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The dietary consumption of probiotics in the form of pharmaceuticals or functional food can improve human health and contribute to disease prevention. However, the biological activity and health potential of food-delivered probiotics can be severely compromised by the stress conditions encountered by the microorganisms throughout the manufacture process, from probiotic preparation to their inclusion into food, subsequent storage and ingestion. Here, we give an account of the stress factors that can have major negative impacts on probiotic viability and functionality, with a focus on food-related environmental adverse conditions. We also describe some of the mechanisms elicited by the microbial cells to counteract these stresses and summarize a few relevant approaches proposed in literature to develop more robust and competitive probiotics by enhancing their stress tolerance, with the aim to improve the efficacy and health value of probiotic functional food.

Lactic acid bacteria (LAB) constitute pivotal microorganisms for the food industry due to their considerable technological and functional potential. Fermented dairy products, that is ancient traditions worldwide, still have been accepted and consumed today for their nutritional and organoleptic attributes. In accordance with the current trends in the market and greater awareness of consumer well-being, a detailed assessment of the health effects of dairy product consumption has been undertaken with particular reference to the production of bioactive metabolites including i.e.,.-aminobutyric acid, conjugated linoleic acid, bioactive peptides, exopoly-saccharides as well as vitamins during LAB fermentation. The long histories of yogurt, kefir, and traditional fermented milk drink as sources of some certain probiotic bacteria as well as the opportunity to bio-fortify the matrix with bioactive microbial compounds in situ make fermented dairy products an attractive model in the efforts to develop sustainable functional food systems. Therefore, this review provides the main biochemical characteristics and biotechnological application of LAB as pro-technological, bioprotective, and health-promoting in the food chain.

SummaryManufacturing of probiotics and functional foods using lactic acid bacteria that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analyzed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for lactic acid bacteria, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~6-fold higher in B2. Mutation B2 could decrease the stability of the aptamer’s regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavine, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Botrytis cinerea, responsible for grey mold, represents the first biological cause of fruit and vegetable spoilage phenomena in post-harvest. Kiwifruit is a climacteric fruit particularly prone to this mold infestation during storage. Lactic acid bacteria (LAB) are food-grade bacteria that can synthesize several metabolites with antimicrobial activity and are, therefore, suggested as promising and eco-friendly resources for the bio-control of molds on fruits and vegetables. In this work, we propose the screening of a collection of 300 LAB previously isolated from traditional sourdoughs for their ability to counteract in vitro the growth of Botrytis cinerea CECT 20973. Only 2% of tested LAB strains belonging to Lactiplantibacillus plantarum species, exerted a strong antagonism against B. cinerea. The cell-free supernatants were partially characterized and results clearly indicated that high levels of lactic acid contributed to the antagonistic activity. PAN01 and UFG 121 cell-free supernatants were investigated as potential bio-control agents in a preliminary in vivo assay using freshly cut kiwifruits as a food model. The application of cell-free supernatants allowed to delay the growth of B. cinerea on artificially contaminated kiwifruits until two weeks. The antagonistic activity was greatly affected by the storage temperature (25 °C and 4 °C) selected for the processed fruits, suggesting the importance to include microbial-based solution in a broader framework of hurdle technologies.

Riboflavin (vitamin B

12 p.-2 fig.-1 tab., Riboflavin (vitamin B2) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic acid bacteria (LAB) can synthesize riboflavin and this capability is strain-dependent. In the last years, a growing interest has focused on the selection of riboflavin-overproducing food-grade LAB for the vitamin biofortification of fermented foods, as well as for the formulation of innovative functional products., In this chapter we report fast and inexpensive techniques in order to (1) screen LAB isolates able to produce riboflavin from different matrices, (2) select spontaneous roseoflavin-resistant riboflavin overproducing strains, and (3) quantify vitamin B2 in culture media by fluorescence detection., These protocols could be useful to select new overproducing strains and/or species from different ecological niches, as well as to optimize the conditions for vitamin bioproduction., Pasquale Russo is the beneficiary of a grant by MIUR in the framework of “AIM: Attraction and International Mobility” (PON R&I2014-2020) (practice code D74I18000190001). This work was supported by the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-097114-B-I00).

Consumers highly appreciate table grapes for their pleasant sensory attributes and as good sources of nutritional and functional compounds. This explains the rising market and global interest in this product. Along with other fruits and vegetables, table grapes are considerably perishable post-harvest due to the growth of undesired microorganisms. Among the microbial spoilers, Botrytis cinerea represents a model organism because of its degrading potential and the huge economic losses caused by its infection. The present review provides an overview of the recent primary physical, chemical, and biological control treatments adopted against the development of B. cinerea in table grapes to extend shelf life. These treatments preserve product quality and safety. This article also focuses on the compliance of different approaches with organic and sustainable production processes. Tailored approaches include those that rely on controlled atmosphere and the application of edible coating and packaging, as well as microbial-based activities. These strategies, applied alone or in combination, are among the most promising solutions in order to prolong table grape quality during cold storage. In general, the innovative design of applications dealing with hurdle technologies holds great promise for future improvements.

The employment of multi-species starter cultures has growing importance in modern winemaking for improving the complexity and wine attributes. The assessment of compatibility for selected species/strains at the industrial-scale is crucial to assure the quality and the safety associated with fermentations. An aspect particularly relevant when the species belong to non-Saccharomyces, Saccharomyces spp. and malolactic bacteria, three categories with different biological characteristics and oenological significance. To the best of our knowledge, the present report is the first study regarding the utilization of a combined starter culture composed of three strains of non-Saccharomyces, Saccharomyces cerevisiae and Lactobacillus plantarum for production of wine at the industrial scale. More in-depth, this work investigated the oenological potential of the autochthonous characterized strains from the Apulian region (Southern Italy), Candida zemplinina (syn. Starmerella bacillaris) 35NC1, S. cerevisiae (NP103), and L. plantarum (LP44), in co-inoculation following a complete scale-up scheme. Microbial dynamics, fermentative profiles and production of volatile secondary compounds were assessed in lab-scale micro-vinification tests and then the performances of the mixed starter formulation were further evaluated by pilot-scale wine production. The above results were finally validated by performing an industrial-scale vinification on 100HL of Negroamaro cultivar grape must. The multi-starter formulation was able to rule the different stages of the fermentation processes effectively, and the different microbial combinations enhanced the organoleptic wine features to different extents. The findings indicated that the simultaneous inoculation of the three species affect the quality and quantity of several volatile compounds, confirming that the complexity of the wine can reflect the complexity of the starter cultures. Moreover, the results underlined that the same mixed culture could differently influence wine quality when tested at the lab-, pilot- and industrial-scale. Finally, we highlighted the significance of employment non-Saccharomyces and L. plantarum, together with S. cerevisiae, autochthonous strains in the design of custom-made starter culture formulation for typical regional wine production with pronounced unique quality.

