Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

A critical issue on microbiological cut-off value of ampicillin resistance in Lactiplantibacillus plantarum.

AIM: Comprehensive evaluation of antibiotic susceptibility patterns in Lactiplantibacillus plantarum strains isolated from grape marc, based on genomic and phenotypic assessment. METHODS AND RESULTS: We assessed the antibiotic resistance-susceptibility patterns of 20 L. plantarum strains for 16 antibiotics. Genomes of relevant strains were sequenced for in silico assessment and comparative genomic analysis. Results showed high MIC values for spectinomycin, vancomycin, and carbenicillin, indicating natural resistance to these antibiotics. Besides, these strains revealed MIC values for ampicillin higher than previously established by the EFSA, indicating the possible presence of acquired resistance genes in the genomes. However, genomic analysis by complete genome sequencing did not reveal presence of ampicillin resistance genes. CONCLUSION: Comparative genomic analysis between our strains and other L. plantarum genomes present in the literature showed several substantial genomic differences, and suggested the need to adjust the cut-off value for ampicillin in L. plantarum. However, further sequence analysis will reveal how these strains have acquired antibiotic resistance.

Assessment of the microbiological origin of blowing defects in Grana Padano Protected Designation of Origin cheese.

Recognized worldwide for its history, flavor, and high nutritional quality, Grana Padano (GP) is one of the most traditional Italian raw-milk, hard-cooked, long-ripened cheese. Throughout GP manufacturing, some well-known and undesired bacterial species, such as clostridia, can proliferate and lead to spoilage defects that mischaracterize the final product; however, little is known about the development of late-blowing defects in hard cheese samples without clostridia. Therefore, in this study we aimed to use metataxonomic analysis to identify bacterial taxa associated with the development of late-blowing defect in GP samples. Furthermore, the presence of several heterofermentative lactobacilli species in defective zones were verified by primer-specific PCR assay. Considering α- and ß-diversity analyses, no statistically significant differences were detected between cheese samples with and without blowing defect. Following taxonomic assignment, Lactobacillus and Streptococcus were the dominant genera, whereas clostridia-related taxa were not detected in any of the 20 analyzed samples. Using EdgeR, the genera Propionibacterium and Acinetobacter were found to be prevalently more abundant in samples categorized as having "big regular holes." In samples with "small regular holes," multiplex PCR amplification revealed differences in terms of Lactobacillus population composition, mainly obligate homofermentative lactobacilli, between defective and non-defective zones of the same cheese wheel. This study demonstrated that GP samples with blowing defects not caused by clostridial development share similar biodiversity indices with GP collected from control zones, but an imbalance of obligate homofermentative lactobacilli was noticed between samples, which requires further analysis to better comprehend the exact mechanism involved in this process.

From the vineyard to the cellar: new insights of Starmerella bacillaris (synonym Candida zemplinina) technological properties and genomic perspective.

A large diversity of yeasts can be involved in alcoholic fermentation; however, Starmerella bacillaris strains have gained great attention due to their relevant and particular characteristics. S. bacillaris is commonly known as an osmotolerant, acidogenic, psychrotolerant, and fructophilic yeast. Most strains of this species are high producers of glycerol and show low ethanol production rates, being highlighted as promising alternatives to the manufacture of low-alcohol beverages. The increased production of high alcohols, such as benzyl alcohol that has antifungal and antibacterial properties, highlights S. bacillaris potential as a biocontrol agent. After harvest, antifungal yeasts become part of the must microbiota and may also improve the fermentation process. Moreover, during the fermentation, S. bacillaris releases important molecules with biotechnological properties, such as mannoproteins and glutathione. Considering the potential biotechnological properties of S. bacillaris strains, this review presents an overview of recent trends concerning the application of S. bacillaris in fermented beverages. KEY POINTS: •S. bacillaris as an alternative to the production of low-alcohol beverages. •S. bacillaris strains present biocontrol potential. •Molecules released by S. bacillaris may be of great biotechnological interest.

The impact of CUP1 gene copy-number and XVI-VIII/XV-XVI translocations on copper and sulfite tolerance in vineyard Saccharomyces cerevisiae strain populations.

