Multi-strain indigenous Yeast and Bacterial starters for ‘Wild-ferment’ Wine production

Final Report Summary - WILDWINE (Multi-strain indigenous Yeast and Bacterial starters for ‘Wild-ferment’ Wine production)

Executive Summary:
Aforetime, wines were produced by the resident grape/winery microbiota. Currently commercial Saccharomyces cerevisiae and Oenococcus oeni starter cultures are widely used to ensure a manageable process. Despite advantages, this may lead to sensory resemblance of wines from diverse origins, whereas exotic starters may fail to take over fermentation. Nowadays, the competitive nature of global wine market urges for the production of premium wines with regional character. Consumers also call for safe wines made according to natural and organic procedures. To this end, the use of indigenous S. cerevisiae or non-Saccharomyces (wild) yeasts is a tool to create wine complexity and authenticity, while selected lactic acid bacteria (LAB) may effectively control malolactic fermentation and thereby eliminate biogenic amines (BA).
The innovative scope of this project was to combine native S. cerevisiae with wild species and native O. oeni with other LAB in the development of peculiar yeast and bacterial blend starters, respectively, able to fulfill all the essential and desirable winemaking properties to serve as starters in induced wild fermentations for the production of specialty organic or conventional wines. For this purpose, the biodiversity of key EU viticultural areas screened to identify strains of enological importance as per their phenotypic characters and genetic traits. Indeed in the context of WILDWINE, more than five thousand yeasts and two thousand five hundred bacteria were isolated from grape musts at different stages of spontaneous alcoholic and malolactic fermentations, from various grapevine cultivars and vineyards in France, Greece, Italy and Spain and identified. Twenty one species of yeasts were identified while the occurrence of diverse species during fermentation varied among the different countries and samples tested. In most cases, Saccharomyces cerevisiae was the dominant population at the end of fermentation courses. Among the bacteria, Oenococcus oeni, Lactobacillus sp. and L. plantarum were the most abundant species recovered from must samples.
Genotyping of S. cerevisiae isolates showed that clustering of strains according to genetic proximity correlate well with their geographical origin, suggesting that isolates from different zones differ in terms of genetic content. This distribution argues in favor of a genetic signature of strains found in specific PDO zones.
Most S. cerevisiae and bacterial strains showed interesting technological properties such as ethanol and SO2 tolerance, little foam production, etc. Characterization of non-Saccharomyces yeasts revealed huge technological differences depending on species and strains.
The genetic stability and gene repertoires of candidate starter strains suggest that: 1) S. cerevisiae strains are considerably stable and probably well-adapted to the fermentation environment, 2) the copy number of genes of interest varies from strain to strain, with interesting differences between commercial starters and vineyard isolates, 3) genes involved in production of biogenic amines are present in a number of lactic acid bacteria isolated from Greece (tyramine and putrescine), absent in isolates from France and Spain, 4), all the lactic acid bacteria tested were genetically stable.
Finally microbial culture collections of oenological yeasts and bacteria, typed at the species and strain levels, from PDO zones have been generated to serve as a reservoir of enological strains for future needs and applications of the wine industry. Their fermentation potential was evaluated in pure micro-culture vinifications. S. cerevisiae strains showed a relative high variation in their ability to rapidly initiate fermentation. Nonetheless, the majority of strains were characterized by a good fermentation capacity.
Their eligibility has been validated in plant-scale fermentations and wines have been evaluated by sensory analysis and consumer acceptance testing. A survey that was carried out in order to assess the consumers’ perception and acceptance of “wild-ferment” wines, showed the intention of consumers in all countries to choose wines produced from non-commercial yeasts, although the quality and price of these wines are critical for the decision of purchasing such wines, while tasting showed higher preference for wild ferment wines.
At the end of the project strains of yeast and bacteria or blends have been selected by each participating group in the corresponding region. The associations and companies have been actively involved, their personnel has been trained, and decisions have been taken regarding the plan for use and exploitation of the results. Initially, the wineries may consider applying the selected strains in a small part of the produce by the “pied de cuve” method. Depending on the results, the wineries will consider the production of the starters in dry form by a company.
Eight (8) publications in peer reviewed journals, 13 papers in conference proceedings, 12 thesis and > 150 dissemination activities have been performed (website, press releases/ articles in local magazines & websites, posters and presentations in workshops/conferences, MSc and PhD theses, flyers, TV interviews, cordis, video clip.

Project Context and Objectives:
Background
Over the last years, the emerging ‘New World’ wine-producing countries have led to fierce competition for wine market share. In addition, consumers’ sophisticated preferences call for superior wines of distinct regional characteristics (terroir wines), as well as for wines made through natural and organic procedures. There is also an increasing interest from consumers for biogenic amine-free wines. Those trends are mirrored by the raise in the production of organic wine in the EU, the increased marketability of wines obtained without the addition of
commercial yeast, the shift from table to superior wines and the introduction of limits on biogenic amines content in wine. This raises an opportunity for ‘Old World’ wine-producing countries with peculiar terroirs and ancient tradition, like many European countries, to make a dynamic comeback with the production of novel wines of ultra-premium quality that will be created according to natural operations. The use of indigenous Saccharomyces or non-Saccharomyces yeasts and lactic acid bacteria (LAB) may offer a great potential in addressing the aforementioned critical issues in modern wine making.
Yeasts and bacteria form the core of wine making. Yeasts conduct the alcoholic fermentation, and also have a prominent role in shaping wine quality. Different yeast species or strains impart vitally diverse organoleptic profiles to wines, adding to the complexity and richness of wine aroma and flavor. Currently, winemakers worldwide add commercial Saccharomyces cerevisiae as starter cultures, to ensure a predictable, reproducible and controlled fermentation. However, the generalized use of prescribed starter cultures that obscure the native microbiota results in the resemblance of analytical and sensory properties of wines, depriving them of variability, complexity and personality. On the other hand, the use of indigenous S. cerevisiae or non- Saccharomyces (wild) yeasts is a tool to create authenticity. Indigenous yeasts can assure the evolvement of the typical sensory properties of wine from a given region while the influence of wild yeasts marks the ‘wildferment’ character of wines.
The addition of industrial yeast starters also conflicts markets’ needs for wines that are produced under more natural and/or organic practices. This trend is reflected by an increase in the organically farmed grapes during the last years, which are required for the production of organic wine in the EU (FiBL, 2010). Although the establishment of specific rules at the stage of winemaking is still debated, it is expected that organic wine production will be further subjected to specific constraints. In this respect, specific label claims (e.g. ‘obtained without the addition of commercial yeast’ or ‘produced by indigenous yeasts’) will probably be anticipated under the common market organisation for organic wine. In addition, several national or private established standards already impose strict regulations for organic wine production, including the use of indigenous yeasts instead of industrial starters to conduct the fermentation.
To increase the influence of the native microbiota in local wine production many winemakers abolish or reduce the size of the inoculum (starter culture) recommended by yeast manufacturers, but this practice may result in uncontrolled fermentations and the production of unmarketable wines. Thus, there is a need to isolate enologically competent indigenous yeasts to be used as inocula for the production of ‘wild-ferment’ conventional or organic wines, but to accomplish this goal a previous thorough description and evaluation of isolates’ performance in winemaking is required.
In wine making, a secondary fermentation, referred to as the malolactic fermentation (MLF), is typically carried out after alcoholic fermentation in red wines and in certain whites of high acidity. This process is conducted by lactic acid bacteria (LAB), mainly strains of Oenococcus oeni. MLF results in a decrease in total acidity and an increase of soft mouth feel, flavour and microbiological stability of the final product. MLF may also have some undesirable effects on wine quality, due to the production of off-flavours, reduction in colour and formation of biogenic amines. The overall effect of MLF is principally dependent on the species and strains that perform the fermentation.
Traditionally, malolactic fermentations were conducted spontaneously by the native wine bacteria. This practice is still applied in several wineries worldwide. However, spontaneous MLF is highly unpredictable, in that the onset, the rate, the completion and the production of off-flavours and biogenic amines cannot be controlled.
To overcome these obstacles, the use of selected MLF starter cultures has been applied during the last four decades. Starter cultures can be selected on the basis of improving flavor and aroma, ensuring control of the time and the rate of MLF and reducing the potential for spoilage by other bacteria. Among LAB species, strains of O. oeni are preferably used because of their resistance to alcohol concentration, pH and SO2 content of wine.
Despite the clear benefits that selected MLF starters can deliver to the wine industry, only a small number of commercial starter cultures have been shown to successfully perform MLF, after the first O. oeni starter strain ML 34 was introduced in 1984. On the same time there is an increasing demand for new MLF starters with defined technological and flavouring properties, which will also meet proposed regulatory safety issues on biogenic amine production.