The influence of riboflavin (B2)-overproducing lactobacilli on the antioxidant status, isoflavone conversion, off-flavor reduction, amino acid profile, and viscosity of B2-bio-enriched fermented soymilk was investigated. Results showed that B2 in fermented soymilk was notably increased from 0.2 to 3.8 μg/mL for Lactobacillus fermentum UFG169 and to 1.9 μg/mL for Lactobacillus plantarum UFG10. The apparent viscosity significantly changed with rising acidity and agglutination of protein. The off-flavor volatile substances (hexanal and nonanal) were significantly reduced in fermented soymilk. Furthermore, a large amount of glucoside form isoflavones was deglycosylated into bioactive aglycones after 4 h up to 32 h. B2 content and isoflavones significantly improved the antioxidant status of soymilk. Partial least squares regression analysis correlated the strain activity and fermentation time with the improved nutritional and functional soymilk qualities. This study demonstrated the strategy for strain development for B2-bio-enriched fermentation to extend the health-promoting benefits of soymilk and soy-related foods. KEY POINTS: • B2-enriched fermentation enhanced the nutrition and functional status of soymilk. • Fermentation time significantly affected the apparent viscosity of fermented soymilk. • Off-flavor volatile substances were significantly reduced or even diminished. • Increased B2and bioactive isoflavones contributed to improved antioxidant potential.

Which is the microbial diversity behind the production of biogenic amines in foods and beverages? What is the food-related significance of this specific subset of micro-biodiversity? Which is the influence of physical, chemical and biological determinants associated to food and beverages matrices that can, directly and indirectly, modulate the concentration of these low molecular weight organic bases? This chapter aims to summarize several aspects that appear to be crucial for a renewed management of the levers susceptible to minimize the content of biogenic amines in edible matrices, highlighting the present main trends and possible future perspectives in the field.

The enterovirus Coxsackievirus B4 (CV-B4) can infect different human tissues and provoke abnormal function or destruction of various organs and cells. Moreover, its infections have been linked to the onset of type 1 diabetes. Coxsackievirus B4 is classified as a "challenging virus", due to the intense yet vain efforts to find effective prevention and therapeutic agents, especially within biological compounds. Lactobacillus plantarum is a lactic acid bacterium that is endowed with probiotic properties, and holds great potential for applications in medical and food industry sectors. Several compounds produced by this microorganism have been associated with various benefits including antimicrobial activity. In this work, we investigated the possible antiviral abilities of two Lb. plantarum strains and their derivatives against CV-B4. The different assays carried out (e.g. pre-incubation, competition and post-infection, using HEp-2 cells as human cell model) suggest that the tested microorganisms and their derivatives have an in vitro inhibiting activity against CV-B4. This is the first report showing the anti-CVB4 activity of Lb. plantarum strains and their derivatives.

The aim of this investigation has been the design and validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeasts, 5 lactic bacteria and 3 acetic acid bacteria species. Furthermore, several strains belonging to these species has been found to produce undesirable compounds for wine consumers. Oligonucleotide probes specific for each microorganism were designed to target the intergenic spacer regions (ISR) between18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consensus primer. Each oligonucleotide-probes exclusively recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 (Saccharomyces cerevisiae), 160 (Lactobacillus brevis) and 124 (Gluconobacter oxydans) cells, three species chosen as experimental models for the three studied microbial classes. Moreover, a novel procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The obtained results confirm that the microarray assay is able to detect specifically different spoilage microorganisms present in mixture in contaminated wines. For the first time the microarray methodology has been applied for the simultaneous identification of different mixed population of spoilage yeast and bacteria directly isolated from wine, thus indicating the practicability of oligonucleotide microarrays as a contamination control in wine industry.

Selected biological resources used as raw materials in beer production are important drivers of innovation and segmentation in the dynamic market of craft beers. Among these resources, local/regional ingredients have several benefits, such as strengthening the connection with territories, enhancing the added value of the final products, and reducing supply costs and environmental impacts. It is assumed that specific ingredients provide differences in flavours, aromas, and, more generally, sensory attributes of the final products. In particular, of interest are ingredients with features attributable and/or linked to a specific geographical origin. This review encompasses the potential contribution and exploitation of biodiversity in the main classes of beer inputs, such as cereals, hops, microbes, and adjuncts, with a specific emphasis on autochthonous biological resources, detailing the innovative paths already explored and documented in the scientific literature. This dissertation proposes an overview of the impact on beer quality for each raw material category, highlighting the benefits and limitations that influence its concrete applications and scale-up, from the field to the stain. The topics explored promote, in the sector of craft beers, trends already capitalised in the production of other alcoholic beverages, such as the preservation and revalorisation of minor and autochthonous varieties, the exploitation of yeast and bacteria strains isolated from specific sites/plant varieties, and the valorisation of the effects of peculiar terroirs on the quality of agricultural products. Finally, the examined tendencies contribute toward reducing the environmental impacts of craft beer manufacturing, and are in line with sustainable development of food systems, increasing the economic driver of biodiversity preservation.

Brettanomyces bruxellensis is a common and significant wine spoilage microorganism. B. bruxellensis strains generally detain the molecular basis to produce compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols that derive from the sequential bioconversion of specific hydroxycinnamic acids such as ferulate and p-coumarate. Although B. bruxellensis can be detected at any stage of the winemaking process, it is typically isolated at the end of the alcoholic fermentation (AF), before the staring of the spontaneous malolactic fermentation (MLF) or during barrel aging. For this reason, the endemic diffusion of B. bruxellensis leads to consistent economic losses in the wine industry. Considering the interest in reducing sulfur dioxide use during winemaking, in recent years, biological alternatives, such as the use of tailored selected yeast and bacterial strains inoculated to promote AF and MLF, are actively sought as biocontrol agents to avoid the “Bretta” character in wines. Here, we review the importance of dedicated characterization and selection of starter cultures for AF and MLF in wine, in order to reduce or prevent both growth of B. bruxellensis and its production of volatile phenols in the matrix.

