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Microbiome science as an interdisciplinary research field has evolved rapidlyover the past two decades, becoming a popular topic not only in the scientificcommunity and among the general public, but also in the food industrydue to the growing demand for microbiome-based technologies that provideadded-value solutions. Microbiome research has expanded in the contextof food systems, strongly driven by methodological advances in different-omics fields that leverage our understanding of microbial diversity and function.However, managing and integrating different complex -omics layersare still challenging. Within the Coordinated Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), a project supported by theEuropean Commission, the workshop “Metagenomics, Metaproteomics andMetabolomics: the need for data integration in microbiome research” gathered70 participants from different microbiome research fields relevant to foodsystems, to discuss challenges in microbiome research and to promote a switchfrom microbiome-based descriptive studies to functional studies, elucidatingthe biology and interactive roles ofmicrobiomes in food systems. A combinationof technologies is proposed. This will reduce the biases resulting from eachindividual technology and result in a more comprehensive view of the biologicalsystem as a whole. Although combinations of different datasets are stillrare, advanced bioinformatics tools and artificial intelligence approaches cancontribute to understanding, prediction, and management of the microbiome,thereby providing the basis for the improvement of food quality and safety.

Our study investigated the potential of Annona squamosa (L.) fruit as a reservoir of yeasts and lactic acid bacteria having biotechnological implications, and phenolics capable of modifying the ecology of microbial consortia. Only a single species of lactic acid bacteria (Enterococcus faecalis) was identified, while Annona fruit seemed to be a preferred niche for yeasts (Saccharomyces cerevisiae, Hanseniaspora uvarum), which were differentially distributed in the fruit. In order to identify ecological implications for inherent phenolics, the antimicrobial potential of water- and methanol/water-soluble extracts from peel and pulp was studied. Pulp extracts did not show any antimicrobial activity against the microbial indicators, while some Gram-positive bacteria (Staphylococcus aureus, Staphylococcus saprophyticus, Listeria monocytogenes, Bacillus megaterium) were susceptible to peel extracts. Among lactic acid bacteria used as indicators, only Lactococcus lactis and Weissella cibaria were inhibited. The chemical profiling of methanol/water-soluble phenolics from Annona peel reported a full panel of 41 phenolics, mainly procyanidins and catechin derivatives. The antimicrobial activity was associated to specific compounds (procyanidin dimer type B [isomer 1], rutin [isomer 2], catechin diglucopyranoside), in addition to unidentified catechin derivatives. E. faecalis, which was detected in the epiphytic microbiota, was well adapted to the phenolics from the peel. Peel phenolics had a growth-promoting effect toward the autochthonous yeasts S. cerevisiae and H. uvarum.

The enormous potential of multi-omics approaches to unravel microbiome-related links between food quality, sustainability and safety still requires experimental work and extensive data integration to increase knowledge and understand the biological and ecological processes involved in the assembly and dynamics of microbial communities along the production chains. Data spanning from DNA sequences to transcripts and metabolites need to be integrated in order to be translated at industrial level and literature showed several successful examples. The application of microbiome studies in food systems has shown the potential to improve food quality. Nevertheless, classical microbiological methods are still highly relevant even if isolation and characterization of strains in pure culture is often laborious and time-consuming and requires the use of several specific growth media that take into the account microbial growth characteristics as well as food characteristics. Studies on microbiomes have become a popular topic in the food industry since it can be used as a tool to improve quality and safety in the food chain.

Fungi and oomycetes found in vineyards cause diseases such as powdery and downy mildew. Consequently, conventional and alternative agronomical practices are widely used prior to harvest to protect grapes. Alternative products are considered more eco-friendly and environmentally sustainable in comparison to conventional chemical products. However, the effect of these alternative products on yeast ecology, from the vineyard to the winery, is poorly understood. This study compared the effect of alternative and conventional chemical antifungal compounds (copper and sulphur based) on grapes' mycobiota in the vineyard and during subsequent winery fermentation using culture-dependent and -independent approaches. Culture-dependent data indicated a treatment-dependent effect on the load and diversity of yeast populations on grapes. It was found that the population of Hanseniaspora uvarum was higher on grapes previously treated with laminarin and copper, compared to the other levels registered on grapes previously treated with the rest of antifungal products tested in this study (including the untreated and conventional treatment controls). Concerning, wine quality, the chemical composition was not correlated to the application of antifungal treatment in the vineyard. Understanding the effect of different antifungal products on grape and wine microbial communities may help in setting up guidelines for wine grape production. These guidelines, can be used to guarantee quality in the pursuit of a sustainable competitive advantage in the market.