The main concept of the WildWine project was to exploit the indigenous microbial diversity in the development of original starter cultures to be used in the production of ‘wild-ferment’ terroir wines. The local biodiversity of important European viticultural regions will be thoroughly screened to identify strains of enological importance as per the phenotypic characters (e.g. aroma metabolome) and genetic traits. The innovative scope of the proposed project is to combine indigenous Saccharomyces with non-Saccharomyces cultures and indigenous O. oeni with LAB species in the formation of peculiar yeast and bacterial blends, respectively. These formulations will be carefully designed to fulfill all the essential and desirable winemaking properties, thus to serve as starter cultures in induced wild fermentations. Our goal was to assign a set of indigenous, novel and privileged strains to the respective PDO European districts to serve as “microbial signature” in the production of organic wines. Novel ‘wild-ferment’ wines will be produced using important grapevine cultivars that are among the most valuable and reputable varieties for high quality red or white wine production in Europe. The ultimate goal is to enable the SME-AGs and their members to diversify and deliver innovative, safe and consumer driven wines of premium quality that will confront the forthcoming regulations on organic wine production and biogenic amine content for national and global markets. By these means the project will assist the SME-AGs from the leading wine producing countries in Europe (France, Greece, Italy, Spain) to enhance their marketing abilities towards a more competitive and sustainable wine industry.

Scienti fic & Technological Objectives

To accomplish the goals of this project, the following scientific and technological objectives were established:
a) Assessment of the indigenous yeast and bacterial diversity involving the isolation of the local yeast and bacterial biodiversity associated with the respective terroirs, the identification of isolates at the species level and the typing of isolates at the strain level with advanced molecular techniques (PCRRFLP, gene sequencing, PFGE, microsatellites, PCR-DGGE, mtDNA restriction analysis). The regionspecific biodiversity will be extensively screened, thus a sufficient number of different indigenous S. cerevisiae, non-Saccharomyces and LAB species/strains will have been isolated by month 9 to serve as a basis for further starter development (Milestone 1). Significant knowledge will be gained in aspects concerning: (a) the biogeography of indigenous yeast and bacterial microbiota, (b) the molecular polymorphism among strains and (c) protocols and molecular identification schemes for non-Saccharomyces yeasts.
b) Pre-selection of strains with enological potential based on the evaluation of the enological characteristics of yeasts and bacteria by in vitro tests (plate analysis, TLC, challenge tests), the assessment of the genetic stability of strains with enological potential through karyotype analysis and the evaluation of the key enological traits of yeasts and bacteria by genetic and molecular analyses (real time quantitative PCR (qPCR) assays or genome sequencing). Transcriptional analysis of genes, whose upregulation has been directly linked with increased production of various desirable aroma compounds, will be also conducted. The generation of an enological yeast and bacteria culture collection for the respective viticultural zones will be conducted. By correlating key enological phenotype with genetic traits information on the ‘commercial wine strain signature’ list will be established. Various yeasts and bacterial strains bearing essential enological characteristics, such as high ethanol production and SO2 resistance, is expected to be identified by month 15 (Milestone 2) to be used in subsequent microfermentations.
c) Evaluation of the performance of pre-selected strains during micro-fermentations inoculated individually, successively or simultaneously in sterile grape must (in vivo) and in naturally processed grape must, containing the natural microbiota (in situ), on the basis of fermentation kinetics (using advanced tools), chemical and sensory characteristics of the finished wines. Significant knowledge will be gained for (a) the behavior of indigenous enological strains under winemaking conditions, (b) aroma metabolome of strains during winemaking, (c) yeast-yeast and yeast-bacteria interaction during fermentation and (d) the kinetic and survival patterns of the starter cultures. Based on the microbial dynamics and metabolomics during micro-fermentations of sterile musts the selection of competent enological yeasts and bacteria will be accomplished by month 24 to be further used in starter development (Milestone 3). Strains that prevail over native microbiota and result in the production of quality wines will be finally selected for the formation of starter cultures and will be determined by fermentation kinetics in natural processed grape must and chemical/sensory analyses of producing wines by month 30 (Milestone 4).
d) Implementation of selected strains and blends as starter cultures in pilot scale wine production. During this phase, the performance of indigenous yeast blends, bacterial strains and inoculation protocols will be assessed in pilot-scale production of ‘wild-ferment’ wines at the premises of the wineries. The successful application of the developed starters in industrial wine production will be validated by tracing inoculated strains and evaluating the chemical and sensory profiles of the wines by month 36 (Milestone 5). This knowledge will allow wine industry to apply induced wild fermentations in the development of novel products that are reproducible, premium, attractive to consumers and in accordance with the demands of the global market for natural allergen-free wines.
e) Consumer studies will be implemented for evaluating the market potential for ‘wild-ferment’ terroir wines produced by the selected indigenous yeasts and bacteria.
f) Exploitation, dissemination and training activities will be carried out to support the exploitation and to protect the Intellectual Property Rights of the project results, to disseminate the knowledge resulting from the project both to the members of the SME-AGs of the consortium and beyond to a wider audience and to train the SME-AGs and their SMEs to facilitate the take-up of the project results. All these activities will be conducted with the aim to improve scientific and technological level as well as competitiveness of the European wine sector. All the RTDs will work in close collaboration in WP7, to ensure that each of the components of WildWine work properly once integrated.
g) Management to optimize the applications of resources and to ensure that all aspects of the EU requirements for communication and reporting are met.
Project Results:
State of the art:
In traditional spontaneous fermentations, the microbiota that is present on the surface of the grape skins participate in the so called natural wine fermentation (Pretorius et al., 1999). During crushing, the natural microbiota passes into the grape juice and may be further enriched by resident species in the winery equipment. Non-Saccharomyces yeasts, like Hanseniaspora, Candida, Metschnikowia and Pichia spp., prevail during the early stages of grape must fermentation (Fleet, 1999). However, as the fermentation progresses the non-Saccharomyces spp. become suppressed with increasing alcohol content, leaving S. cerevisiae strains to predominate and complete the process (Fleet, 1999; Lema et al., 1996). Lactic acid bacteria (LAB) are also encountered on grape surfaces, albeit at relatively low populations. Upon crushing, native LAB belonging to Lactobacillus Leuconostoc and Pediococcus pass into the juice, which is further enhanced by winery species (Jackson 2008). There are several species of LAB evolving in fermenting grape must, including L. plantarum, L. hilgardii, L. brevis, P. damnosus, P. pentosaceus, Leuc. mesenteroides and O. oeni (Ribéreau-Gayon et al., 2006). Most of these species do not multiply during alcoholic fermentation (Volschenk et al., 2006). After its completion, the surviving bacterial population may enter an active growth phase. At this stage, MLF will commence when the total population exceeds 106 cfu/mL (Ribéreau-Gayon et al. 2006).
Although, wines produced by spontaneous fermentations can develop distinct regional and other attractive characteristics, the risk of bad vintages that would allow for spoilage species to dominate, leading to stuck fermentations and diminished product quality, may not be excluded (Ciani et al., 2010). Therefore, the common practice to assure a ‘clear’ and efficient alcoholic or malolactic fermentation is to add a sufficiently large dose of a single strain of S. cerevisiae (yeast starter) or O. oeni (bacterial starter) to the grape juice or wine to take over the process. In this case, the inoculated strain becomes dominant early in the process, masking the indigenous microbiota. Industrial strains that are currently used in winemaking are commonly derived from grapes, grape musts, wines or winery equipment (Shinohara et al., 1994). Those strains are carefully chosen on the basis of several characteristics including fermentation rate, fermentation at low temperature, sulphur dioxide tolerance, high sugar and ethanol tolerance, low production of volatile acid, killer activity, low biogenic amine production . Starters are also selected to enhance flavor formation, thus producing more aromatic wines.
At present, there are about 200 strains of S. cerevisiae and several O. oeni commercially available that are used by winemakers worldwide. However, the generalised use of prescribed starter cultures results in the resemblance of analytical and sensory properties of wines, depriving them from regional variability, complexity and personality (Ciani et al., 2010). Therefore, such products do not meet current consumers’ requirements and wine market trend for premium wines that fully express their particular terroir. Moreover, the addition of industrial starters conflicts markets’ needs for wines that are produced under more natural and/or organic practices. This trend is mirrored by an increase in the organically farmed grapes during the last years, which are required for the production of organic wine in the European Union (FiBL, 2010).
Wild microbiota in winemaking:
Recently, there is an increasing interest in the industrial application of non- Saccharomyces yeast species as part of mixed starter formulations, together with S. cerevisiae (Ciani et al., 2010). Similarly, the use of different LAB species other than O. oeni is currently being considered (du Toit et al., 2011). Despite the significant contribution that wild yeasts and LAB may have in the production of superior wines, the concept of their use in wine production is quite new. Only recently Kluyveromyces thermotolerans was the first non-Saccharomyces species to be commercially released for use in winemaking (Anonymous, 2004). This product has been developed for the enhancement of floral and tropical fruit aromas and to provide more complex and rounded flavours to white and red wines, respectively. Since then, a few similar products have been released by the major manufacturers in the field that may combine commercial starters with non-Saccharomyces strain(s). The composition of those yeast starter blends is secret and in all cases is under intellectual property right protection.
In 2011, the first commercially available bacterial co-culture blend was introduced in the market. This starter comprises of Oenococcus oeni and Lactobacillus plantarum and it was developed by a research institute for a company. It is anticipated that the use of MLF starter cultures of LAB strains selected from the wine indigenous microbiota of a given region takes advantage of the natural adaptation of strains to wine characteristics, and may simultaneously maintain regional peculiarities (Izquierdo et al., 2004).
Genetic diversity and identification of yeast/bacteria isolates: In the benefit of preserving the local biodiversity and enhancing regional influence on wine character, grapes or fermenting musts of a region represent an important natural source of yeast and bacteria for starter culture development (Fleet 2008). Europe is believed to hold the world’s oldest, still under continuous cultivation, vineyards and it is highly likely to represent rich reservoirs of yeast strains of enological importance, due to their long-term concerted co-evolution with wine grapes.