Climate change threatens food systems, with huge repercussions on food security and on the safety and quality of final products. We reviewed the potential of food microbiology as a source of biotechnological solutions to design climate-smart food systems, using wine as a model productive sector. Climate change entails considerable problems for the sustainability of oenology in several geographical regions, also placing at risk the wine typicity. The main weaknesses identified are: (i) The increased undesired microbial proliferation; (ii) the improved sugars and, consequently, ethanol content; (iii) the reduced acidity and increased pH; (iv) the imbalanced perceived sensory properties (e.g., colour, flavour); and (v) the intensified safety issues (e.g., mycotoxins, biogenic amines). In this paper, we offer an overview of the potential microbial-based strategies suitable to cope with the five challenges listed above. In terms of microbial diversity, our principal focus was on microorganisms isolated from grapes/musts/wines and on microbes belonging to the main categories with a recognized positive role in oenological processes, namely Saccharomyces spp. (e.g., Saccharomyces cerevisiae), non-Saccharomyces yeasts (e.g., Metschnikowia pulcherrima, Torulaspora delbrueckii, Lachancea thermotolerans, and Starmerella bacillaris), and malolactic bacteria (e.g., Oenococcus oeni, Lactobacillus plantarum).

This study reports the first application of a next generation sequencing (NGS) analysis. The analysis was designed to monitor the effect of the management of microbial resources associated with alcoholic fermentation on spontaneous malolactic consortium. Together with the analysis of 16S rRNA genes from the metagenome, we monitored the principal parameters linked to MLF (e.g., malic and lactic acid concentration, pH). We encompass seven dissimilar concrete practices to manage microorganisms associated with alcoholic fermentation: Un-inoculated must (UM), pied-de-cuve (PdC), Saccharomyces cerevisiae (SC), S. cerevisiae and Torulaspora delbrueckii co-inoculated and sequentially inoculated, as well as S. cerevisiae and Metschnikowia pulcherrima co-inoculated and sequentially inoculated. Surprisingly, each experimental modes led to different taxonomic composition of the bacterial communities of the malolactic consortia, in terms of prokaryotic phyla and genera. Our findings indicated that, uncontrolled AF (UM, PdC) led to heterogeneous consortia associated with MLF (with a relevant presence of the genera Acetobacter and Gluconobacter), when compared with controlled AF (SC) (showing a clear dominance of the genus Oenococcus). Effectively, the SC trial malic acid was completely degraded in about two weeks after the end of AF, while, on the contrary, malic acid decarboxylation remained uncomplete after 7 weeks in the case of UM and PdC. In addition, for the first time, we demonstrated that both (i) the inoculation of different non-Saccharomyces (T. delbrueckii and M. pulcherrima) and, (ii) the inoculation time of the non-Saccharomyces with respect to S. cerevisiae resources (co-inoculated and sequentially inoculated) influence the composition of the connected MLF consortia, modulating MLF performance. Finally, we demonstrated the first findings of delayed and inhibited MLF when M. pulcherrima, and T. delbrueckii were inoculated, respectively. In addition, as a further control test, we also assessed the effect of the inoculation with Oenococcus oeni and Lactobacillus plantarum at the end of alcoholic fermentation, as MLF starter cultures. Our study suggests the potential interest in the application of NGS analysis, to monitor the effect of alcoholic fermentation on the spontaneous malolactic consortium, in relation to wine.

Lactobacilli naturally present in the neonatal gut are believed to be beneficial for the human hosts and are investigated as potential probiotics. In this study, we aimed to characterize six Lactobacillus plantarum strains derived from the feces of a breast-fed infant, for the development of new probiotic cultures. Our attention was focused on L. plantarum in reason of the presence, within such species, of both pro-technological and probiotic strains, i.e., a combination of particular interest to design tailored probiotic starter cultures for developing countries. The bacterial isolates exhibiting lactobacilli-like phenotypic characteristics were identified as members of the L. plantarum group by 16S rRNA gene sequencing, and their diversity was evaluated by randomly amplified polymorphic DNA (RAPD) PCR patterns. The selected strains were screened for probiotic potential through in vitro tests. Firstly, bacterial survival was evaluated in an in vitro system simulating the human oro-gastrointestinal tract, using also milk as a carrier matrix. Besides, physiological traits such as antibiotic susceptibility, antimicrobial activity against selected enteric pathogens, and adhesion to abiotic surfaces and to gastric mucin were studied. Considering the resistance to simulated gastrointestinal digestion and the results from the biofilm and mucin adhesion tests, a strain-denominated L. plantarum LSC3 was selected for further evaluation of in vitro adhesion ability to intestinal mucosa and immunomodulatory activities. L. plantarum LSC3 was able to adhere efficiently to human enterocyte-like cells (Caco-2 cells), and decreased IL-8 transcription while increasing IL-10 mRNA level, as revealed by transcriptional analysis on LPS-stimulated human (THP-1) macrophages. Our results highlight that L. plantarum LSC3 fulfills major in vitro probiotic criteria as well as interesting immunostimulatory properties, and thus may be a promising candidate for further in vivo studies aiming at the development of novel probiotic starter cultures.

For three consecutive years, an Italian winery in Apulia has dealt with sudden alcoholic stuck fermentation in the early stages of vinification process, i.e., typical defects addressable to bacterial spoilage. After a prescreening trial, we assessed, for the first time, the influence of the commercial fungicide preparation Ridomil Gold® (Combi Pepite), containing Metalaxyl-M (4.85%) and Folpet (40%) as active principles, on the growth of several yeasts (Saccharomyces cerevisiae and non-Saccharomyces spp.) and lactic acid bacteria of oenological interest. We also tested, separately and in combination, the effects of Metalaxyl-M and Folpet molecules on microbial growth both in culture media and in grape must. We recalled the attention on Folpet negative effect on yeasts, extending its inhibitory spectrum on non-Saccharomyces (e.g., Candida spp.). Moreover, we highlighted a synergic effect of Metalaxyl-M and Folpet used together and a possible inhibitory role of the fungicide excipients. Interestingly, we identified the autochthonous S. cerevisiae strain E4 as moderately resistant to the Folpet toxicity. Our findings clearly indicate the urgent need for integrating the screening procedures for admission of pesticides for use on wine grape with trials testing their effects on the physiology of protechnological microbes.