In wine production, sulfites are widely used as antimicrobials and antioxidants, whereas copper is associated with fungicides and wine fining treatments. Therefore, wine yeasts are constantly exposed to these agents. Copper tolerance is related to the copy number of the CUP1 gene, encoding for a metallothionein involved in copper detoxification. In wine yeasts, sulfite resistance mainly depends on the presence of the translocation t(XVI;VIII) in the promoter region of the SSU1 gene. This gene encodes for a plasma membrane sulfite pump involved in sulfite metabolism and detoxification. Recently, a new translocation, t(XVI;VIII), was identified. In this work, 253 Saccharomyces cerevisiae strains, representing three vineyard populations from two different continents, were analyzed, along with 20 industrial starters. Copper and sulfites tolerance as well as distribution of CUP1 gene copy-number, t(XVI;VIII)and t(XVI;XV) of SSU1 gene were studied to evaluate the impact of these genomic variations on population phenotypes. The CUP1 gene copy-number was found to be highly variable, ranging from zero to 79 per strain. Moreover it differently impacted the copper tolerance in the populations of the two continents. The diffusion of t(XVI;VIII) and, for the first time, t(XVI;XV) was determined in the three vineyard populations. The correlation between the presence of the translocation and strain sulfite tolerance levels was significant only for the t(XVI;VIII).

Whole-genome sequence and comparative genome analysis of Lactobacillus paracasei DTA93, a promising probiotic lactic acid bacterium.

The complete genome sequence of Lactobacillus paracasei DTA93, isolated from healthy infant feces is reported, along with in silico genetic analyses of its main features. The 3.02 Mb sequenced genome possesses 2990 protein-coding sequences distributed on 341 SEED subsystems. In previous in vitro studies, this strain demonstrated interesting probiotic properties, anti-cancer activity, and anti-bacterial activity. The whole-genome sequencing allowed to identify DTA93 as L. paracasei and to precisely place it within the L. casei group, since it shows the highest number of orthologous genes/proteins in common with the type strain L. paracasei ATCC 25302T. In silico analyses revealed the absence of potentially negative traits, such as presence of prophages, transmissible antibiotic resistance and virulence factors. The results provided here considerably increased the amount of information available on DTA93 in favor of its possible use in food products as health-promoting culture. The complete genome data have been deposited in GenBank under the accession number VTYT00000000.

Synbiotic VSL#3 and yacon-based product modulate the intestinal microbiota and prevent the development of pre-neoplastic lesions in a colorectal carcinogenesis model.

Colorectal cancer is a public health problem, with dysbiosis being one of the risk factors due to its role in intestinal inflammation. Probiotics and synbiotics have been used in order to restore the microbiota balance and to prevent colorectal carcinogenesis. We aimed to investigate the effects of the probiotic VSL#3® alone or in combination with a yacon-based prebiotic concentrate on the microbiota modulation and its influence on colorectal carcinogenesis in an animal model. C57BL/6J mice were divided into three groups: control (control diet), probiotic (control diet + VSL#3®), and synbiotic (yacon diet + VSL#3®). The diets were provided for 13 weeks and, from the third one, all animals were subjected to induction of colorectal cancer precursor lesions. Stool samples were collected to evaluate organic acids, feces pH, ß-glucuronidase activity, and microbiota composition. The colon was used to count pre-neoplastic lesions and to determine the cytokines. The microbiota composition was influenced by the use of probiotic and synbiotic. Modifications were also observed in the abundance of bacterial genera with respect to the control group, which confirms the interference of carcinogenesis in the microbiota. Pre-neoplastic lesions were reduced by the use of the synbiotic, but not with the probiotic. The protection provided by the synbiotic can be attributed to the modulation of the intestinal inflammatory response, to the inhibition of a pro-carcinogenic enzyme, and to the production of organic acids. The modulation of the composition and activity of the microbiota contributed to beneficial changes in the intestinal microenvironment, which led to a reduction in carcinogenesis. KEY POINTS: • Synbiotic reduces the incidence of colorectal cancer precursor lesions. • Synbiotic modulates the composition and activity of intestinal microbiota. • Synbiotic increases the abundance of butyrate-producing bacteria.

Complete Genome Sequence and Carbohydrates-Active EnZymes (CAZymes) Analysis of Lactobacillus paracasei DTA72, a Potential Probiotic Strain with Strong Capability to Use Inulin.