This study aims to discuss the microbial ecology of the broiler gut environment, Campylobacter prevalence across the broiler production chain with a follow-up focus on a possible mitigation strategy, based on the use of bacteriophages. Scientific literature published from the last two decades was reviewed and data were collected to establish the ranges of Campylobacter loads from different samples. Results showed that the pathogen load in the sample is likely to increase from the different stages of the production chain. Contamination of water and feed represents the most notable source of contamination during the primary production, while cross-contamination of broiler carcasses, skin, and meat occurs during the slaughter, dressing, and processing via machinery, work surfaces, water, and air partially due to the leaking of contaminated feces from visceral rupture. Knowledge gaps were identified and included: a lack of studies detecting Campylobacter in broilers in most of the European countries over the last decade and a low number of studies determining the bacterial load in crates used to transport broilers to the slaughterhouse. Determining the prevalence of Campylobacter in the broiler industry will enable us to set critical control points to produce broiler flocks and meat products with a low risk of Campylobacter contamination.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of

A low initial contamination level of the meat surface is the sine qua non to extend the subsequent shelf life of ground beef for as long as possible. Therefore, the short- and long-term effects of a pregrinding treatment with electrolyzed water (EW) on the microbiological and physicochemical features of Piedmontese steak tartare were here assessed on site, by following two production runs through storage under vacuum packaging conditions at 4°C. The immersion of muscle meat in EW solution at 100 ppm of free active chlorine for 90 s produced an initial surface decontamination with no side effects or compositional modifications, except for an external color change that was subsequently masked by the grinding step. However, the initially measured decontamination was no longer detectable in ground beef, perhaps due to a quick recovery by bacteria during the grinding step from the transient oxidative stress induced by the EW. We observed different RNA-based metataxonomic profiles and metabolomic biomarkers (volatile organic compounds [VOCs], free amino acids [FAA], and biogenic amines [BA]) between production runs. Interestingly, the potentially active microbiota of the meat from each production run, investigated through operational taxonomic unit (OTU)-, oligotyping-, and amplicon sequence variant (ASV)-based bioinformatic pipelines, differed as soon as the early stages of storage, whereas microbial counts and biomarker dynamics were significantly distinguishable only after the expiration date. Higher diversity, richness, and abundance of Streptococcus organisms were identified as the main indicators of the faster spoilage observed in one of the two production runs, while Lactococcus piscium development was the main marker of shelf life end in both production runs. IMPORTANCE Treatment with EW prior to grinding did not result in an effective intervention to prolong the shelf life of Piedmontese steak tartare. Our RNA-based approach clearly highlighted a microbiota that changed markedly between production runs but little during the first shelf life stages. Under these conditions, an early metataxonomic profiling might provide the best prediction of the microbiological fate of each batch of the product.

A detailed understanding of the microbiome of cheese and dairy products is key to the optimization of flavour, appearance, overall quality and safety. Microorganisms (including bacteria, yeasts, moulds and viruses, especially bacteriophages) from the environment can enter the dairy supply chain at multiple stages with several implications. The ability to track these microorganisms and to understand their function and interaction can be greatly enhanced by the use of high-throughput sequencing. Depending on the specific production technology, dairy products can harbor several strains and antibiotic-resistance genes that can potentially interact with the gut microbiome, once the product is ingested. Milk-associated or cheese-associated microbial communities with their interaction, function and diversity are a key factor for the dairy industry. Multi-omics approaches have been seldom utilized in literature and they need to be further considered. Studying the role, origin, diversity and function of the microbial species involved in the complex system of dairy production can help improve processes in several fields of application. Integrating an extensive sampling procedure with an extensive culture based methodology is necessary. To this end, local producers, and in general stakeholders, should be guided to discover and maintain their microbial diversity. A better management of microbial resources through precision fermentation processes will in turn reduce overall food losses and increase the possibility to use the microbiome in order to increase the local producers' income.