What is the microbial diversity behind the production of biogenic amines in foods and beverages? What is the food-related significance of this specific subset of micro-biodiversity? What is the influence of physical, chemical and biological determinants associated with food and beverages matrices that can, directly and indirectly, modulate the concentration of these low molecular weight organic bases? This chapter aims to summarize several aspects that appear to be crucial for a renewed management of the levers susceptible to minimize the content of biogenic amines in edible matrices, highlighting the present main trends and possible future perspectives in the field.

Malolactic fermentation (MLF) is a secondary fermentation in wine that usually takes place during or at the end of alcoholic fermentation. Lactobacillus plantarum is able to conduct MLF (particularly under high pH conditions and in co-inoculation with yeasts) and some strains are commercially used as MLF starter cultures. Recent evidences suggest a further use of selected L. plantarum strains for the pre-alcoholic acidification of grape must. In this study, we have carried out an integrated (molecular, technological, and biotechnological) characterization of L. plantarum strains isolated from Apulian wines in order to combine the two protechnological features (MLF performances and must acidification aptitudes). Several parameters such as sugar, pH and ethanol tolerance, resistance to lyophilisation and behaviour in grape must were evaluated. Moreover, the expression of stress gene markers was investigated and was linked to the ability of L. plantarum strains to grow and perform MLF. Co-inoculation of Saccharomyces cerevisiae and Lactobacillus plantarum in grape must improve the bacterial adaptation to harsh conditions of wine and reduced total fermentation time. For the first time, we applied a polyphasic approach for the characterization of L. plantarum in reason of the MLF performances. The proposed procedure can be generalized as a standard method for the selection of bacterial resources for the design of MLF starter cultures tailored for high pH must.

Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2 -like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. IMPORTANCE This study evaluates the role of extracellular polysaccharides that are produced by different strains of Lactobacillus plantarum in the determination of the cell surface properties of these bacteria and their capacity to interact with their environment, including their signaling to human host cells. The results clearly show that the consequences of removal of these polysaccharides are very strain specific, illustrating the diverse and unpredictable roles of these polysaccharides in the environmental interactions of these bacterial strains. In the context of the use of lactobacilli as health-promoting probiotic organisms, this study exemplifies the importance of strain specificity.

Probiotics are microorganisms that confer beneficial effects on the host; nevertheless, before being allowed for human consumption, their safety must be verified with accurate protocols. In the genomic era, such procedures should take into account the genomic-based approaches. This study aims at assessing the safety traits of Bacillus coagulans GBI-30, 6086 integrating the most updated genomics-based procedures and conventional phenotypic assays. Special attention was paid to putative virulence factors (VF), antibiotic resistance (AR) genes and genes encoding enzymes responsible for harmful metabolites (i.e. biogenic amines, BAs). This probiotic strain was phenotypically resistant to streptomycin and kanamycin, although the genome analysis suggested that the AR-related genes were not easily transferrable to other bacteria, and no other genes with potential safety risks, such as those related to VF or BA production, were retrieved. Furthermore, no unstable elements that could potentially lead to genomic rearrangements were detected. Moreover, a workflow is proposed to allow the proper taxonomic identification of a microbial strain and the accurate evaluation of risk-related gene traits, combining whole genome sequencing analysis with updated bioinformatics tools and standard phenotypic assays. The workflow presented can be generalized as a guideline for the safety investigation of novel probiotic strains to help stakeholders (from scientists to manufacturers and consumers) to meet regulatory requirements and avoid misleading information.

This work aims to improve the management of the malolactic fermentation (MLF) in red wines by elucidating the interactions between Starmerella bacillaris and Saccharomyces cerevisiae in mixed fermentations and malolactic bacteria. Two Starm. bacillaris strains were individually used in mixed fermentations with a commercial S. cerevisiae. MLF was performed using two autochthonous Lactobacillus plantarum and one commercial Oenococcus oeni inoculated following a simultaneous (together with S. cerevisiae) or sequential (at the end of alcoholic fermentation) approach. The impact of yeast inoculation on the progress of MLF was investigated by monitoring the viable microbial populations and the evolution of the main oenological parameters, as well as the volatile organic composition of the wines obtained in mixed and pure micro-scale winemaking trials. Our results indicated that MLF was stimulated, inhibited, or unaffected in mixed fermentations depending on the strains and on the regime of inoculation. O. oeni was able to perform MLF under all experimental conditions, and it showed a minimal impact on the volatile organic compounds of the wine. L. plantarum was unable to perform MLF in sequential inoculation assays, and strain-depending interactions with Starm. bacillaris were indicated as factor affecting the outcome of MLF. Moreover, uncompleted MLF were related to a lower aromatic complexity of the wines. Our evidences indicate that tailored studies are needed to define the appropriate management of non-Saccharomyces and malolactic starter cultures in order to optimize some technological parameters (i.e. reduction of vinification time) and to improve qualitative features (i.e. primary and secondary metabolites production) of red wines.