The whole genome sequence of Lactobacillus paracasei DTA72, isolated from healthy infant feces, is reported, along with the Carbohydrates-Active enZymes (CAZymes) analysis and an in silico safety assessment. Strain DTA72 had previously demonstrated some interesting potential probiotic features, such as a good resistance to gastrointestinal conditions and an anti-Listeria activity. The 3.1 Mb sequenced genome consists of 3116 protein-coding sequences distributed on 340 SEED subsystems. In the present study, we analyzed the fermentation capability of strain DTA72 on six different carbohydrate sources, namely, glucose, fructose, lactose, galactose, xylose, and inulin by using phenotypical and genomic approaches. Interestingly, L. paracasei DTA72 evidenced the best growth performances on inulin with a much shorter lag phase and higher number of cells at the stationary phase in comparison with all the sugars tested. The CAZyme analysis using the predicted amino acid sequences detected 80 enzymes, distributed into the five CAZymes classes. Moreover, the in silico analysis revealed the absence of blood hemolytic genes, transmissible antibiotic resistances, and plasmids in DTA72. The results described in this study, together with those previously reported and particularly the strong capability to utilize inulin as energy source, make DTA72 a very interesting potential probiotic strain to be considered for the production of synbiotic foods. The complete genome data have been deposited in GenBank under the accession number WUJH00000000.

Whole genome comparison of two Starmerella bacillaris strains with other wine yeasts uncovers genes involved in modulating important winemaking traits.

Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low-ethanol and high-glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole-genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non-Saccharomyces yeasts produced a detailed reconstruction of the pan-genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen-activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high-quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol-3-phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol-3-phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.

Differences in Carbohydrates Utilization and Antibiotic Resistance Between Streptococcus macedonicus and Streptococcus thermophilus Strains Isolated from Dairy Products in Italy.

Streptococcus thermophilus and S. macedonicus are the only two species of the genus related to food productions so far known. In the present study, eight S. thermophilus and seven S. macedonicus strains isolated from dairy environments in Italy were compared in order to evidence possible species-specific technological characteristics. Their capability to use lactose, galactose, fructose, and glucose, sugars commonly present in foods and two carbohydrates considered as prebiotics, xylose and inulin, along with the respective growth kinetics were studied. Results showed a luxuriant growth on lactose and different behaviors on galactose, glucose, and fructose. No growth on inulin and xylose was recorded, which is a positive feature for strains intended to be used as starter cultures. Growth parameters, namely, λ, µmax, and Nmax, were estimated by using the Gompertz model. Antibiotic resistance to 14 drugs revealed an overall similar behavior between the two species with only a marked difference regarding gentamycin. Antimicrobial activity was also tested against six deleterious bacterial strains, but none of the strains evidenced inhibitory capabilities. The results presented here could be helpful to compare technological potentialities of the two species and to choose strains of the most suitable species for selected microbiological food transformations.

Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin.

Within the genus Streptococcus, S. thermophilus and S. macedonicus are the 2 known species related to foods. Streptococci are widely used as starter cultures to rapidly lower milk pH. As S. macedonicus has been introduced quite recently, much less information is available on its technological potential. Because temperature is an important factor in fermented food production, we compared the growth kinetics over 24 h of 8 S. thermophilus and 7 S. macedonicus strains isolated from various dairy environments in Italy, at 4 temperatures, 30°C, 34°C, 37°C and 42°C. We used the Gompertz model to estimate the 3 main growth parameters; namely, lag phase duration (λ), maximum growth rate (µmax), and maximum cell number at the stationary phase (Nmax). Our results showed significant differences in average growth kinetics between the 2 species. Among the strains tested, 37°C appeared to be the optimal temperature for the growth of both species, particularly for S. macedonicus strains, which showed mean shorter lag phases and higher cell numbers compared with S. thermophilus. Overall, the growth curves of S. macedonicus strains were more similar to each other whereas S. thermophilus strains grew very differently. These results help to better define and compare technological characteristics of the 2 species, in view of the potential use of S. macedonicus in place of S. thermophilus in selected technological applications.

Genetic variability and physiological traits of Saccharomyces cerevisiae strains isolated from "Vale dos Vinhedos" vineyards reflect agricultural practices and history of this Brazilian wet subtropical area.

Vale dos Vinhedos appellation of origin has a very recent history as industrial wine making region. In this study we investigated the genetic and phenotypic variability of Saccharomyces cerevisiae strains isolated from South-Brazilian vineyards in order to evaluate strain fermentation aptitude and copper and sulphites tolerance. Merlot grape bunches were collected from three vineyards and yeast isolation was performed after single bunch fermentation. High genotypic variability was found and most of the genotypes revealed to be vine-specific. No industrial strain dissemination was present in the sampled vineyards, although it has been wildly reported in traditional winemaking countries. From the phenotypic traits analysis these Brazilian native strains showed good fermentation performances, good tolerance to sulphites and, in particular, a high copper tolerance level. Copper is the most important metal in the formulation of fungicides against downy mildew (Plasmopara viticola), one of the most harmful disease of the vines, and other fungal pests. The high tolerance to copper suggests an environmental adaptation to the strong use of copper-based fungicides, requested by the wet subtropical climate.

