420 ± 19
Berry weight (g)3.1 ± 0.4
Berries per bunch (n°)135 ± 14
Berry skin (%)16 ± 1.10
Table
Table 2. Phenological stage dates for Vitis vinifera L. cv. Nero Antico di Pretalucente vines.
Table 2. Phenological stage dates for Vitis vinifera L. cv. Nero Antico di Pretalucente vines.
Phenological StageDate 2019Date 2020Date 2021
Budburst
(green shoot tips clearly visible = 50% of total buds)		18 April20 April17 April
Flowering
(50% of flowers open and caps fallen)		7 June8 June5 June
Veraison
(50% of berries had changed color)		13 September16 September11 September
Ripening
(100% berries completely black) 		19 October20 October18 October
Table
Table 3. Allele sizes in base pair of nine SSR loci of Vitis vinifera L. cv. Nero Antico di Pretalucente.
Table 3. Allele sizes in base pair of nine SSR loci of Vitis vinifera L. cv. Nero Antico di Pretalucente.
AccessionSSR Locus
VVS2VVMD5VVMD7VVMD27VrZAG62VrZAG79VVMD25VVMD28VVMD32
Nero Antico di Pretalucente133:151234:234241:247183:183190:190240:248254:262233:257251:271
Table
Table 4. Main oenological parameters determined at different times (T0, T7, and at the end of fermentation—T20).
Table 4. Main oenological parameters determined at different times (T0, T7, and at the end of fermentation—T20).
Oenological ParametersT0T7T20
Alcohol (% v/v)-11.44±1.3412.75±2.07
Residual sugars (g/L)213.63 ± 12.7818.97 ± 2.080.71 ± 0.08
pH3.41 ± 0.563.37 ± 1.143.48 ± 0.24
Total acidity (g/L) *5.10 ± 0.095.11 ± 0.435.28 ± 0.14
Volatile acidity (g/L) **0.07 ± 0.010.38 ± 0.030.54 ± 0.03
Malic acid (g/L)2.52 ± 0.122 ± 0.050.17 ± 0.02
Lactic acid (g/L)-0.3 ± 0.011.31 ± 0.05
Glycerol (g/L)-8.09 ± 1.998.56 ± 1.98
Anthocyanins (mg/L)32 ± 5.7885 ± 12.92266 ± 6.64
Polyphenols (GAE/L)368 ± 8.93728 ± 7.88977 ± 12.72
Intensity 3.8 ± 0.65
Tonality 0.76 ± 0.17
* expressed as tartaric acid. ** expressed as acid acetic.
Table
Table 5. Volatile compounds detected in Nero Antico di Pretalucente wines.
Table 5. Volatile compounds detected in Nero Antico di Pretalucente wines.
Higher Alcoholsmg/L
(S)-3,4-Dimethylpentanol0.24 ± 017
1-Butanol, 2-methyl4.15 ± 0.12
1-Butanol, 3-methyl27.12 ± 1.99
1-Hexanol0.24 ± 0.03
1-Pentanol2.13 ± 0.32
2,3-Butanediol0.21 ± 0.023
2-Heptanol0.77 ± 0.11
Phenylethyl alcohol20.37 ± 4.78
1-butanol3.23 ± 0.98
1-octanol0.65 ± 0.06
1-nonanol0.77 ± 0.05
1-dodecanol3.08 ± 0.84
1-propanol2.88 ± 0.56
TOT65.84
Esters
Isoamyl acetate9.05 ± 1.02
Phenethyl acetate0.03 ± 0.01
Ethyl isovalerate0.23 ± 0.18
Pentanoic acid, 2-methyl-, butyl ester4.67 ± 0.94
Pentanoic acid, 2,2-dimethyl-, methyl ester2.66 ± 1,03
Pentanoic acid, 3-methyl-, ethyl ester2.88 ± 0.06
Pentanoic acid, 4-methyl-, ethyl ester1.98 ± 0.05
Undecanoic acid, 11-bromo-ethylester0.77 ± 0.12
2-pheylethyl ethanoate3.55 ± 0.05
Hexyl-ethanoate2.93 ± 0.66
Ethyl phenyl acetate3.54 ± 1.12
Ethyl decanoate 6.63 ± 2.14
Ethyl 9-decenoate0.03 ± 0.01
Ethyl acetate14.77 ± 1,32
Ethyl hexanoate1.12 ± 0.25
Ethyl octanoate2.34 ± 2.76