The management of the alcoholic fermentation (AF) in wine is crucial to shaping product quality. Numerous variables (e.g., grape varieties, yeast species/strains, technological parameters) can affect the performances of this fermentative bioprocess. The fact that these variables are often interdependent, with a high degree of interaction, leads to a huge ‘oenological space’ associated with AF that scientists and professionals have explored to obtain the desired quality standards in wine and to promote innovation. This challenge explains the high interest in approaches tested to monitor this bioprocess including those using volatile organic compounds (VOCs) as target molecules. Among direct injection mass spectrometry approaches, no study has proposed an untargeted online investigation of the diversity of volatiles associated with the wine headspace. This communication proposed the first application of proton-transfer reaction-mass spectrometry coupled to a time-of-flight mass analyzer (PTR-ToF-MS) to follow the progress of AF and evaluate the impact of the different variables of wine quality. As a case study, the assessment of VOC variability associated with different combinations of Saccharomyces/non-Saccharomyces was selected. The different combinations of microbial resources in wine are among the main factors susceptible to influencing the content of VOCs associated with the wine headspaces. In particular, this investigation explored the effect of multiple combinations of two Saccharomyces strains and two non-Saccharomyces strains (belonging to the species Metschnikowia pulcherrima and Torulaspora delbrueckii) on the content of VOCs in wine, inoculated both in commercial grape juice and fresh grape must. The results demonstrated the possible exploitation of non-invasive PTR-ToF-MS monitoring to explore, using VOCs as biomarkers, (i) the huge number of variables influencing AF in wine, and (ii) applications of single/mixed starter cultures in wine. Reported preliminary findings underlined the presence of different behaviors on grape juice and on must, respectively, and confirmed differences among the single yeast strains ‘volatomes’. It was one of the first studies to include the simultaneous inoculation on two non-Saccharomyces species together with a S. cerevisiae strain in terms of VOC contribution. Among the other outcomes, evidence suggests that the addition of M. pulcherrima to the coupled S. cerevisiae/T. delbrueckii can modify the global release of volatiles as a function of the characteristics of the fermented matrix.

Donkey milk is receiving increasing interest due to its attractive nutrient and functional properties (but also cosmetic), which make it a suitable food for sensitive consumers, such as infants with allergies, the immunocompromised, and elderly people. Our study aims to provide further information on the microbial variability of donkey milk under cold storage conditions. Therefore, we analysed by high-throughput sequencing the bacterial communities in unpasteurized donkey milk just milked, and after three days of conservation at 4 °C, respectively. Results showed that fresh donkey milk was characterized by a high incidence of spoilage Gram-negative bacteria mainly belonging to Pseudomonas spp. A composition lower than 5% of lactic acid bacteria was found in fresh milk samples, with Lactococcus spp. being the most abundant. The occurrence of microbial species belonging to risk group 2 was found in fresh milk. After three days of cold storage, the bacterial biodiversity of donkey milk was strongly reduced, since about 93% of the bacterial communities were identified as different species of psychrotrophic Pseudomonas. In conclusion, we report a preliminary description of the microbial diversity of donkey milk by using a metagenomic approach and encouraging a correct exploitation of this high-value niche product.

There is an error in the original publication of this paper. The incorrect author name was captured as "Djamel Dridier" instead of "Djamel Drider". The original article has been corrected.

For 15 years, non-Saccharomyces starter cultures represent a new interesting segment in the dynamic field of multinationals and national companies that develop and sell microbial-based biotechnological solutions for the wine sector. Although the diversity and the properties of non- Saccharomyces species/strains have been recently fully reviewed, less attention has been deserved to the commercial starter cultures in term of scientific findings, patents, and their innovative applications. : Considering the potential reservoir of biotechnological innovation, these issues represent an underestimated possible driver of coordination and harmonization of research and development activities in the field of wine microbiology. After a wide survey, we encompassed 26 different commercial yeasts starter cultures formulated in combination with at least one non-Saccharomyces strain. The most recent scientific advances have been explored delving into the oenological significance of these commercial starter cultures. Finally, we propose an examination of patent literature for the main yeasts species commercialised in non-Saccharomyces based products. : We highlight the presence of asymmetries among scientific findings and the number of patents concerning non-Saccharomyces-based commercial products for oenological purposes. Further investigations on these microbial resources might open new perspectives and stimulate attractive innovations in the field of wine-making biotechnologies.

Lactic acid bacteria (LAB), a heterogeneous group of bacteria that produce lactic acid as the main product of carbohydrate degradation, play an important role in the production and protection of fermented foods. Moreover, beside the technological use of these microorganisms added to control and steer food fermentations, their beneficial healthy properties are largely overt. Thus, numerous LAB strains have obtained the probiotic status, which entails the ability to maintain and promote a good health of consumers. In particular, increasing consideration is being focused on probiotic microorganisms that can improve the human immune response against dangerous viral and fungal enemies. For such beneficial microbes, the term "immunobiotics" has been coined. Together with an indirect host-mediated adverse effect against undesirable microorganisms, also a direct antagonistic activity of several LAB strains has been largely demonstrated. The purpose of this review is to provide a fullest possible overview of the antiviral and antifungal activities ascribed to probiotic LAB. The interest in this research field is substantiated by a large number of studies exploring the potential application of these beneficial microorganisms both as biopreservatives and immune-enhancers, aiming to reduce and/or eliminate the use of chemical agents to prevent the development of pathogenic, infectious, and/or degrading causes.

We report the first polyphasic characterization of native Saccharomyces cerevisiae in order to select candidate strains for the design of starter cultures tailored for Apulian sparkling wines obtained from local grape variety. In addition, it is the first survey in our region that propose the selection of autochthonous starter cultures for sparkling wine i) including a preliminary tailored genotypic and technological screening, and ii) monitoring analytical contribution during secondary fermentation in terms of volatile compounds (VOCs). Furthermore, we exploit the potential contribute of autochthonous cultures throughout the productive chain, including the possible improvement of base wine. One representative strain from each cluster was characterized i) for tolerance to abiotic and biotic stressors peculiar of sparkling wine fermentation, ii) for the performances in base wine production, and iii) for the aptitudes to promote in-bottle secondary fermentation in white and rose sparkling wines, both obtained from Apulian grape varieties. Genetic characterization led to group 164 S. cerevisiae in 16 genetic clusters based on interdelta profiles. Stress tolerance assays shown a certain correlation with fermentative attitude. Our evidences demonstrated a different fermentative behavior and release of VOCs of the different strains in association with primary and secondary fermentations and as function of wine and rose sparkling wine. Furthermore, performances in white/rose sparkling wines have been found to be strain-dependent characters. Overall, we propose different strains as biotechnological resources suitable to improve the quality of regional sparkling wines and to provide a driver of innovation/segmentation in the market.