Genome comparison and physiological characterization of eight Streptococcus thermophilus strains isolated from Italian dairy products.

Eight Streptococcus thermophilus strains of dairy origin isolated in Italy were chosen to investigate autochthonous bacterial diversity in this important technological species. In the present study a comparative analysis of all the 17 S. thermophilus genomes publicly available was performed to identify the core and the variable genes, which vary among strains from 196 to 265. Additionally, correlation between the isolation site and the genetic distance was investigated at genomic level. Results highlight that the phylogenetic reconstruction differs from the geographical strain distribution. Moreover, strain M17PTZA496 has a genome of 2.15 Mbp, notably larger than that of the others, determined by lateral gene transfer (including phage-mediated incorporation) and duplication events. Important technological characters, such as growth kinetics, bacteriocin production, acidification kinetics and surface adhesion capability were studied in all the Italian strains. Results indicate a wide range of variability in adhesion properties that significantly clustered strains into four groups. Genomic differences among strains in relation to these characters were identified but a clear correlation between genotype and phenotype was not always found since most of the genomic modifications arise from single nucleotide polymorphisms. This research represents a step forward in the identification of strains-specific functions in Streptococcus thermophilus and it has also the potential to provide valuable information to predict strain specific behaviors in industrial processes.

Different mechanisms of resistance modulate sulfite tolerance in wine yeasts.

From a technological point of view, yeast resistance to sulfite is of great interest and represents an important technological character for winemaking. Several mechanisms are involved, and strain-dependent strategies to obtain SO2 resistance can deeply influence wine quality, although this choice is less relevant in determining the technological performance of the strain during fermentation. In this study, to better understand the strain-specific mechanisms of resistance, 11 Saccharomyces cerevisiae strains, whose genomes have been previously sequenced, were selected. Their attitude towards sulfites, in terms of resistance and production, was evaluated, and RNA-sequencing of four selected strains was performed during fermentation process in synthetic grape must in the presence of SO2. Results demonstrated that at molecular level, the physical effect of SO2 triggered multiple stress responses in the cell and high tolerance to general enological stressing condition increased SO2 resistance. Adaptation mechanism due to high basal gene expression level rather than specific gene induction in the presence of sulfite seemed to be responsible in modulating strain resistance. This mechanism involved higher basal gene expression level of specific cell wall proteins, enzymes for lipid biosynthesis, and enzymes directly involved in SO2 assimilation pathway and efflux.

Potential use of scotta, the by-product of the ricotta cheese manufacturing process, for the production of fermented drinks.

In the present work, the use of scotta as substrate for bacterial fermentation was studied with the objective of obtaining a drink from transformation of this by-product. Scotta retains most of the lactose of the milk and it is normally colonized by a natural microbiota. A treatment was devised to reduce the autochthonous microbial populations in order to reduce competition towards the inoculated bacterial strains. Nine lactic acid bacteria (LAB) were assessed for their capability to develop in scotta. They evidenced different behaviors regarding growth rate, acidification capability and nitrogen consumption. A co-inoculum of three LAB, namely a Streptococcus thermophilus, a Lactobacillus delbrueckii subsp. bulgaricus and a Lb. acidophilus strains, chosen among those giving the best performances in single-strain fermentation trials, gave abundant (close to 10(9) cfu/ml) and balanced growth and lowered pH to 4.2, a value similar to that of yogurt. These results show that scotta may have potential as a substrate for bacterial growth for the production of a fermented drink. Further studies are needed to optimize the organoleptic aspects of the final product.

Outlining a selection procedure for Saccharomyces cerevisiae isolated from grape marc to improve fermentation process and distillate quality.