In winemaking industry, good cleaning and sanitization practices are essential in bottle filling process to preserve quality and avoid subsequent alterations after bottling, when microbes find environment favourable for their development. Devices connected by pipelines, like wine bottling machines, are usually cleaned using Cleaning-in-Place (CIP) method, generally requiring a high consumption of water and the use of chemical cleaning detergents with a negative impact on the environment. Ozone has recently attracted attention due to its efficacy against a broad spectrum of microorganisms and its ability to clean leaving no residues on treated surfaces, protecting the environment and human health. This study aimed to investigate the impact of aqueous (3.5 mg/L for 15 and 30 min of contact time) and gaseous ozone (30 μL/L for 30 and 60 min of contact time) treatments in comparison with usual sanitizing treatment with peracetic acid (1% for 15 min of contact time) on six wine related microorganisms of oenological significance for their potential proliferation in the bottled wine. To this end, an artificially contaminated wine was used to fill rigid and flexible stainless-steel pipes and a bottling machine. The effectiveness of each treatment was evaluated using culture-dependent approach. The microorganisms showed different sensibilities to the treatments, dependent on the sanitization method used. The exposure to aqueous ozone for 30 min was the most effective treatment for pipes cleaning, followed by peracetic acid. On the other hand, when considering the bottling machine, the use of peracetic acid as sanitizing agent led to a complete removal of the cells, while aqueous ozone for a contact of 30 min was able to eliminate all microorganisms except S. cerevisiae.

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Spoilage by Brettanomyces bruxellensis is a major concern for the wine industry. The negative effect of this yeast on wine quality, that is, potentially large associate economic losses and the production of phenolic off-flavors, requires application of specific control measures. We investigated the ability of ozone to control the B. bruxellensis population on treated Barbera winegrape berries, and the impact on subsequent winemaking or fermentation. To further explore the ability of ozone treatments to reduce the B. bruxellensis population, a mix of three different strains was artificially inoculated onto the surface of grape berries. Grape berries were ozone-treated either in aqueous (6 or 12 min) or gaseous form (12 or 24 hr), crushed, then fermented to evaluate the effect of these treatments on B. bruxellensis and Saccharomyces cerevisiae growth dynamics and wine composition. Microbiological analysis revealed a significant reduction in B. bruxellensis of ~2.2 log colony forming units (CFU)/mL after treatment with gaseous ozone for 24 hr. The wines produced from grape berries previously exposed to gaseous ozone for 24 hr contained the lowest levels of acetic acid. Moreover, 4-ethylphenols were detected in wines produced from grape berries treated with water (6 or 12 min), in which B. bruxellensis population reached 5.0 log CFU/mL at the end of fermentation. Molecular analysis suggested that the three tested strains had similar sensitivity to the ozone treatments applied. This study shows the first results about the ability of ozone to control the population of different B. bruxellensis strains within the same species in the same manner.

Aims The complex mycobiota that colonizes traditional fermented sausages plays an important role in the organoleptic properties of such products. The aim of the present study was to investigate fungal diversity and mycotoxin production during maturation of PGI Salame Piemonte. Methods and results Casing and meat samples were collected at five sampling times from three different batches produced in the same factory and analysed using culture-dependent and independent approaches. Penicillium nalgiovense, which was deliberately inoculated, and Debaryomyces hansenii were the most dominant taxa in casings. Several other fungi mainly belonging to Penicillium crustosum, Penicillium glabrum, Penicillium nordicum, Cladosporium spp., Candida sake, Candida zeylanoides and Yarrowia divulgata were also identified. The casing mycobiota was compared to that of the meat using a metataxonomic approach and a higher fungal diversity was observed in meat as compared to casings. Mycotoxins and penicillin G were monitored using QTOF LC-MS and only trace amounts of roquefortine C were detected in two batches. Conclusions The present study highlighted the diversity of Salame Piemonte mycobiota and the important contribution of autochthonous fungi to its diversity. The absence of mycotoxins and penicillin G confirmed the high hygienic quality of the studied product regarding fungal and mycotoxin contamination. Significance and impact of the study For the first time, this study provides insights about Salame Piemonte mycobiota, which together with the bacterial microbiota and Salame Piemonte process specifications, are responsible for the product organoleptic properties.