Diethyl succinate8.18 ± 1.93
Ethyl lactate2.77 ± 0.07
TOT68.11
Organic acids
n-Decanoic acid0.06 ± 0.01
Hexanoic acid0.12 ± 0.05
Butanoic acid0.55 ± 0.04
Propanoic acid0.24 ± 0.03
Acetic acid0.06 ± 0.02
Octanoic acid1.12 ± 0.09
TOT2.15
Ketones
β-ionone0.02 ± 0.01
2,3-butanedione0.06 ± 0.02
3-hexanone0.03 ± 0.01
TOT0.11
Terpenes
Nerol0.41 ± 0.02
Terpinen-4-ol0.03 ± 0.01
TOT0.44
Aldehydes
Benzaldehyde0.34 ± 0.06
Decanal0.44 ± 0.03
Nonanal1.12 ± 0.05
TOT1.90
Table
Table 6. Volatile compounds with OAV >1.
Table 6. Volatile compounds with OAV >1.
Higher AlcoholsOTV (mg/L)Odour DescriptionOAV
2-Heptanol0.2 **Fresh lemon, grass, herbal, sweet, floral, fruity 3.85
Phenylethyl alcohol14 **Sweet, floral, fresh, bready, rose, honey 1.46
1-octanol0.12 ***Waxy, green, aldehydic and floral with a sweet, fatty, coconut nuance5.42
1-dodecanol1 **Earthy, soapy, waxy, fatty, honey, coconut 3.08
Esters
Isoamyl acetate0.16 **Sweet, banana, fruity, ripe, estery 56.56
Ethyl isovalerate0.001 **Fruity, sweet, apple, pineapple230
Ethyl decanoate0.2 *Sweet, waxy, fruity, apple, grape, oily, brandy33.15
Ethyl acetate12.26 **Ethereal, fruity, sweet, weedy, green 1.20
Ethyl hexanoate0.014 *Sweet, fruity, pineapple, waxy, green, banana78.57
Ethyl octanoate0.005 *Fruity, wine, sweet, apricot, banana, brandy, pear468
Diethyl succinate1.2 **Mild, fruity, cooked, apple, ylang6.82
Ketons
β-ionone0.00009 *Floral, woody, sweet, fruity, berry, tropical, violet 222
Terpenes
Nerol0.40Neroli, citrus, magnolia1.03
Aldehydes
Decanal0.001 **Sweet, aldehydic, waxy, orange, peel, citrus, floral 440
Nonanal0.015 **Waxy, aldehydic, rose, fresh, orange, peel 74.67
* Gomez-Mıguez et al. [58]. ** Welke et al. [59]. *** Fracassetti et al. [60].
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Perpetuini, G.; Rossetti, A.P.; Giordano, L.; Pulcini, M.; Dufrusine, B.; Battistelli, N.; Zulli, C.; Arfelli, G.; Palliotti, A.; Dainese, E.; et al. Characterization of Nero Antico di Pretalucente Wine and Grape Fungal Microbiota: An Expression of Abruzzo Region Cultivar Heritage. Fermentation 2023, 9, 150. https://doi.org/10.3390/fermentation9020150

AMA Style

Perpetuini G, Rossetti AP, Giordano L, Pulcini M, Dufrusine B, Battistelli N, Zulli C, Arfelli G, Palliotti A, Dainese E, et al. Characterization of Nero Antico di Pretalucente Wine and Grape Fungal Microbiota: An Expression of Abruzzo Region Cultivar Heritage. Fermentation. 2023; 9(2):150. https://doi.org/10.3390/fermentation9020150

Chicago/Turabian Style

Perpetuini, Giorgia, Alessio Pio Rossetti, Lucia Giordano, Marta Pulcini, Beatrice Dufrusine, Noemi Battistelli, Camillo Zulli, Giuseppe Arfelli, Alberto Palliotti, Enrico Dainese, and et al. 2023. "Characterization of Nero Antico di Pretalucente Wine and Grape Fungal Microbiota: An Expression of Abruzzo Region Cultivar Heritage" Fermentation 9, no. 2: 150. https://doi.org/10.3390/fermentation9020150

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