The feasibility studies related to this project show that grape must is an untapped reservoir of yeast species, encompassing both S. cerevisiae and wild yeasts strains, which can dominate at some stage during fermentation.
Although indigenous S. cerevisiae is encountered at low numbers in fresh musts, in most cases it becomes dominant as alcohol accumulates during winemaking. (Fleet 1999; Nisiotou et al., 2007).
The addition of commercial S. cerevisiae at dosages suggested by manufacturers obscures wild yeasts, as well as native S. cerevisiae strains. In this case, the contribution of natural microbiota to wine characteristics is diminished. Thus, the isolation of suitable indigenous S. cerevisiae and wild yeasts to be used as starters can achieve natural fermentation characteristics, in a controlled and reproducible manner. The indigenous S. cerevisiae can assure the completion of fermentation, while non-Saccharomyces strains may confer the ‘wildferment’ character to regional wines. Isolates will be pre-selected on the basis of desirable enological properties, such as valuable enzyme production, low H2S formation, ‘killer’ phenotype etc. Final selection of yeasts has to focus on fermentation performances. Organoleptic and biochemical properties during winemaking, like production of desired aroma compounds, has to be considered. For monitoring yeast succession, a rapid and efficient methodology for strain detection and enumeration during the alcoholic fermentation course needs to be applied.
Previous studies show that native lactic acid bacteria, which bear genes for biogenic amine formation, reach populations of 106 cfu/ml during spontaneous MLF, whilst levels of 103 cfu/ml are required for production of 1 mg/ml of biogenic amines (Lucas et al., 2008). Winemaking trials show that the use of selected bacteria as starter cultures for conducting malolactic fermentation can inhibit the growth of undesirable bacteria and the production of biogenic amines (Nannelli et al., 2008). Different bacterial strains have been also found to deliver distinct organoleptic attributes to wine through the production of diacetyl, esters, polyols and b-glucosidase.
Thus, the selection of competent indigenous bacteria for use as starter cultures can achieve MLF in a controlled and reproducible manner, eliminate biogenic amines and enhance the sensory qualities of the wine. Bacteria need to be pre-selected on the basis of desirable enological properties, such as valuable enzyme production, ethanol and SO2 tolerance, growth temperature range, low biogenic amines formation etc. Final selection of starter cultures needs to focus on MLF performance and production of biogenic amines during winemaking. Additionally, organoleptic and biochemical properties, like production of desired aroma compounds, needs to be also considered. For monitoring bacterial strains, a rapid and efficient methodology for strain detection and enumeration during the malolactic fermentation course has to be applied.
The main S & T results/foregrounds in the respective WP of the project are:

WP2. Assessment of the indigenous yeast & bacterial diversity
Yeasts and bacteria were isolated from grape musts at different stages of spontaneous alcoholic and malolactic fermentations. Samples were obtained from various grapevine cultivars and vineyards in France, Greece, Italy and Spain. Identification of yeast isolates at the species level was conducted based on PCR-RFLP and sequence analyses of the 5.8S-ITS or D1/D2 region of rDNA. From a total of 5336 yeasts isolated(1250 in France, 1580 in Greece, 1100 in Italy and 1406 in Spain), 3946 strains were so far assigned to 21 species, such as: Aureobasidium pullulans, Candida incommunis, C. zemplinina, Issatchenkia orientalis, Hanseniaspora uvarum, H. guilliermondii, Pichia anomala, P. kluyveri, Sacch. cerevisiae, Saccharomycodes ludwigii, Torulaspora delbruecki and Zygosaccharomyces cidri/fermentati. The occurrence of diverse species during fermentation varied among the different countries and samples tested. S. cerevisiae was the dominant population at the end of fermentation course in most cases (D2.1).
For bacteria identification purposes, the amplified ribosomal DNA restriction analysis (ARDRA) combined with sequence analysis of a partial 16S rDNA region, species-specific PCR or the variable number of tandem repeat (VNTR) PCR analysis was applied. Out of total 2680 bacteria isolated so far (975 in France, 279 in Greece, 350 in Italy and 1076 in Spain), 1734 strains were identified. Oenococcus oeni, Lactobacillus sp. and L. plantarum were the most abundant speciesrecovered from wine samples at different stages of spontaneous malolactic fermentations.
Genotyping of S. cerevisiae isolates was conducted by interdelta-pcr or microsatellites markers. A total of 2,430 S. cerevisiae isolates, 231 from France, 1,333 from Greece, 636 from Italy and 230 from Spain were analysed and 192, 144, 96 and 7 distinct molecular patterns were identified, respectively. The genetic diversity of S. cerevisiae isolates within different countries was relatively high, except for Spanish isolates. Although there was no clear clustering of Greek isolates accordingto the vineyard of origin, analysis of molecular variance (AMOVA) showed that the vineyard populations from Nemea differed significantly from those of Peza. The analysis of genetic structure conducted so far for the French isolates points to a genetic link between strains isolated from Saint-Emilion and Sauternes regions.
For genotyping non-Saccharomyces yeasts different methods have been applied such as TRtRNA-PCR, RAPD, SAU-PCR or microsatellite analysis (D2.2). Overall, similarly to S. cerevisiae, numerous highly diverse molecular patterns were detected for various
non-Saccharomyces species. For Greek isolates, there was not an obvious clustering of banding patterns according to the sampling point or the vineyard of origin. Nevertheless, in most cases, a clear structure between Nemea and Peza vineyard populations was detected. The numerous distinct molecular patterns identified between the two zones could be utilised as genetic signatures of terroir strains. Most of the Spanish isolates were different strains of H. uvarum species, without any clear grouping according to their origin. Furthermore, higher biodiversity was observed in Priorat’s vineyards with ecological handling than in those with traditional one.
Results from LAB genotyping showed that clustering of strains according to genetic proximity correlated well with their geographical origin and only few strains were detected simultaneously in several PDO zones, suggesting that isolates from different zones differ in terms of genetic content. This distribution argues in favor of a genetic signature of strains found in specific PDO zones.