Metagenomics is a powerful tool to study and understand the microbial dynamics that occur during food fermentation and allows to close the link between microbial diversity and final sensory characteristics. Each food matrix can be colonized by different microbes, but also by different strains of the same species. In this study, using an innovative integrated approach combining culture-dependent method with a shotgun sequencing, we were able to show how strain–level biodiversity could influence the quality characteristics of the final product. The attention was placed on a model food fermentation process: Salame Piemonte, a Protected Geographical Indication (PGI) Italian fermented sausage. Three independent batches produced in February, March and May 2018 were analysed. The sausages were manufactured, following the production specification, in a local meat factory in the area of Turin (Italy) without the use of starter cultures. A pangenomic approach was applied in order to identify and evaluate the lactic acid bacteria (LAB) population driving the fermentation process. It was observed that all batches were characterized by the presence of few LAB species, namely Pediococcus pentosaceus, Latilactobacillus curvatus and Latilactobacillus sakei. Sausages from the different batches were different when the volatilome was taken into consideration, and a strong association between quality attributes and strains present was determined. In particular, different strains of L. sakei, showing heterogeneity at genomic level, colonized the meat at the beginning of each production and deeply influenced the fermentation process by distinctive metabolic pathways that affected the fermentation process and the final sensory aspects.

Aliarcobacter butzleri is an emerging pathogen that may cause enteritis in humans, however, the incidence of disease caused by this member of the Campylobacteriaceae family is still underestimated. Furthermore, little is known about the precise virulence mechanism and behavior during infection. Therefore, in the present study, through complementary use of comparative genomics and physiological tests on human gut models, we sought to elucidate the genetic background of a set of 32 A. butzleri strains of diverse origin and to explore the correlation with the ability to colonize and invade human intestinal cells in vitro. The simulated infection of human intestinal models showed a higher colonization rate in presence of mucus-producing cells. For some strains, human mucus significantly improved the resistance to physical removal from the in vitro mucosa, while short time-frame growth was even observed. Pangenome analysis highlighted a hypervariable accessory genome, not strictly correlated to the isolation source. Likewise, the strain phylogeny was unrelated to their shared origin, despite a certain degree of segregation was observed among strains isolated from different segments of the intestinal tract of pigs. The putative virulence genes detected in all strains were mostly encompassed in the accessory fraction of the pangenome. The LPS biosynthesis and in particular the chain glycosylation of the O-antigen is harbored in a region of high plasticity of the pangenome, which would indicate frequent horizontal gene transfer phenomena, as well as the involvement of this hypervariable structure in the adaptive behavior and sympatric evolution of A. butzleri. Results of the present study deepen the current knowledge on A. butzleri pangenome by extending the pool of genes regarded as virulence markers and provide bases to develop new diagnostic approaches for the detection of those strains with a higher virulence potential.

Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two Starmerella bacillaris strains displaying metabolic properties, compared to the reference yeast Saccharomyces cerevisiae. The analysis of growth and fermentation parameters provided a comprehensive picture of S. bacillaris preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed with S. cerevisiae regarding rates, final population, and CO(2) production. In particular, Lys and His supported substantial S. bacillaris growth and fermentation contrary to S. cerevisiae, while only 3 nitrogen sources, Arg, NH(4)(+), and Ser, promoted S. cerevisiae growth more efficiently than that of S. bacillaris strains. Furthermore, S. bacillaris strains displayed a higher fermentative activity than S. cerevisiae during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of S. bacillaris regarding nitrogen source preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species. IMPORTANCE Mixed fermentations combining non-Saccharomyces and S. cerevisiae strains are increasingly implemented in the wine sector, as they offer promising opportunities to diversify the flavor profile of end products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of S. bacillaris, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources and, consequently, potential issues during fermentation. The data of this study highlight the importance of appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-Saccharomyces yeasts.