Cereal-based functional beverages represent social, economic, and environmental sustainable opportunities to cope with emerging trends in food consumption and global nutrition. Here we report, for the first time, the polyphasic characterization of three cereal-based kefir-like riboflavin-enriched beverages, obtained from oat, maize and barley flours, and their comparison with classical milk-based kefir. The four matrices were successfully fermented with commercial starters: i) milk-kefir and ii) water-kefir, proving the potential of cereal ingredients in the formulation of dairy-like fermented beverages with milk-kefir starter behavior better in these matrices. In the light of their potentiality, seven riboflavin-producing Andean Lactic Acid Bacteria (LAB) were tested for tolerance to food stresses commonly encountered during food fermentation. Moreover, the LAB strains investigated were screened for spontaneous riboflavin overproducing derivatives. Lactobacillus plantarum M5MA1-B2 with outstanding response to stress, was selected to improve riboflavin content in an in situ fortification approach. The combination of L. plantarum M5MA1-B2 riboflavin overproducing strain with milk kefir starter in oat, lead to cover, for one serving of 100 g, 11.4% of Recommended Dietary Allowance (RDA). Besides, addition of L. plantarum M5MA1-B2 improved performance of water kefir in oat and maize matrices. Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) analysis provided the on-line Volatile Organic Compounds profiles supporting the best combination of starter, LAB and cereal matrix for novel functional foods development.

Minimally processed fruits are an ideal alternative to dairy products to deliver probiotic microorganisms. At the same time, several innovative employments of lactic acid bacteria (LAB) have been proposed in the food industry, including bio-fortification with nutritional compounds and bio-protection against foodborne pathogenic bacteria. In this study, probiotic riboflavin over-producing Lactobacillus plantarum B2 and Lactobacillus fermentum PBCC11.5 were inoculated on fresh-cut cantaloupe by immersion in a dipping solution. The viability of probiotic microorganisms and the main physico-chemical parameters of melon pieces, including the riboflavin content, were monitored for 11 days of storage under refrigerated conditions. Finally, both probiotics were tested for their antagonistic effect against different concentrations of an isolate of Listeria monocytogenes from fruit origin. Overall, high viability of both probiotics species was found at the end of the shelf life. The main technological and nutritional parameters of the fruits were unaffected by probiotic-enrichment, except some sensorial attributes when melons were inoculated with L. plantarum B2. The riboflavin content increased about two-fold in probiotic cantaloupe. Moreover, L. plantarum B2 and L. fermentum PBCC11.5 showed a good ability to reduce the level of L. monocytogenes on artificially contaminated melons. In conclusion, the results of this work encourage further implementation of new foods with multifunctional properties.

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

International audience; Aims To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in ‘Nero di Troia’ wine using a complete autochthonous microbial regime. Methods and Results Oenococcus oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety ‘Uva di Troia’ were isolated, selected and characterized. Multilocus sequence typing and variable number tandem repeat analysis were used to investigate intraspecific diversity. Oenococcus oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from ‘Nero di Troia’ wine. After a preliminary screening using co-inoculation regime, the O. oeni strains were grouped in reason of the different behaviour in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Conclusions Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. Significance and Impact of the Study The ‘microbial terroir’ of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work, we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety.

Analytical tools for the identification and quantification of volatile organic compounds (VOCs) produced by microbial cultures have countless applications in an industrial and research context which are still not fully exploited. The various techniques for VOC analysis generally arise from the application of different scientific and technological philosophies, favoring either sample throughput or chemical information. Proton transfer reaction-mass spectrometry (PTR-MS) represents a valid compromise between the two aforementioned approaches, providing rapid and direct measurements along with highly informative analytical output. The present paper reviews the main applications of PTR-MS in the microbiological field, comprising food, environmental, and medical applications.

The aim of this study was to investigate the prevalence and virulence factors of Staphylococcus aureus isolates from sheep and goat dairy products using end-point PCR. A total of 90 samples were analyzed and 37 (41.1%) resulted positive for S. aureus. S. aureus genes were found in 7 (18.9%) out of 37 isolates: sec was the most frequently recovered gene (42.8%), followed by seh (28.6%), sea (14.3%) and see (14.3%). The mecA gene was detected in one isolate (2.7%), and this strain was also positive for the presence of seh. PCR revealed 35 isolates (94.6%) as positive for the icaA gene. No S. aureus isolate carried lukF-PV and lukS-PV genes. The presence of these potentially harmful bacteria is a potential public health risk, therefore processing innovation in the ovine and goat dairy industry is required in order to reduce the microbiological contamination of these foods.

Among the innovative trends in the wine sector, the continuous exploration of enological properties associated with wine microbial resources represents a cornerstone driver of quality improvement. Since the advent of starter cultures technology, the attention has been focused on intraspecific biodiversity within the primary species responsible for alcoholic fermentation (Saccharomyces cerevisiae) and, subsequently, for the so-called ‘malolactic fermentation’ (Oenococcus oeni). However, in the last decade, a relevant number of studies proposed the enological exploitation of an increasing number of species (e.g., non-Saccharomyces yeasts) associated with spontaneous fermentation in wine. These new species/strains may provide technological solutions to specific problems and/or improve sensory characteristics, such as complexity, mouth-feel and flavors. This review offers an overview of the available information on the enological/protechnological significance of microbial resources associated with winemaking, summarizing the opportunities and the benefits associated with the enological exploitation of this microbial potential. We discuss proposed solutions to improve quality and safety of wines (e.g., alternative starter cultures, multistrains starter cultures) and future perspectives.

The winemaking process includes two traditional steps performed by microorganisms, alcoholic fermentation (AF) and malolactic fermentation (MLF). The AF is essential; sugars from grape must are converted into ethanol. This fermentation is carried out by yeast, mainly by Saccharomyces cerevisiae, however is not the only yeast involved in the process. A few specific non-Saccharomyces species, as Hanseniaspora uvarum have been proved to positively modify the wine chemical composition, especially contribute to the sensory characteristics of wines [1]. Interactions between different microbial populations present in grape must are interesting from the point of view of their impact on the final quality of the wine. The main interaction occurs among Saccharomyces spp. yeasts, non-Saccharomyces yeasts, and lactic acid bacteria (LAB) [2]. In the light of the rising request for autochthonous starters tailored for given 'terroir'[3], the objective of this work was to select the best inoculation time of autochthonous of Oenococcus oeni strains in combination with two S. cerevisiae strains and one H. uvarum strain isolated from Apulian wines. Direct interactions were also investigated monitoring cell viability on specific solid culture media. Microvinifications were performed using must from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and H. uvarum, were co-inoculated and O. oeni strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. This research was supported by the Apulian Region Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA."