WP3: Pre-selection of strains with enological potential
Different Saccharomyces and non-Saccharomyces yeasts and lactic acid bacteria strains that were isolated from PDO zones of Greece, Italy, France and Spain during activities reported in WP2 were characterized by RTD performers to evaluate their technological performance by in vitro tests (see Del. 3.1). Partners have analyzed strains from their respective regions following a number of phenotypic tests including, for yeasts: ethanol tolerance, SO2 resistance, H2S production, volatile acidity production, growth temperature range, maximum population size, biogenic amines formation, foam generation, killer activity, flocculation phenotype, enzymatic activities (b-glucosidase, esterase, pectinase, protease) and alcoholic fermentation kinetics, and for bacteria: ethanol tolerance, SO2 resistance, pH resistance, copper resistance, biogenic amines formation, resistance to freeze, survival to inoculation in wine and malolactic fermentation kinetics. Partners have analyzed several dozens or hundreds of Saccharomyces and non-Saccharomyces species and a few tens of Oenococcus and non-Oenococcus bacteria.
Most S. cerevisiae strains showed interesting technological properties such as ethanol and SO2 tolerance, little foam production, etc., which will make it possible to select candidates for the next steps in the development of multi-strains starters. Characterization of non-Saccharomyces yeasts revealed huge technological differences depending on species and strains and some of them were of interest for the development of multi-strain starter in the next steps. For example resistance to ethanol was important in a number of Lachancea thermotolerans and Candida zemplinina strains, whereas yeasts of other species were generally more sensitive, strains of Candida zemplinina and Pichia kluyveri were the most important producers of H2S, Hanseniaspora uvarum yeasts could not finish the alcoholic fermentation. With respect to bacterial technological properties, Lactobacilli and Pediococci did not tolerate high acidity or ethanol content, whereas some strains of O. oeni proved to resist to different wine conditions, particularly after an acclimatization step. Due to the lack of sufficient numbers of yeasts and bacteria strains isolated from samples of vintage 2012, partners have performed a second round of isolation/identification/typification of microorganisms collected in 2013.
In Del 3.2 the RTD partners DEMETER, UNITO, URV and UB2 have examined the genetic stability and gene repertoires of candidate starter strains from their respective PDO regions. Not all strains were fully characterized in order to give priority to the preparation of necessary industrial trials (WP4, WP5) in the 2014 vintage, which will be the last one covered by the project. Strains that performed the best in the vintage 2014 investigated deeper to unravel the genetics of specific signatures that would be relevant for the industrial application of the starter cultures developed in the project. Genetic characterizations performed suggest that: 1) S. cerevisiae strains are considerably stable and probably well-adapted to the fermentation environment, 2) the copy number of genes of interest varies from strain to strain, with interesting differences between commercial starters and vineyard isolates (DEMETER), 3) genes involved in production of biogenic amines are present in a number of lactic acid bacteria isolated from Greece (tyramine and putrescine), absent in isolates from France and Spain, 4), all the lactic acid bacteria tested to date are genetically stable.
Finally (see Del. 3.3) microbial culture collections of oenological yeasts and bacteria from their respective PDO zones have been generated: Peza and Nemea (Greece, DEMETER), Asti-Monferrato and Piedmont (Italy, UNITO), Priorat (Spain, URV), Saint-Emilion and Sauternes (France, UB2). Microorganisms were typed at the species and strain levels by molecular methods whenever possible and then stored in deep-frozen (-80°C) as pure cultures. The total number of strains in collections is:
- Saccharomyces cerevisiae : 55 (DEMETER), 96 (UNITO), 13 (URV), 26 (UB2)
- Non-Saccharomyces yeasts : 42 (DEMETER), 464 isolates (UNITO), 30 (URV), 141 (UB2)
- Oenococcus oeni : 8 (DEMETER), 16 (UNITO), 47 (URV), 47 (UB2)
- Non-Oenococcus lactic acid bacteria : 19 (DEMETER), 0 (UNITO), 30 (URV), 0 (UB2)
These collections served as a reservoir of enological strains for future needs and applications of the wine industry.
Additional experiments for O. oeni : Due to the absence of Oenococcus oeni from vineyard samples, DEMETER and URV performed additional experiments during 2013 vintage. To increase the probability of isolating O. oeni, samples were taken from winery tanks performing spontaneous MLF in Nemea and Priorat.
Lactic acid bacteria (LAB) were recovered from 8 out of the 9 tanks in Nemea (Greece) examined at populations ranging between 4-7 log CFU/ml. Eighty-eight isolates were further identified as Oenococcus oeni, Lactobacillus hilgardii and Pediococcus parvulus. Isolates were clustered by RAPD analysis into 38 groups of distinct molecular patterns. Different strains were further subjected to technological characterization. Most of the isolates tested resisted ethanol and SO2 up to 14% and 30 ppm, respectively. O. oeni and P. parvulus isolates were able to grow at low pH (i.e. at 3 or 3.5) in the presence of 7 % ethanol. LAB strains were also examined for the presence of genes involved in biogenic amine (BA) production. All O. oeni and P. parvulus strains were negative for the existence of BA producing genes. However, all three Lactobacillus hilgardii strains originating from the same winery tank were positive for the presence of tyramine and/or putrescine producing genes.
Due to the poor recovery of LAB in Spain (some 20 Lactobacillus and almost any Oenococcus) in the experimental set up from grapes in the vintage 2012, samples of 28 wines doing MLF from 7 different wineries were also taken in the same vintage. From these, a total of 965 isolates of O. oeni were obtained. In vintage 2013, in order to get more isolates with a wider genetic diversity, other 18 samples from grapes and 19 from wines were obtained. In this way, a total of 1902 isolates was obtained from both vintages, with near 1400 O. oeni and circa 500 of other species, mainly from Lactobacillus. After identification and molecular typing of all these isolates, 91 strains of O. oeni and several strains of 4 different species Lactobacillus and Fructobacillus were recognized. So, the assessment of indigenous bacterial diversity was finished at the end of 2013 (WP2).
During 2014, in vitro tests of performance of malolactic fermentation and resistance to different pH and ethanol concentrations in wine-like medium were carried out with the 25 most predominant strains. With these results and those of genetic analysis, a preselection of strains with oenological potential was done (WP3) and the three best strains of O. oeni were chosen for the assays of micro fermentations with real wine.
As the tasks of WP5 of production of wines with selected strains had to be carried out in the last vintage 2014, we decided to test these pre-selected strains of O. oeni directly inoculating them in industrial wines in this vintage and so, some aspects of WP4 will be covered at the same time that those of WP5. We have coinoculated together the three pre-selected strains in oak barrels and we are verifying which strain has been imposed, and final wines will be analyzed chemically and their sensorial profiles will be assessed. This will cover the aspects of evaluation of performance of strains in vivo signaled in WP4.
WP4: Development of starter cultures in micro-fermentations
The fermentation potential of several species of Saccharomyces, Non-Saccharomyces, Oenococcus oeni and other lactic acid bacteria (LAB) was evaluated in pure micro-vinifications (Del. 4.1). The number of strains tested by each RTD partner was: Saccharomyces (DEMETER 42, UNITO 96, UB 16, URV 17), Non Saccharomyces (DEMETER 25, UNITO 1, UB 8, URV 15) and Oenococcus oeni/LAB (DEMETER 23, UNITO 7, UB 10).
S. cerevisiae strains showed a relative high variation in their ability to rapidly initiate fermentation. Nonetheless, the majority of strains were characterized by a good fermentation capacity. Interspecies variations on the fermentation kinetics were observed for non-Saccharomyces strains. L. thermotolerans and T. delbrueckii strains appeared to have the highest capacity for prompt and rapid initiation of growth in grape must, while C. zemplinina and T. delbrueckii strains achieved the highest CO2 production. Significant intra- and interspecies variations in total and volatile acidity were observed in the final products.
Selected strains belonging to C. zemplinina, H. uvarum, L. thermotolerans, M. pulcherrima and T. delbrueckii were evaluated in various combinations with indigenous S. cerevisiae strains under different inoculation and nutritive supplementation scenarios. The wild yeast species applied also influenced the efficiency of sequential and simultaneous fermentations.
The ability of different vineyard and winery-associated LAB to conduct malolactic fermentation (MLF) was initially tested in synthetic wine. Strains belonging to Lactobacillus plantarum, Pediococcus parvulus and Weissela uvarum failed to survive in synthetic wine medium. On the contrary, O. oeni populations were able to persist for extended time periods and to successfully degrade malic acid.
Selected autochthonous Saccharomyces cerevisiae and non-Saccharomyces strains were evaluated in fermentations of sterile and non-sterile grape must of Agiorgitiko, Vilana and Kotsifali/Mantilari varieties. Inoculation protocols included single-strain inoculations of autochthonous (ASc) or commercial S. cerevisiae (CSc) and simultaneous (CoI) or sequential (SeqI) mixed inoculations with native S. cerevisiae/non-Saccharomyces mix. In sterile grape musts ASc, CSc and CoI showed higher fermentation rates and faster completion of the fermentation than SeqI. In all cases, CoI showed similar fermentation kinetics to ASc. In the case of Vilana and Kotsifali/Mandilari grape musts fermentation rates were higher for CSc than ASc. In Agiorgitiko grape must CSc and ASc showed similar fermentation rates. In general, commercial and native S. cerevisiae populations were higher in CSc, ASc and CoI than in SeqI, while the highest population for non-Saccharomyces species was observed in SeqI. As in the case of sterile, in non-sterile grape musts, ASc showed higher fermentation rates and faster completion of the fermentation than SeqI. Similarly, S. cerevisiae populations were higher in CSc or CoI than in SeqI.