Worldwide the interest for biological control of food spoilage microorganisms has significantly increased over the last decade. Wine makes no exception to this trend, as consumer demands for wines free of preservatives that are considered negative for human health, increase. Biological control during wine fermentation aims at producing high quality wines, while minimizing, or even eliminating, the use of chemical additives. Its success lies in the inoculation of microorganisms to prevent, inhibit or kill undesired microbes, therefore maintaining wine spoilage at the lowest level. The food industry already makes use of this practice, with dedicated commercial microbes already on the market. In winemaking, there are commercial microbes currently under investigation, particularly with the aim to reduce or replace the use of sulphur dioxide. In this review, the potential of wine yeasts and lactic acid bacteria as bioprotection agents and their mechanisms of action during wine fermentation are presented.

Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two Starm. bacillaris strains displaying metabolic properties, compared to the reference yeast S. cerevisiae The analysis of growth and fermentation parameters provided a comprehensive picture of Starm. bacillaris preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed in S. cerevisiae regarding rates, final population and CO2 production. In particular, Lys and His supported substantial Starm. bacillaris growth and fermentation contrary to S. cerevisiae, while only 3 nitrogen sources, Arg, NH4+ and Ser, promoted S. cerevisiae growth more efficiently than that of Starm. bacillaris strains. Furthermore, Starm. bacillaris strains displayed a higher fermentative activity than S. cerevisiae during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of Starm. bacillaris regarding nitrogen sources preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species.IMPORTANCE Mixed fermentations combining non-Saccharomyces and S. cerevisiae strains are increasingly implemented in the wine sector as they offer promising opportunities to diversify the flavour profile of end-products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of Starm. bacillaris, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation, through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources, and consequently potential issues during fermentation. The data of this study highlight the importance of an appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-Saccharomyces yeasts.

Microbial complexity and contamination levels in food processing plants heavily impact the final product fate and are mainly controlled by proper environmental cleaning and sanitizing. Among the emerging disinfection technologies, ozonation is considered an effective strategy to improve the ordinary cleaning and sanitizing of slaughterhouses. However, its effects on contamination levels and environmental microbiota still need to be understood. For this purpose, we monitored the changes in microbiota composition in different slaughterhouse environments during the phases of cleaning/sanitizing and ozonation at 40, 20, or 4 ppm. Overall, the meat processing plant microbiota differed significantly between secondary processing rooms and deboning rooms, with a greater presence of psychrotrophic taxa in secondary processing rooms because of their lower temperatures. Cleaning/sanitizing procedures significantly reduced the contamination levels and in parallel increased the number of detectable operational taxonomic units (OTUs), by removing the masking effect of the most abundant human/animal-derived OTUs, which belonged to the phylum Firmicutes. Subsequently, ozonation at 40 or 20 ppm effectively decreased the remaining viable bacterial populations. However, we could observe selective ozone-mediated inactivation of psychrotrophic bacteria only in the secondary processing rooms. There, the Brochothrix and Pseudomonas abundances and their viable counts were significantly affected by 40 or 20 ppm of ozone, while more ubiquitous genera like Staphylococcus showed a remarkable resistance to the same treatments. This study showed the effectiveness of highly concentrated gaseous ozone as an adjunct sanitizing method that can minimize cross-contamination and so extend the meat shelf life. IMPORTANCE Our in situ survey demonstrates that RNA-based sequencing of 16S rRNA amplicons is a reliable approach to qualitatively probe, at high taxonomic resolution, the changes triggered by new and existing cleaning/sanitizing strategies in the environmental microbiota in human-built environments. This approach could soon represent a fast tool to clearly define which routine sanitizing interventions are more suitable for a specific food processing environment, thus limiting the costs of special cleaning interventions and potential product loss.