In the last years, the interactions among different microbial populations have been studied by the scientific community in the aim of understanding their impact on the quality of wines. The main interactions during the vinification process occur among the yeast Saccharomyces cerevisiae, non-Saccharomyces yeasts species and lactic acid bacteria (LAB). A special interest has been dedicated to investigate the role non-Saccharomyces yeasts as they are responsible for much of the aromatic complexity and its presence improves the final organoleptic characteristics of the wine. Concerning LABS, selected strains belonging to Lactobacillus plantarum species have been recently investigated to produce starter cultures for malolactic fermentation in wine. The aim of the present study is to design mixed starter culture to simultaneously carry out Alcoholic fermentation (AF) and malolactic fermentation (MLF), thus obtaining wine denoted by complex organoleptic properties in a shorter period of time. We report the evaluation of the best inoculation time of L. plantarum strains in combination with autochthonous S. cerevisiae strains and non-Saccharomyces (Hanseniaspora sp.). Microvinifications were performed using must derived from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and non-Saccharomyces strains were simultaneously or sequentially co-inoculated during the AF with the L. plantarum strains. The sequential co-inoculation was carried out at different point, when the concentration of the produced ethanol was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. The obtained results demonstrated that L. plantarum cell viability after AF was strain-dependent. L. plantarum strains showed the highest viability when they were co-inoculated with S. cerevisiae and H. uvarum strains. This research was supported by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

Sparkling wines contain high amount of CO2 and are the result of re-fermentation of a still wine, usually called base wine. Several ingredients, such as sucrose, selected yeasts, bentonite and some nutrients, are added to base wine in order to induce the re-fermentation. Then wines can be bottled, fermented and aged for a long period (about 9-12 months). These wines represents an important percentage of the high-quality wine market and have an important economic impact due to its high added value [1-2], justifying the increasing attention to product improvements. Modern biotechnologies can be used to improve quality of sparkling wines, and also to reduce their production time and cost. Starter cultures selected for sparkling wine production must satisfy several characteristics, in particular considering that re-fermentation is carried out in hostile environment due to several factors, such elevated ethanol content, low pH and increasing carbon dioxide pressure [3]. In addition starter cultures should have other important characteristics, such as autolysis (important for aging in close contact with lees) and flocculation (another important selection criterion that aim lees removal) [4]. Authochtonous starter cultures have an important rule on wine quality, because autochthonous yeasts i) might be well adapted to specific enviromental conditions and ii) might allow to obtain wines with typical organoleptic properties. In these context this study investigated the use of select autochthonous resources for sparkling wine production. We genetically characterized about 200 Saccharomyces cerevisiae autochthonous strains isolated from "Nero di Troia" spontaneous fermentation. After the evaluation of intraspecific diversity using interdelta analyis, we selected one represtative strain for each of the 15 genetic cluster. These strains were technological characterized at lab scale and, successively, tested in winery for both induce alcoholic fermentation in base wine and re-fermentation of sparkling wine. This research was supported by the Apulian Region Project "Innovazioni di processo e di prodotto nel comparto dei vini spumanti da vitigni autoctoni pugliesi" (IProViSP).

Biocontrol strategies for the limitation of undesired microbial developments in foods and beverages represent a keystone toward the goal of more sustainable food systems. Brettanomyces bruxellensis is a wine spoilage microorganism that produces several compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols. To control the proliferation of this yeast, sulfur dioxide is commonly employed, but the efficiency of this compound depends on the B. bruxellensis strain; and it is subject to wine composition and may induce the entrance in a viable, but nonculturable state of yeasts. Moreover, it can also elicit allergic reactions in humans. In recent years, biological alternatives to sulfur dioxide such as the use of yeasts and lactic acid bacteria starter cultures as biocontrol agents are being investigated. The controlled inoculation of starter cultures allows secure, fast and complete alcoholic and malolactic fermentations, limiting the residual nutrients that B. bruxellensis utilizes to survive and grow in wine. The current study is focused on the assessment of the effect of autochthonous yeasts and bacterial strains from the Apulia Region on the development of B. bruxellensis in wine, in terms of both growth and volatile phenols' production. The investigation evidences the positive role of indigenous mixed cultures in the control of this spoilage yeast, either co-inoculating different strains of Saccharomyces cerevisiae, S. cerevisiae/non-Saccharomyces or co-inoculating S. cerevisiae/Oenococcus oeni. Our findings expand the existing knowledge of the application of protechnological microbial diversity and of non-Saccharomyces as a biocontrol agent in oenology. We report a further demonstration of the interest in selecting indigenous strains as a strategic tool for winemakers interested in the improvement of regional wines.

International audience; The number of studies claiming probiotic health effects of Lactobacillus plantarum is escalating. Lb. plantarum is a lactic acid bacterium found in diverse ecological niches, highlighting its particular capabilities of adaptation and genome plasticity. Another function that needs to be underlined is the capabilities of Lb. plantarum to produce diverse and potent bacteriocins, which are antimicrobial peptides with possible applications as food preservative or antibiotic complementary agents. Taken together, all these characteristics design Lb. plantarum as a genuine model for academic research and viable biological agent with promising applications. The present review aims at shedding light on the safety of Lb. plantarum and run through the main studies underpinning its beneficial claims. The mechanisms explaining probiotic- related features are discussed.

In order to evaluate if the reuse of food industry treated wastewater is compatible for irrigation of food crops, without increased health risk, in the present study a cropping system, in which ground water and treated wastewater were used for irrigation of tomato and broccoli, during consecutive crop seasons was monitored. Water, crop environment and final products were monitored for microbial indicators and pathogenic bacteria, by conventional and molecular methods. The microbial quality of the irrigation waters influenced sporadically the presence of microbial indicators in soil. No water sample was found positive for pathogenic bacteria, independently from the source. Salmonella spp. and Listeria monocytogenes were detected in soil samples, independently from the irrigation water source. No pathogen was found to contaminate tomato plants, while Listeria monocytogenes and E. coli O157:H7 were detected on broccoli plant, but when final produce were harvested, no pathogen was detected on edible part. The level of microbial indicators and detection of pathogenic bacteria in field and plant was not dependent upon wastewater used. Our results, suggest that reuse of food industry wastewater for irrigation of agricultural crop can be applied without significant increase of potential health risk related to microbial quality.