WP5: Implementation of selected strains and blends as starter cultures in pilot scale wine production
Selected strains of native non-Saccharomyces, Saccharomyces cerevisiae and Oenococcus oeni were used for the production of ‘wild ferment’ wines at the premises of the participating wineries (Del. 5.1). Grape musts from Peza (Kotsifali/Mandilari cv. and Vilana cv.) and from Nemea (Agiorgitiko cv.) regions (Greece) were inoculated with a single native or commercial S. cerevisiae strain and with the native non-Saccharomyces/S. cerevisiae mix (simultaneously or sequentially) by partners DEMETER, PEZA and NEMEA and CAVINO. A mixture of native Oenococcus oeni strains was used for the production of wild ferment Agiorgitiko wines. Fermentations were followed by gravimetric, chemical and microbiological analysis.
Partners SVBA, IFV, GUIRAUD and BELLEVUE (France) tested different strains and blends depending on the type of wine (red or sweet white wines) and the expected aromatic profile. Yeast creams and frozen bacteria were provided by partner UB2. Pilot scale wine productions were performed in the cellars of Chateau Guiraud and Chateau Bellevue. Partner IFV has performed small scale wine productions in its experimental cellar. The wines were produced from Merlot, Semillon and Sauvignon grape varieties. A total of 1750 kg and 3500 kg of grapes were harvested manually at Chateau Guiraud and Chateau Bellevue. Vinifications were performed in 225-l oak barrels or 30-l stainless steel vats (IFV) using the procedures existing in the wineries, except that fermentations were carried out with the selected yeasts and bacteria or with control strains. This has resulted in the production of 6x 225-liters barrels of Saint Emilion wine, of 13 barrels of Sauternes wine and 14x 30-liters vats of both types of wines.
Partner URV produced selected yeast as well as Oenococcus oeni strains in the lab and used them in industrial production of ‘wild-ferment’ wines at the premises of FERRER BOBET in DOQ Priorat (Spain). Alcoholic fermentations were carried out in inox tanks (1050 litres) according to winery practices. For malolactic fermentations, wines were transferred into 250 l oak barrels. Experiments were performed with two different grape varieties: Carignan (CA) and Grenache (GR). Inoculated strains were followed during fermentation and commercial strains were used as a control. The interaction with the competitive must microbiota was examined and the effect of the added starter itself on the fermentation process was defined. In all the cases the inoculated strains were recovered during fermentations and dominated them. The final wines were chemically analysed and sensory evaluated. Most of the Wild-ferment wines were different from the organoleptic point of view, although no main preferences were detected, except in one case, were the Carignan wines with the mixed alcoholic fermentation and the wildwine malolactic fermentation was preferred.
Saccharomyces and non-Saccharomyces strains were isolated from spontaneously fermented grape musts from Asti-Monferrato regions (Italy) and after careful genetic and technological characterization were inoculated in pilot scale production of ‘wild-ferment’ wines by Partner UNITO, ARALDICA and ASTI MONFERATO. Two kind of alcoholic fermentations were carried out: the first in 70L of must and the other in 15 hL of must. Lactic acid bacteria belonging to the species O. oeni were also isolated and characterized and a total of 2 strains inoculated into two wines obtained with two different strains of selected S. cerevisiae.
In Del. 5.2 the microbial kinetics in the above pilot plant fermentations with chemical & sensory characteristics of wines were followed. Inoculation protocols included single-strain inoculations of autochthonous (ASc), commercial S. cerevisiae (CSc) or O. oeni (COo) and simultaneous (CoI) or sequential (SeqI) mixed inoculations with native S. cerevisiae/non-Saccharomyces or O. oeni (AOo) mix. In Agiorgitiko and Kotsifali/Mantilari grape must ASc, CSc and CoI showed higher fermentation rates and faster completion of the fermentation than SeqI. Non-Saccharomyces yeasts population decreased at a faster rate and reached lower levels in the case of single-strain compared to mixed inoculations. Non-Saccharomyces persisted longer in sequential compared to simultaneous inoculation. In the case of Vilana grape must, fermentation kinetics were rather similar among the different inoculation scenarios. Genotypic analysis confirmed the dominance of native S. cerevisiae and non-Saccharomyces yeasts. Autochthonous Oenococcus oeni mixture of two strains inoculated in Agiorgitiko wines after completion of alcoholic fermentation efficiently actualized MLF. The use of diverse inoculation protocols differentially affected the chemical composition of the wines. Aroma compounds were generally found at higher concentrations in wines inoculated with autochthonous S. cerevisiae strains than with commercial starters and with sequentially compared to simultaneously inoculated mixture. In all cases, volatile acidity, acetic acid and acetaldehyde were produced at levels of little sensory importance, while, organic acids and higher alcohols were produced at concentrations that positively influence the ‘’fermentation bouquet’’. Wines were evaluated for their appearance, aroma and palate by a trained panel of experts. The sequential and simultaneous inoculation of non-Saccharomyces and S. cerevisiae seems to improve the organoleptic characteristics of the produced wines. Generally, the assessors panel showed higher preference for the sequentially- than the simultaneously- or singly-inoculated wines. For wines subjected to MLF, AOo was the most preferred from the panel.
BioAquitaine, UB2 and IFV tested the selected yeast strains for pilot scale production of red and sweet wines at Chateau Bellevue and Chateau Guiraud, respectively. Selected bacteria were tested only at Chateau Bellevue for the malolactic fermentation of red wines.
For the red wine, 1 selected Saccharomyces cerevisiae and 2 selected non-Saccharomyces yeasts were tested in pure culture or blends of 2 strains at chateau Bellevue and IFV. They were compared with indigenous fermentation and a commercial strain. No difference was detected in terms of kinetics, sensory evaluation and analytical parameters, including the esters content of the obtained wines.
For the sweet wine, 2 selected Saccharomyces cerevisiae and 1 selected non-Saccharomyces yeast were tested in pure culture or blends at chateau Guiraud and IFV. They were compared with indigenous fermentation and commercial Saccharomyces cerevisiae yeast or blend of commercial S. cerevisiae and non-Saccharomyces strains. Differences were observed in terms of kinetics. The commercial yeast and commercial yeast blends performed the best, followed by the pure and blend of selected strains, whereas the spontaneous fermentation performed by indigenous strains was rather bad. The same tendency was observed for the production of volatile acidity. Important variations in esters content were determined between the trials. It was also note that the presence of a Torulaspora strain impact significantly on the production of volatile thiols. The sensory evaluation revealed some differences of bitterness, the preference being given to the commercial strain ST.
For bacteria, the implantation controls performed in trials performed at Chateau Bellevue and IFV showed that the selected strains failed to perform the malolactic fermentation. Therefore it was not possible to evaluate the sensory properties of wines produced with these wild blends.
URV used selected yeast as well as Oenococcus oenii strains produced in the lab in industrial production of ‘wild-ferment’ wines at the premises of the Ferrer Bobet winery in DOQ Priorat. Alcoholic fermentations were carried out in inox tanks (1050 litres) according to winery practices. For malolactic fermentations, wines were transferred into 250 l oak barrels. Experiments were performed with two different grape varieties: Carignan (CA) and Grenache (GR). Inoculated strains were followed during fermentation and commercial strains were used as a control. The interaction with the competitive must microbiota was examined and the effect of the added starter itself on the fermentation process was defined. In all the cases the inoculated strains were recovered during fermentations and dominated them.
The final wines were chemically analysed and sensory evaluated. Most of the Wild-ferment wines were different from the organoleptic point of view, although no main preferences were detected, except in one case, were the Carignan wines with the mixed alcoholic fermentation and the wildwine malolactic fermentation was preferred.

WP6: Consumer studies
A survey was carried out (Del. 6.1) in order to assess the consumers’ perception and acceptance of “wild-ferment” wines, which are influenced and shaped by vineyard-originated wine yeast strains, and to estimate their market potential. For this purpose a questionnaire was designed and completed by panels of wine consumers. Apart from the questions regarding the core of the survey, we included a few introductory questions in order to provide the unfamiliar audience with some basic knowledge about the role of wine yeasts in wine making. The experimental wines were also evaluated for their appearance, aroma and palate by a panel of consumers.
Partner DEMETER (Greece) distributed the questionnaire to wine consumers of at least 20 years old. It is worth-noting that most of Greek consumers were adequately informed about the existence/effect of the indigenous yeasts to the organoleptic characteristics of wines. The survey clearly showed the intention of consumers to choose wines produced from non-commercial yeasts. Partner Demeter also designed special score-sheets for the sensory evaluation of the pilot plant produced wines by the panel of consumers. The evaluation was based on the visual, taste and aroma attributes of wines. In general, consumers showed higher preference for mixed than singly inoculated wines.
Partner BioAquitaine (France) distributed the questionnaire to app. 30 persons. The results revealed that there is a real interest of consumers for wild microorganisms and “wild-made” wines, although the quality and price of these wines are critical for the decision of purchasing such wines. Two tasting sessions of wild fermented wines produce during WP5 were organized in the framework of important French exhibitions: the “Salon de l’Agriculture” and “Millesime Bio”. In both sessions, 42 to 50 persons have evaluated the proposed panel of wines which consisted in red wines produced from Merlot grapes of Chateau Bellevue and Sauternes wines from Chateau Guiraud. Wild-fermented wines were compared with control wines produced using commercial microorganisms or classical indigenous fermentation. No significant preference was noticed for the red wines, but consumers have preferred sweet wines obtained with a wildwine selected blend of Saccharomyces cerevisiae and Torulaspora delbrueckii strains.
Partner URV (Spain) distributed the questionnaire to specialized public and found that the majority of the responders were aware of the wine production by commercial strains and of the fact that the presence of «autochthonous» microorganisms could define a different wine. However, the quality and price of “wild-ferment” wines are critical for the decision of purchasing such wines. URV asked 33 vine growers, cellar workers and oenologists to evaluate the pilot plant wines. No significant differences were observed among the different wines. Thus, it should be emphasized the high quality of all of them, because all the tasters considered the wines clearly different.
Partner UNITO (Italy) distributed the questionnaire to 53 persons and found that 32% would prefer to buy a wine made with “wild” microbes, even if it was at higher price than the conventional one. The 43% of the panel thought that “wild wines” may provide an extra regional signature for European wines to compete in the global market. All the wines produced in the project were classified like the high quality product, and the most appreciated from all the tasters was the wine fermented with S. cerevisiae Sc 25.