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

International audience; Recently the use of electrolyzed water (EW) attracted much attention as a high-performance, new technology for its potential use in the food industry. The aim of this work was to investigate the impact of grape EW treatments, applied at different time intervals prior to harvest, on the indigenous yeast populations of grape surface (Chenin blanc and Cabernet franc) and the occurrence of 2,4,6-trichloroanisole (TCA) in Cabernet franc wine. In addition, the evolution of inoculated and spontaneous fermentations on treated and non-treated grapes was also considered. The yeast population present on grape berries surface was influenced in a grape variety and EW treatment time-dependent way, since only Chenin blanc grapes treated with EW 7 days prior to harvest had significantly lower yeast population levels, compared to the respective control. Concerning the yeast diversity in the grape samples, a dominance of Aureobasidium pullulans was observed in treated grapes, independently of the grape variety. At the end of alcoholic fermentation, 2,4,6-trichloroanisole was detected in wine when the EW solution was applied at one or two weeks before harvest time. After wine storage, 2,4,6-trichloroanisole and chlorophenols contents generally exhibited a loss relative to initial values. The results showed that EW treatments tended to slightly increase the TCA concentration in final wine and did not affect the fermentation performances and chromatic properties of resulting wine. On the other hand, absorption or desorption phenomena by wine lees could be involved in the change of 2,4,6-trichloroanisole concentration in wine during storage time.

Bacterial communities and Volatile Organic Compounds (VOCs) profile of deepwater rose shrimp (Parapenaeus longirostris) stored at 0 °C (ice) and 4 °C were investigated using 16S amplicon based sequencing and Solid phase micro-extraction (SPME) - Gas chromatography/mass spectrometry (GC/MS), respectively. The shelf-life of shrimps determined by sensory assessment was 5 and 2 days at 0 °C and 4 °C, respectively. Based on 16S analysis (culture-independed), the initial microbiota of shrimps mainly consists of Photobacterium, Candidatus Hepatoplasma, Psychrobacter, Acinetobacter and Delftia. Psychrobacter and Carnobacterium dominated during storage at both temperatures. Psychrobacter was the most dominant taxon at the end of shelf-life of chill-stored shrimps. A minor microbial population composed by Brevundimonas, Stenotrophomonas, Staphylococcus, Legionella, Acinetobacter, Bacillus, Escherichia-Shigella, Enterococcus, Enterobacter, Klebsiella was also detected. Those taxa may be originated from the environment due to an inadequate hygienic practice during fishing, handling and icing. VOCs such as ethanol, 3-methyl-1-butanol, 2-ethyl-1-hexanol, 3-hydroxy-2-butanone, indole etc., were found to be associated with shrimps at 4 °C, while acetone and dimethyl sulfide with shrimps in ice. Some VOCs, from microbial or chemical origin, increased in shrimps either at 0 °C (i.e. 1-octen-3-ol, trans-2-octenal) or at 4 °C (i.e. 3-methyl-1-butanol, indole), while 2-methylbutanal and 3-methylbutanal increased in both temperatures. A positive correlation between Psychrobacter with 2-ethyl-1-hexanol and Carnobacterium with 3-methyl-1-butanol was also observed. Concluding, we suggest the reinforcement of Good Hygiene Practices on fishing boats during fishing/handling, the rapid onboard icing and keeping shrimps iced avoiding even small increase of storage temperature that affects quality parameters (e.g. microbial population level, synthesis of microbiota, VOCs profile) in order to provide a product of the highest quality and safety in the market.

Simultaneous inoculation of yeast and lactic acid bacteria (LAB) is considered a state-of-the-art strategy to reduce overall vinification time and improve microbiological stability of wines. This inoculation protocol sparked interest in selecting yeast and LAB strains to modulate malic acid consumption rate and wine composition. The study presented here addresses the impact on malic acid consumption and metabolite production of combining Saccharomyces cerevisiae strains with different fermentation rates and nutrition demands with Lactobacillus plantarum and Oenococcus oeni strains. S. cerevisiae strains in pure culture fermentations without LAB inoculation exhibited different patterns of malic acid consumption rate and metabolite production. Simultaneous S. cerevisiae and LAB inoculation influenced the kinetics of lactic acid production and titratable acidity in a LAB-strain dependent manner. Wines undergoing malolactic fermentation with L. plantarum ML Prime finished faster and contained more L-lactic acid than similar wines inoculated with O. oeni Lalvin VP41; however, the degree of acidification depended on the S. cerevisiae strain used to conduct alcoholic fermentation. This study reveals new knowledge about the use of L. plantarum in winemaking and shows the effect of S. cerevisiae strains with different enological characteristics, with or without LAB co-inoculation, on wine composition.