Nowadays grape marc represents one of the main by-product of winemaking. Many South Europe countries valorize this ligno-cellulosic waste through fermentation and distillation for industrial alcoholic beverage production. The storage of marcs is a crucial phase in the distillation process, due to the physicochemical transformations ascribed to microbial activity. Among the methods adopted by distillers to improve the quality of spirits, the use of selected yeasts has not been explored so far, therefore in this work we evaluated the selection criteria of Saccharomyces cerevisiae strains for grape marc fermentation. The proposed selection procedure included three steps: characterization of phenotypical traits, evaluation of selected strains on pasteurised grape marc at lab-scale (100 g) and pilot-scale fermentation (350 kg). This selection process was applied on 104 strains isolated from grape marcs of different origins and technological treatment. Among physiological traits, ß-glucosidase activity level as quality trait seems to be only partially involved in increasing varietal flavour. More effective in describing yeast impact on distillate quality is the ratio higher alcohols/esters that indicates strain ability to increase positive flavours. Finally, evaluating grape marc as source of selected yeasts, industrial treatment rather than varietal origin seems to shape strain technological and quality traits.

The impact of genomic variability on gene expression in environmental Saccharomyces cerevisiae strains.

Environmental Saccharomyces cerevisiae strains are crucially important, as they represent the large pool from which domesticated industrial yeasts have been selected, and vineyard strains can be considered the genetic reservoir from which industrial wine strains with strong fermentative behaviour are selected. Four vineyard strains with different fermentation performances were chosen from a large collection of strains isolated from Italian vineyards. Their genomes were sequenced to identify how genetic variations influence gene expression during fermentation and to clarify the evolutionary relationship between vineyard isolates and industrial wine strains. RNA sequencing was performed on the four vineyard strains, as well as on the industrial wine yeast strain EC1118 and on the laboratory strain S288c, at two stages of fermentation. We showed that there was a large gene cluster with variable promoter regions modifying gene expression in the strains. Our results indicate that it is the evolvability of the yeast promoter regions, rather than structural variations or strain-specific genes, that is the main cause of the differences in gene expression. This promoter variability, determined by variable tandem repeats and a high number of single-nucleotide polymorphisms together with 49 differentially expressed transcription factors, explained the strong phenotypic differences in the strains.

Metagenomic analysis of the microbial community in fermented grape marc reveals that Lactobacillus fabifermentans is one of the dominant species: insights into its genome structure.

Grape marc used for the production of distilled beverages undergoes prolonged storage which allows alcoholic fermentation to occur. Harsh conditions including low pH, limited oxygen and nutrients, temperature fluctuations, and high ethanol concentrations imposed by that environment create a strong selective pressure on microorganisms. A detailed characterization of the bacterial community during two time points of the fermentation process was performed using high-throughput sequencing of the V3-V6 16S rDNA hypervariable regions. The results revealed a marked reduction in the number of bacterial species after 30 days of incubation and made it possible to identify those species that are able to grow in that extreme environment. The genome sequence of Lactobacillus fabifermentans, one of the dominant species identified, was then analyzed using shotgun sequencing and comparative genomics. The results revealed that it is one of the largest genomes among the Lactobacillus sequenced and is characterized by a large number of genes involved in carbohydrate utilization and in the regulation of gene expression. The genome was shaped through a large number of gene duplication events, while lateral gene transfer contributed to a lesser extent with respect to other Lactobacillus species. According to genomic analysis, its carbohydrate utilization pattern and ability to form biofilm are the main genetic traits linked to the adaptation the species underwent permitting it to grow in fermenting grape marc.

Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances.

We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Exploring the use of Saccharomyces cerevisiae commercial strain and Saccharomycodes ludwigii natural isolate for grape marc fermentation to improve sensory properties of spirits.

In Mediterranean countries the most diffuse practice to obtain the valorization of grape marc, the main by-product from winemaking, is the production of spirits. During this process, marc storage for sugar fermentation represents a crucial step, since side-fermentations leading to off-flavours production can very easily occur. In this study we evaluated the effect of the addition of two yeast strains, inoculated separately at the beginning of the storage period, into marcs from two Italian grape varieties with the aim to control the development of autochthonous microbiota and to improve spirit quality. The presence of the inoculated strains was monitored by means of PCR-based approaches. A commercial Saccharomyces cerevisiae strain, chosen as this species is notably the best ethanol producer, showed excellent ability to dominate the autochthonous microflora and to reduce off-flavours as demonstrated by chemical analysis and sensory evaluation. A Saccharomycodes ludwigii strain, chosen for increasing varietal compounds thus enhancing spirit aroma, showed a level of implantation not sufficient to assure a clear beneficial effect on quality. The implantation level of this strain was affected by S. cerevisiae competition since the highest level was found in grape marc with lower sugar content, where indigenous S. cerevisiae were less persistent.