Potential Impact:
The Consortium expected a number of potentially exploitable results which are presented together with their type of exploitation by each association or company in the following parts, as described in the Annex I –“Description of work” the grant agreement 315065. The exploitation of the project’s results after the training of the SME-AGs key personnel, and after the training of SMEs involved was the most significant impact of this project. Our first aim was to protect the IPR owned by SME-AGs, as well as to release non-confidential research findings from this project into the public domain, though oral presentations at conferences and in peer reviewed journal articles or in the website. Making the project outputs available in this way, this maximizes the impact of the project.
Management of knowledge and intellectual property:
The Consortium has chosen the default Intellectual Property Rights regime for projects for the benefit of SME-AGs. This means, giving full ownership of all project results and intellectual property rights to the SME-AGs participants which will use them for their benefit while their SMEs will have license to use them. The RTD performers are remunerated for their work. The Consortium agreement precisely states how the partners associations share or jointly own these results according to the following rules as described in the CA.
According to this “The consortium should be given at least 45 days prior notice for any planned dissemination activity and have 30 days to object. Intellectual Property Rights (IPRs) will be protected by patent applications filed by one or more participants who have generated the results to be patented. They will be considered as the authors, while the SME-AGs will be the owners. All the partners agreed on critical concerns related to the Consortium Agreement:
- All the publications are identified as the results of a research project, which is supported by REA, FP7-SME programme. All experimental results obtained by participants in this project for which none of the participants can claim any feasible commercialization or patenting interest will be made public. Unrestricted results will be submitted to national and international scientific and trade journals for publication.
- Every IPR issued from the project results will be the ownership of the SME-AGs. The French, Italian and Spanish SME-AGs (BIO-AQUITAINE, ASTI-MONFERRATO and DOQPRIORAT) will have each full ownership (100%) to two of the eight project’s results and license for the rest six. The Greek SME-AGs (PEZA and NEMEA) will have each the ownership of the results 4, and 3 respectively and license for the rest seven. The participating SMEs (CAVINO, BELLEVUE, GUIRAUD, BOBET and ARALDICA) will have a competitive advantage as they will have license to use the project’s results for 2 years after the end of the project. Members of the SME-AGs will be also granted licenses at request.
- The SME-AGs and SMEs have full access to all new knowledge generated in the project and non-discriminatory access to pre-existing know-how. Background owned by the RTD performers or the SME partners that is or will be found necessary for the implementation of the project will be granted royalty free to all partners. After the end of the project SMEs, SME-AGs and RTD performers will grant royalty free access to background needed to use the foreground.
- The RTDs will inform the SME-AGs and SMEs promptly of any invention arising from the project.
- Any possible patentable procedure or product will be registered by the SME-AGs but the RTD partners will be the authors, but this circumstance will not give them any rights on exploitation.
- SME-AGs will be primarily responsible for filing of patent applications in their own name provided the conditions above mentioned are satisfied.
- No participant will sell or transfer to third parties the project exploitation rights or licenses for any knowledge developed in the framework of the present project outside the present Agreement, if not previously agreement with all the other participants is reached.
- RTD performers are free to use the results for further research (non commercial exploitation) if those results are not identified as confidential. Besides the RTD performers can publish the results after obtaining the authorization by SME-AGs.
- All the publications are identified as the results of a research project which is supported by European Commission (FP7-SME).
- All experimental results obtained by participants in this project for which none of the participants can claim any feasible commercialization or patenting interest will be made public. Unrestricted results will be submitted to national and international scientific and trade journals for publication.
- There are no existing anticipated business agreements that may impose limitations on the subsequent exploitation or information or inventions generated as a result of the project.

Access rights
The elements of background have been expressly specified in the Consortium Agreement and access rights to the background are granted both for project execution and foreground use purposes, on royalty-free conditions. In particular, the RTD performers have agreed to grant access rights to their background as necessary for the SME-AGs to exploit WildWine on royalty-free conditions. Access rights to foreground for project execution purposes have been granted on royalty-free conditions to and by all participants, as established in the FP7 rules. The RTD performers may be granted royalty-free access rights to the project foreground exclusively for further research purposes.

In order to ensure that the SMEAGs are capable to take-up the results of Wildwine, the RTD performers provided training to the SME-AGs and to the SMEs of the project on how to use the new strains.

The following activities have been performed for the exploitation of results:
1. Collection of data regarding the training and knowledge transfer needs of the participating SMEs. Training activity regarding the personnel of the participating SMEs.
2. Activities for the development and launch of new products where needed, that will use the new multi-strain mixture (yeast blends) and LAB-strain starter technology. The activities, intending to increase firm competitiveness in domestic and foreign markets include:
a) a chain of meetings with the directors of the participating firms. These will initiate discussions, trigger brainstorming and generate ideas for the development and launch of new products that will use the new technology across domestic and foreign markets.
b) a chain of telephone conversations with the principal clients/ agents of the participating firms in their export countries. The purpose of these conversations was the exchange of information for precise identification of the appropriate features of the new food products as export target country markets may have different requirements.
3. Attempt to incorporate information about the new products in the marketing and advertising campaigns of the participating companies to ameliorate innovativeness profile of the new product across the wider public.
4. A public relations/press campaign targeting the wine sector. The aim will be to ameliorate company reputation intra-sector and possibly trigger imitating actions by competitors in other EU countries
5. Training actions to educate young & experienced wine and food scientists on the new product technologies.
There are no existing anticipated business agreements which may impose limitations on the subsequent exploitation or information or inventions generated as a result of the project.

Project results
At the end of the project strains of yeast and bacteria or blends have been selected by each participating group in the corresponding region. More specifically:
In France:
Project result No 1 & 2: French Yeast and Bacterial Starters & Blends:
Three Saccharomyces yeasts: strain Be15 for red wine chateau Bellevue and strains Sc 48 and Sc 86 for sweet wines of Chateaux Guiraud. Three non-Saccharomyces yeasts, a Torulaspora delbrueckii and a Metschnikowia pulcherrima strain for red wines of Chateau Bellevue. A Torulaspora delbrueckii strain Td63 for sweet wine of chateau Guiraud. Three Oenococcus oeni bacteria strains, two strains were «specific» for Chateau Bellevue and 1 strain «specific» for Aquitaine.
The selected microorganisms were:
- 3 Saccharomyces yeasts. Strain Be15 selected from red wine chateau Bellevue showed similar fermentation qualities as available commercial yeasts. Strains Sc 48 and Sc 86 isolated from sweet wines of Chateaux Guiraud Saccharomyces showed performance a little lower than the classic commercial yeast used for sweet wine in terms of fermentation kinetics and volatile acidity production.
- 3 non-Saccharomyces yeasts. A Torulaspora delbrueckii and a Metschnikowia pulcherrima strains were selected for red wines produced at Chateau Bellevue. A vinification trial performed at the pilot scale in the cellar of the Chateau Bellevue showed no difference with trials performed with commercial yeast or with the selected yeasts Be15 alone, in terms of fermentation kinetics, analytical composition or sensory evaluation by professionals or consumers. A Torulaspora delbrueckii strain Td63 was also selected for sweet wine of chateau Guiraud. Pilot scale vinifications performed in the winery showed differences in terms of fermentation kinetics and volatile acidity between the trials, the best performances being attributed to the commercial Saccharomyces strain alone and to the commercial blend of S. cerevisiae/T. delbrueckii. The trials were not differentiated by professionals during a sensory evaluation but a panel of consumers preferred the wine produced by the selected mixed culture.
- 3 Oenococcus oeni bacteria strains. Two strains were « specific » from Chateau Bellevue and 1 strain « specific » from Aquitaine. They were tested only during one vintage for the pilot scale production of a red wine at chateau Bellevue. All of the three strains failed to conduct the malolactic fermentation in this wine, probably because the vintage conditions were favorable for the development of indigenous bacteria. That put in evidence the difficulty to select microorganisms during the short period of the project.
In term of production of microorganisms, 16l of yeast creams were produced to inoculate 8 Hl of wine at chateau Bellevue and 6l to inoculate 4 Hl for a pilot scale trial at IFV. Each bacterial strain was produced in app. 3 weeks from a 2-litres culture to inoculated barrels of 2.5 Hl.

Plan for exploitation: The Bordeaux University (UB2) in France will produce a book dedicated to organic winemaking. The commercial exploitation of the selected strains is not envisaged by the association of BioAquitaine for several reasons, particularly because the project has not revealed the existence of terroir strains for the region and the selected yeast and bacteria strains were less efficient than existing commercial strains. The duration of the project was probably too short to allow for an efficient selection to be made. However, yeast strains selected for sweet wine production at chateau Guiraud were well appreciated by the winemaker and they could give some added-value to this wine at the marketing level. It is possible to propose to winemakers to select, produce and use strains from their own wineries as a marketing argument to increase the sales. The main difficulty will be the costs of strain selection and production that are excessive and possibly higher than the expected profits.
The industrial production of the selected strains and blends is not yet scheduled for several reasons. 1/ Yeasts selected for red wine fermentations did not perform better than available commercial strains and indigenous fermentation. 2/ Although they were isolated from the partner winery, the results have shown that these strains are not specific for the terroir. Yeasts (and bacteria) strains isolated from a restricted area can be detected in distantly related regions, suggesting that there are not genetic families of strains specific for a terroir. 3/ The duration of the project was too short to allow an efficient selection of bacterial strains. Additional pilot scale trials will be necessary to validate or not their efficiency.
These arguments do not encourage for the industrial production and distribution of the selected strains. However, a blend of selected Saccharomyces/non-Saccharomyces yeasts used for sweet wines production at chateau Guiraud was much more efficient than indigenous strains in spontaneous fermentation. Although it was a little bit less efficient than a commercial blend, it could represent a good alternative. At the level of winery, the selection, production and use of its own strains could give a marketing argument for increasing its sales. The main constraint to this development is the cost of the small-scale production of the selected strains, which is presently much higher than commercial strains. A winery that would choose to select and use its own strains would have to consider the cost of selection and production in its business plan. This possible outcome of the project will be disseminated to winemakers of the region of Bordeaux.

In Greece:
Project result No 3. Greek Yeast and Bacterial Starters & Blends from NEMEA region: Lachancea thermotolerans E13NL1 / Saccharomyces cerevisiae E27NW12, a yeast blend and Oenococcus oeni 24NK12 / Oenococcus oeni O11L5 bacterial blend for Agiorgitiko cultivar

Project result No 4. Greek Yeast and Bacterial Starters & Blends from PEZA region: Lachancea thermotolerans E427PL4 / Saccharomyces cerevisiae E321PW6, a yeast blend for Vilana cultivar and Candida zemplinina E348PL4 / Saccharomyces cerevisiae E347PW1, a yeast blend for Kotsifali/Mandilari cultivar.