Yeasts are generally recognized as contaminants in the production of white-brined cheeses, such as Feta and Feta-type cheeses. The most predominant yeasts species are Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Kluyveromyces lactis, Rhodotorula mucilaginosa, and Trichosporon spp. Although their spoilage potential varies at both species and strain levels, yeasts will, in case of excessive growth, present a microbiological hazard, effecting cheese quality. To evaluate the hazard and trace routes of contamination, the exact taxonomic classification of yeasts is required. Today, identification of dairy yeasts is mainly based on DNA sequencing, various genotyping techniques, and, to some extent, advanced phenotypic identification technologies. Even though these technologies are state of the art at the scientific level, they are only hardly implemented at the industrial level. Quality defects, caused by yeasts in white-brined cheese, are mainly linked to enzymatic activities and metabolism of fermentable carbohydrates, leading to production of metabolites (CO2, fatty acids, volatile compounds, amino acids, sulfur compounds, etc.) and resulting in off-flavors, texture softening, discoloration, and swelling of cheese packages. The proliferation of spoilage yeast depends on maturation and storage conditions at each specific dairy, product characteristics, nutrients availability, and interactions with the co-existing microorganisms. To prevent and control yeast contamination, different strategies based on the principles of HACCP and Good Manufacturing Practice (GMP) have been introduced in white-brined cheese production. These strategies include milk pasteurization, refrigeration, hygienic sanitation, air filtration, as well as aseptic and modified atmosphere packaging. Though a lot of research has been dedicated to yeasts in dairy products, the role of yeast contaminants, specifically in white-brined cheeses, is still insufficiently understood. This review aims to summarize the current knowledge on the identification of contaminant yeasts in white-brined cheeses, their occurrence and spoilage potential related to different varieties of white-brined cheeses, their interactions with other microorganisms, as well as guidelines used by dairies to prevent cheese contamination.

The use of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae is gaining attention in recent years due to their ability to modulate the metabolites production of enological interest. In the present study, four of the most popular planted red grape varieties (Cabernet sauvignon, Merlot, Pinot noir and Shiraz) were fermented using the aforementioned species and two different inoculation protocols (inoculation of S. cerevisiae after 24 and 48 h from the Starm. bacillaris inoculation), in order to evaluate their impact on the volatile composition and chromatic characteristics of wines. Analysis from chemical composition showed that titratable acidity and glycerol content exhibited marked differences among wines after fermentation. For volatile compounds, mixed fermented wines using an inoculation delay of 48 h led to reduction of volatile compounds (mainly esters). A shorter 24 h delay produced wines with higher values of color intensity than pure fermented wines. The differences observed between the inoculation protocols can be explained by the growth dynamics of both species during fermentation. These findings suggest that mixed fermentations posed a great potential in reducing metabolites which are considered negative for wine quality (mainly ethyl acetate and volatile fatty acids) and with an improvement of the chromatic profile of the wines.

In this study, we investigated the possible effect of electrolyzed water (EW), aqueous ozone (WO) and gaseous ozone (GO) on Brettanomyces bruxellensis DSM 7001 strain artificially inoculated on the grape surface and on its evolution during the subsequent, inoculated must fermentation. Culture-dependent and -independent techniques were used to evaluate the effectiveness of treatments against B. bruxellensis, as well as its presence during fermentation. Particularly, GO treatment of 24 h decreased its presence by about 2.1 Log, making it possible to reduce significantly the concentration of ethylphenols in the wine in relation to the control wine. EW and WO treatments caused less relevant reductions. The results showed that all the treatments reduced the presence of this yeast on grapes. However, in these experimental conditions it was not possible to achieve a complete removal of this undesirable yeast. Industrial relevance Brettanomyces spp. is considered a wine spoilage yeast due to its ability to produce off-flavors (described as Brett character) and high levels of acetic acid. Broad disinfectant action against microorganisms, eco-friendliness and easiness of on-site application are among the main advantages of the ozone and the electrolyzed water. This study demonstrated the antimicrobial potential of the EW, WO and GO treatments against B. bruxellensis inoculated on post-harvest grapes.