In general, aroma compounds were found at higher concentrations in wines inoculated with autochthonous S. cerevisiae strains than with commercial starters and with sequentially compared to simultaneously inoculated mixture. In all cases, volatile acidity, acetic acid and acetaldehyde were produced at levels of little sensory importance, while organic acids and higher alcohols were produced at concentrations that positively influence the ‘’fermentation bouquet’’. In most cases, the sequential and simultaneous inoculation of non-Saccharomyces and S. cerevisiae seems to improve the organoleptic characteristics of the produced wines. For wines subjected to MLF, the native O. oeni blend was the most preferred from the panel.
Although not apparent in pilot plant fermentations, probably due to the high indigenous yeast populations of grape must, strain Lachancea thermotolerans E427PL4 significantly enhanced the total acidity of the final product in experimental fermentations. Therefore, this strain can be used by PEZA for the biological acidification of the wine. Commercial and native yeast fermented wines were differentiated by the sensory panel. Assessors showed higher preference for the wine fermented by the native yeast blend, which appeared to have better mouth aroma and higher aroma intensity than the commercial starter.
In the case of Kotsifali/Mandilari cultivar, the use of native yeasts blend diversified the final product. More precisely, the use of native yeast blend, inoculated either sequentially or simultaneously, increased the total acidity, glycerol content, organic acids, total fusel alcohols and esters. Commercial and native yeast fermented wines were differentiated by the sensory panel. Assessors showed higher preference for the commercial yeast, albeit with little differences with the sequentially added native yeast blend.
In the case of Agiorgitiko cultivar, the native yeast blend increased total fusel alcohols and fruity esters. Assessors showed higher preference for the sequentially inoculated native yeast blend for its fruity aroma, the aroma duration and the good balance. Autochthonous Oenococcus oeni mixture efficiently actualized MLF in Agiorgitiko wines and was preferred by the assessors over the commercial strain or the spontaneous MLF conducted by NEMEA.
Plan for expoitation: The associations of PEZA and NEMEA showed great interest in the yeast and bacterial strains isolated by DEMETER. The strains are kept in the culture collection of DEMETER and are available to the associations for further use. Initially, the wineries of the associations may consider applying the selected strains in a small part of the produce by the “pied de cuve” method. Depending on the results, the wineries will consider the production of the starters in dry form by a company.

In Italy
Project result No 5 & 6: Italian Yeast and Bacterial Starters & Blends: Two strains of Saccharomyces serevisiae, Sc35 and Sc38, were selected in Italy.
Within the project several hundreds of Saccharomyces cerevisiae were isolated and after several trials, starting from in vitro, for then in pilot scale and finally in industrial scale, three strains were selected for further implementation.
The members of the Consorzio Asti and Monferrato showed an important interest in the project and for this reason throughout the collaboration of UNITO and the Consorzio, it was proposed to ask a biotechnology company to propagate the selected yeasts and used them in the 2015 harvest. More specifically, two strains of Saccharomyces serevisiae, Sc35 and Sc38, were used to produce yeast inocula to be used in real trials in three wineries. The volumes inoculated ranged from 50 to 100 hl. At the time this report was written, the fermentations initiated and proceeded correctly.

In Spain
Project result No 7 & 8: Spanish Yeast and Bacterial Starters & Blends: URV in Spain has selected 7 wine yeast strains: Candida zemplinina (CECT 13129), Hanseniaspora uvarum (CECT 13130), Metschnikowia pulcherrima (CECT 13131), Saccharomyces cerevisiae (CECT 13132, CECT 13133, CECT 13134), Torulaspora delbrueckii (CECT 13134), Oenooccus oeni (CECT 8893, CECT 8894, CECT 8895)
In Spain Ferrer Bobet winery has produced wines with different combinations of yeast and Lactic Acid Bacteria blends. The results show that the wines are significantly different, although the consumer preferences are not significantly better in any case. However, in internal tasting among the owners and oenologists of the company, the wines produced with a complete blend of autochthonous Saccharomyces and Non-Saccharomyces as well as with the Lactic Acid Bacteria were clearly preferred. This preference has introduced the interest for wines produced with these blends.
Furthermore, it was observed that the wines produced with the complete blend of yeast underwent the malolactic fermentation faster and more efficiently.
The Ferrer Bobet winery has applied the in the form of “pied de cuve” method, which could be the preferred method for using these blends.

Dissemination of project’s results.
The exploitation of the project’s results was the most significant impact. It was our intention to protect the IPR owned by SME-AGs, as well as to release non-confidential research findings from this project into the public domain, though press releases, flyers, oral presentations at conferences and in peer reviewed journal articles or in the website (www.wildwine.eu). Making the project outputs available in this way, we maximized the impact of the project.
The dissemination activities, included general publications, conferences, workshops, webpage, press releases, flyers, etc. Promotion of the project and its final products has been achieved through participation in food and wine festivals, commercial expos, etc. It provides a list of the scientific publications relating to foreground. Its contents can be made available in the public domain. The following sentence was added to all publications of this project: "The research leading to these results has received funding from the European Union's Seventh Framework Programme managed by REA - Research Executive Agency FP7/2007-2013 under grant agreement no [315065]".
Dissemination activities were aimed to broadly diffuse awareness on the scientific achievements of the project, raise interest on the application possibilities it opens in induced wild fermentations and promote wild-ferment wines to the consumers. The objective was to disseminate the learned outcomes about the use of multi-strain mixtures (yeast and bacterial blends) as starters and the effective and reliable quality optimization of ‘wild’ fermentation, that allow SMEs to launch ‘wild-ferment’ wines into the market. Our aims included the transfer of knowledge and best practice to wine industry and the scientific community for:
(i) advancement in the technological process of induced wild alcoholic and malolactic fermentations by using indigenous starter cultures towards a properly controlled, predictable and reliable process,
ii) innovation in the production of a novel, organic, safe wines for the consumers, and
(iii) promotion of multi- strain starters and wild wines. The main results of this research are expected to improve the competitiveness of the European wine industry.
Dissemination activities were accomplished via presentations at national and international workshops seminars organized by the SME-AGs for their members. Dissemination activities aim at ensuring and facilitating consumer awareness about the high quality wines by reaching an audience as wide and relevant as possible across Europe, with the active participation of all consortium members. Project’s results also were disseminated through publications in international journals, national workshops and international conferences.

The dissemination activity of WildWine has been primary promoted by the SME-AGs. SME-AGs were in charge of spreading within the local grape/wine sector the information gathered during the development of the project and to allow and speed up the take up of the results by SMEs and by the market. To carry out such activities they have used their website. In addition, they organized specific meetings to inform their members of the project evolution and possibilities. The SME-AGs promoted the project and its results through participation in food and wine festivals, commercial expos, etc., organizing wine taste events, gathering questionnaires etc.
The RTDs collaborated and were also involved in these activities, being more used and competent in scientific dissemination. Dissemination towards the scientific community by means of publications on high impact journal will be considered after careful consideration of the issues concerning the protection of Intellectual properties by the SME-AGs and their authorisation. All the publications were identified as results of a research project that is supported by European Commission (FP7-SME).

The work during the first period has been mainly devoted to diffusing the project content to the members of the participating Associations (PEZA, NEMEA, BIOAQUITAINE, ASTI-MONFERRATO, DOQPRIORAT). This information was also disseminated through participation in different Congresses and Symposia as well as at wine festivals, food or wine expositions. News with respect to the objectives of the project have been also published in newspapers of marked diffusion in regional and local areas, in magazines, in electronic sites.
Many press releases for the activities of the project have been published in every participating country. It must be emphasized that the project and its objectives are nowadays very well known in all participating countries.
A wider diffusion has been achieved thanks to the website of the project (http://www.wildwine.eu). The construction of this site made possible world diffusion through Internet, the most powerful diffusion tool. The website has been constructed for the project WILDWINE 315065 to disseminate to the sector of interest and to the wider public, the project, its targets and objectives and its expected outcomes. The information is available in 5 languages (English, Greek, Italian, French and Spanish, the main languages of the participants) in order to disseminate the information to people from the interested countries that speak only their native language. The page is updated regularly and will be active even after the end of the project. A visitor counter of the Website (ClusterMaps) has counted more than 1000 visits from all over the world in the first year of the active webpage showing the great interest for the project’s objectives indicating also the possible impact that it will have. Links with this web page has been inserted in all the organizations participating in the project. News and press releases relevant to the project are published regularly through this website.
Numerous activities were performed also during the 2nd period of the project to disseminate its objectives and results to the industry, the scientific community and to a wider public. Dissemination activities included publications, organization of conferences/workshops, presentations, exhibitions, flyers, theses, posters etc. These actions will be continued well after the end of the project. A publication in Cordis just after the end of the project summarizes the successful implementation of the project and the achieved results (http://cordis.europa.eu/news/rcn/123846_en.html )
A video clip containing project activities has been prepared and submitted as Deliverable 7.5 at the end of the project (July 2015) (https://vimeo.com/142903881).
In total: Eight (8) publications in peer reviewed journals, 13 papers in conference proceedings, 12 thesis and > 150 dissemination activities have been performed (website, press releases/ articles in local magazines & websites, posters and presentations in workshops/conferences, MSc and PhD theses, flyers, TV interviews, cordis, video clip.

Future prospects: new publications and conferences will be produced after the end of the project to communicate the last results. Specific information to organic winemakers will be provided by BioAquitaine. A book dedicated to organic winemaking is in preparation under the supervision of BioAquitaine. It will present the obtained results. The results will be also transferred to bachelor and master students in oenology of the ISVV during courses in wine microbiology. The results will be also included in the courses provided at ISVV to professionals of the wine industry.

Additional results and Impact:
The assessment of the indigenous yeast and bacterial diversity residing in different viticultural areas of Europe will contribute to a better description and preservation of the regional biodiversity. The description of the phenotypic diversity of isolates with particular emphasis on key characteristics of technological importance as well as the analysis of the chromosomal stability of isolates will constitute a valuable microbial collection to be used in various applications of the wine industry (fermentations of different grape varieties, production of sweet wines, etc). or in the fermentation of other food commodities, not only in the context of this project but also for future generations.
The results from the microbiological assessments of the starters in micro-fermentations, with respect to strain combinations and time of addition, in order to identify the kinetics and the metabolome throughout the complete procedure, will allow for the development of novel starter cultures, that will enable the wine industry to benefit from real wild fermentations and avoid economic losses by unmarketable wines due to uncontrolled processes.
The application of the results by the associations and the SMEs will play a major role in validating the performance of mixed cultures (starter blends) in industrial scale fermentation processes. This will provide to SMEs advancement in the technological process of wild alcoholic and malolactic fermentations by using indigenous starter cultures towards a properly controlled, predictable and reliable process. Such blends will be specially designed to produce high quality wines by enhancing the influence of the particular “terroir” on wine character. At the same time the inclusion of selected LAB strains will prevent the accumulation of biogenic amines, thus providing the SMEs the advantage of producing safe wines.
The data obtained on consumer perception and acceptance of ‘wild-ferment’ wines are very essential for the marketing, advertisement and sales of the possibly developed products. It will be a strong point in the exploitation of the results.

Expected impact for the participating RTDs: The project has promoted scientific knowledge that has been disseminated through the website, publications and national and international conferences in the field of wine science and technology, in new and emerging technologies that can be applied to the wine industry and in the improvement of existing technologies.
For the participating SME-AGs and the end users: Effective up taking of new technologies has been ensured by their active involvement in the project. The SMEs in the project are wine producers, so they can immediately benefit as the results of this project contribute to: i) innovative, natural, consumer-driven wines fermented by the indigenous microbiota ii) increased wine quality iii) establishing processing conditions which reassure high levels of safety to consumers. Benefits for the participating companies also include the enhancement of their reputation/brand equity intra-sector and among the wider public as well as the development of new products destined towards both the domestic and international markets. All these aspects will consolidate and expand foreseeable immediate sales increase.
Thus the project will help to: create new jobs and/or maintain the already existing ones; improve marketing activities; promote the SME-AGs in the field of product development, quality assurance and consumer research; transfer scientific knowledge; broaden the European wine industry to other countries; support the agricultural sector and the industrial development. Having participated in the project they have a competitive advantage as they can have an added economic benefit from licensing the expected development of the new products. Benefits for the participating SME-AGs and their members also include the enhancement of their reputation/brand equity intra-sector and among the wider public as well as the development of new products destined towards both the domestic and international markets. All these aspects can consolidate and expand foreseeable immediate sales increase.

The development and implementation of indigenous starters in induced wild fermentations will enable wine makers to produce elegant wines that will fully reflect their sense of place and will be compatible with current and forthcoming regulations for organic wine production and biogenic amine content. The production of high quality, safe products is one of the determining factors in the future growth and increased competitiveness of European wine producers. The proposed “point-of-care” technology offers a solution to a need identified by consortium SME-AGs as common, not only to their members, but to the wine sector as a whole in Europe and worldwide, including:
 The implementation of induced wild fermentation technology in a controlled and reproducible manner
 The launch of innovative, terroir wines
 Establishment of ‘signature’ strains correlated with specific viticultural regions
 Compliance with organic wine market rules
 Healthy wines free of amines
 Competent MLF cultures with defined technological and flavouring properties, which will also meet regulatory safety issues on biogenic amine production.

The WildWine project made other contributions, including:
 The preservation of the microbial natural biodiversity in situ and by raising the public concern.
 Preservation and expansion of the local vineyards within viticultural regions towards a more environmentally sustainable development.
 The procedures developed in this project contribute a simple and convenient standardized methodology for advancing detection of biogenic amines and fermentation control schemes and develop the best practice guidelines for SMEs in this area. Inspection agencies, national and transnational authorities as well as companies and laboratories that provide diagnostic services can also benefit from these techniques by curbing public costs in time and analyses.
 Increased benefit of consumers and EU citizens in terms of health and safety.
 Contribution to employment and development in rural areas by helping wine producers protect traditional income sources. The employment in research institutions, educational organizations and the wine industry in the EU has also been promoted.

Contribution to advancement of knowledge / technological progress

Industrial yeast or bacterial starters often violate the originality of regional wines by conferring comparable aromatic profile to different terroirs. In turn, spontaneous fermentations by the indigenous microbiota are highly risky, and may deliver wines that are unmarketable or unsafe, due to high content of biogenic amines. The project results can allow SME-AGs a level of control in spontaneous or wild type fermentations, through the development of yeast and bacterial starter blends composed of indigenous, privileged strains, that can ensure production of elegant wines reflecting sense of place and compatible with terroir-driven approaches. This is the
first time to develop starter cultures for particular terroirs, for the benefit of the participating SME-AGs. To our knowledge no other similar project has been funded.
In the context of the project a set of novel privileged strains, serving as “microbial signature”, have been assigned to important PDO European zones and knowledge established in aspects concerning: (a) the biogeography of indigenous yeast and bacterial microbiota, (b) the behavior of indigenous enological strains under winemaking conditions, (c) aroma metabolome of strains during winemaking (c) genetic traits of enologically important novel species/strains, and (d) the molecular polymorphism among strains. This knowledge allows wine industry to apply induced wild fermentations in the development of novel products that are reproducible, premium, attractive to consumers and in accordance with the demands of the global market for natural safe wines .
The WildWine project offered to the SME-AGs and their members i) advancement in the technological process of spontaneous or wild alcoholic and malolactic fermentations by using indigenous starter cultures towards a properly controlled, predictable and reliable process and ii) innovation in the production of a novel,organic, safe product for the consumers.
In the context characterized by several food crises in the ten last years, consumers are more and more aware of the quality of products and their impact on health. The improvement of citizens’ health through the availability of new generation of healthy foods is one of the main perspectives of the project in terms of public health. On this side, the project allows members of SME-AGs to valorize their products on the market and thus reinforce their standard of competitiveness by putting at the disposal of European citizens some scientific data for a better wine fermentation control and for the development of allergen-free wines.

Contribution to regulation and standards
The project is in accordance to two out of the three major goals of the reform of the common market organization (CMO) for wine, adopted by the EU in 2008 (http://ec.europa.eu/agriculture/markets/wine/index_en.htm ), which are:
 “making EU wine producers even more competitive - enhancing the reputation of European wines and regaining market share both in the EU and outside”
 “preserving the best traditions of European wine growing and boosting its social and environmental role inrural areas”.
Although the introduction of precise regulation for organic wine production at the stage of winemaking is still debated, it is expected that it will follow certain limitations. In this respect, specific label claims (e.g. ‘obtained without the addition of commercial starters’ or ‘produced by indigenous microbiota’) will probably be anticipated under the common market organisation for organic wine. In addition, several national or private established standards already impose strict regulations for organic wine production, including the use of indigenous yeasts or bacteria instead of industrial starters to conduct the fermentation. In the WildWine project competent strains isolated from vineyards within the wine production zone have been selected to serve as starters, thereby enabling wineries to conform to restrictions for organic wine production. The companies involved may try to obtain if they like certification of the proposed technology as ‘Zero-input’ winemaking approach for organic wine production.

It is anticipated that the addition of selected non-Saccharomyces yeasts and LAB species to the grape must allows winemakers to take advantage of wild fermentations in the improvement of wine quality and to avoid the risks that a spontaneous fermentation entails. With respect to the production of natural terroir wines, the selected indigenous Saccharomyces, non-Saccharomyces and LAB species can serve as a source of authenticity, by conferring distinct regional and desirable characteristics to wines.
The presence of biogenic amines in wines is currently not regulated worldwide. However, because of their potential health implications, EU intends to include biogenic amines under similar regulations proposed for allergens. Wines with high concentrations are already rejected from certain markets, like Canada. The project can assist in the formulation of regulations regarding biogenic amines content in wines by providing scientific knowledge to food safety authorities in each participating country.

List of Websites:
www.wildwine.eu https://vimeo.com/142903881

Final Report Summary - WILDWINE (Multi-strain indigenous Yeast and Bacterial starters for ‘Wild-ferment’ Wine production)

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