Starmerella bacillaris (synonym Candida zemplinina ) is a non- Saccharomyces yeast that has been proposed as a co-inoculant of selected Saccharomyces cerevisiae strains in mixed culture fermentations to enhance the analytical composition of the wines. In order to acquire further knowledge on the metabolic interactions between these two species, in this study we investigated the impact of oxygen addition and combination of Starm. bacillaris with S. cerevisiae strains on the microbial growth and metabolite production. Fermentations were carried out under two different conditions of oxygen availability. Oxygen availability and strain combination clearly influenced the population dynamics throughout the fermentation. Oxygen concentration increased the survival time of Starm. bacillaris and decreased the growth rate of S. cerevisiae strains in mixed culture fermentations, whereas it did not affect the growth of the latter in pure culture fermentations. This study reveals new knowledge about the influence of oxygen availability on the successional evolution of yeast species during wine fermentation.

Yeasts represent the most important group of microorganisms to winemakers, because they are responsible for the transformation of sugar-rich musts into high quality wines. Among them Starmerella bacillaris , have been indicated to affect the chemical composition of the musts and wines by producing various metabolites of enological interest. This species has been isolated from grapes, winery environments and during spontaneous fermentations in different countries in the world. Its use together with selected Saccharomyces cerevisiae in mixed culture fermentations has been proposed to increase wine particular characteristics. This review includes the latest reports covering the biotechnological characteristics of this non- Saccharomyces species, as well as its application in winemaking industry.

Background and Aims The aim of this work was to assess and compare the chemical composition and colour characteristics of Barbera red wines obtained after partial alcohol reduction using three promising methodologies for implementation at the industrial level. Methods and Results Alcohol reduction was achieved by: (i) pre-fermentation addition of liquid derived from grape must (reverse osmosis by-product); (ii) mixed fermentations with strains of Starmerella bacillaris and Saccharomyces cerevisiae; and (iii) dealcoholisation of wine post-fermentation with a polypropylene membrane. The microbiological approach enabled the production of wines with a slightly lower alcohol concentration (−0.2 to −0.3% v/v), while facilitating the release of anthocyanin and some esters of fatty acids (ethyl hexanoate and ethyl dodecanoate) that could contribute positively to wine aroma with pleasant nuances. The low impact of the partial replacement of grape juice on the chemical composition and chromatic characteristics of Barbera wines makes this technique a good option for reducing the ethanol concentration by up to 1.0–2.0% v/v. In contrast, the use of a polypropylene membrane influenced negatively the composition of red wines by reducing significantly the concentration of esters (−60%) and anthocyanin (−17%), independently of the dealcoholisation level (up to −2% v/v). Conclusions The alcohol reduction strategies can greatly influence the volatile and phenolic composition of the wine. The choice of either a technological or microbiological approach in the wine industry is dependent on the alcohol reduction required. Significance of the Study This is the first comparative study of three strategies to reduce the alcohol concentration on the same batch of must/wine.

Recently the use of ozone as sanitizing agent has been proposed on winegrapes in order to control mycobiota after harvest. The aim of this work was to investigate possible indirect physico-chemical effects of ozone treatment on berry skin phenolic composition and extractability. Vitis vinifera L. cv Nebbiolo and Barbera, chosen for their different anthocyanin profiles, were post-harvest treated for 24 and 72 h with gaseous ozone (30 μL/L). Skin anthocyanin and flavanol extractability was assessed during maceration (6, 24, 48, 96, 168 and 240 h) using a wine-like solution. In our experimental conditions, ozone did not affect significantly the final extraction yield of anthocyanins (TA), proanthocyanidins (PRO), and flavanols reactive to vanillin (FRV) in Barbera, although TA and FRV extractabilities were higher in control samples than in ozone-treated samples during the first stages of maceration. In Nebbiolo, the final TA extraction yield was positively influenced by the ozone treatment (68.6, 64.2, and 59.9% for 24 h ozone-treated berries, 72 h ozone-treated berries and control samples, respectively). Final PRO and FRV extractability also increased in both ozone-treated samples compared to the control (+ 8.6–9.1% for PRO and + 7.3–11.7% for FRV). No significant differences were found among treatments for individual anthocyanins in both cultivars at the end of maceration. Therefore, the use of ozone as sanitizing agent in red varieties prior to winemaking process can be considered because it did not negatively affect the extractability of skin anthocyanins and flavanols.