The interactions among different microbial populations have interested the wine microbiology community with the final aim of understanding the impact on wines' quality. The main interaction occurs between yeast Saccharomyces cerevisiae, yeast non-Saccharomyces spp. and lactic acid bacteria (LAB) [1]. In last years, there is a special interest in the study of non-Saccharomyces yeasts as they are responsible for much of the aromatic complexity and its presence improves the final organoleptic characteristics of the wine [2]. Interactions between yeasts and LAB have been studied always combining S. cerevisiae with some LAB and in almost all cases Oenococcus oeni, but poor evidences have been reported for interactions between non-Saccharomyces and LAB in the wine environment. Lactobacillus plantarum selected strains have been receiving increasing attention as starter cultures for malolactic fermentation in wine. With the aim to design of mixed culture to perform AF and MLF simultaneously in a short period of time, with complex organoleptic properties, and also to control the indigenous microbiota and to protect wine against spoilage microorganisms, in this work we report the evaluation of the best inoculation time of L. plantarum strains (both commercial and autochthonous Apulian strains) in combination with autochthonous S. cerevisiae strains and non-Saccharomyces (Hanseniaspora sp. and Candida sp.). Interactions were also investigated monitoring cell viability on specific solid culture media. Microvinifications were performed using must from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and non-Saccharomyces, were co-inoculated and L. plantarum strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. This research was supported by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

The aromatic impact of bakery yeast starters is currently receiving considerable attention. The flavor characteristics of thedough and the finished products are usually evaluated by gas chromatography and sensory analysis. The limit of both tech-niques resides in their low-throughput character. In the present work, proton-transfer-reaction mass spectrometry (PTR-MS),coupled to a time-of-flight mass analyzer, was employed, for the first time, to measure the volatile fractions of dough andbread, and to monitor Saccharomyces cerevisiae volatile production in a fermented food matrix. Leavening was performed onsmall-scale (1g) dough samples inoculated with different commercial yeast strains. The leavened doughs were then baked,and volatile profiles were determined during leavening and after baking. The experimental setup included a multifunctionalautosampler, which permitted the follow-up of the leavening process on a small scale with a typical throughput of 500 distinctdata points in 16h. The system allowed to pinpoint differences between starter yeast strains in terms of volatile emissionkinetics, with repercussions on the final product (i.e. the corresponding micro-loaves). This work demonstrates the applicabilityof PTR-MS for the study of volatile organic compound production during bread-making, for the automated and online real-timemonitoring of the leavening process, and for the characterization and selection of bakery yeast starters in view of theirproduction of volatile compounds. Copyright © 2014 John Wiley & Sons, Ltd.Keywords: bakery; PTR-MS; leavening; yeast; VOCs

Pseudoalteromonas haloplanktis, a psychrotrophilic marine bacterium of biotechnological interest, shows anti-biofilm properties and is particularly relevant for cold storage of vacuum packed seafood. We focused our interest on the activation of cholesterol metabolism in this bacterium as the presence in its genome of a putative 3-ketosteroid-Δ(1) -dehydrogenase. This study reports GC-MS and LC-MS/MS profiles of sterols/steroids and their derivatives found in cell extracts of P. haloplanktis grown in a medium with a low content of cholesterol. Here, for the first time, we suggest that P. haloplanktis produces some intermediates of cholesterol catabolism, putatively identified as 24-hydroxycholest-1,4-dien-3-one-26-oic acid, chol-1,4-dien-3-one-24-oic acid, 26-hydroxycholest-4-en-3-one, and pregn-4-en-3-one-20-carboxylic acid, a finding already reported in other microorganisms. The presence of these compounds, also considered steroid precursors, produced by P. haloplanktis in vacuum packed seafood could be of interest for healthy of consumers, as well as, for biotechnological applications in pharmaceutical industry.

Currently, the majority of prebiotics in the market are derived from non-digestible oligosaccharides. Very few studies have focused on non-digestible long chain complex polysaccharides in relation to their potential as novel prebiotics. Cereals β-glucans have been investigated for immune-modulating properties and beneficial effects on obesity, cardiovascular diseases, diabetes, and cholesterol levels. Moreover, β-glucans have been reported to be highly fermentable by the intestinal microbiota in the caecum and colon, and can enhance both growth rate and lactic acid production of microbes isolated from the human intestine. In this work, we report the effects of food matrices containing barley β-glucans on growth and probiotic features of four Lactobacillus strains. Such matrices were able to improve the growth rate of the tested bacteria both in unstressed conditions and, importantly, after exposure to in vitro simulation of the digestive tract. Moreover, the effect of β-glucans-containing food on bacterial adhesion to enterocyte-like cells was analyzed and a positive influence on probiotic-enterocyte interaction was observed.

In this work we evaluated the effect of a perturbation on nitrification performance in a wastewater treatment plant (WWTP) treating urban and saline thermal bath wastewater, which regularly occurred during summer months. We wanted to find out if this related to changes in the ammonia oxidizing communities. The bacterial and ammonia-oxidizing bacterial (AOB) community from three different basins of the WWTP were evaluated using PCR-DGGE and cloning and sequencing of 16S rRNA gene fragments, over a six month survey. Both eubacterial and AOB communities underwent continuous change over time, with a particularly prominent shift between the third and fourth month of monitoring for eubacteria and the fourth month and fifth month for AOB. At the same time, reduction of nitrification performance was observed in the WWTP. The AOB community in the activated sludge was dominated by clones with high 16S rRNA sequence identity to halophilic-halotolerant organisms from the Nitrosomonas oligotrohpa and Nitrosomonas marina clusters. A significant correlation (R = 0.97) was detected between the structure of the AOB community and environmental parameters, indicating that the AOB community structure changed in line with the environmental changes that took place over the sampling period. This study reports a clear association of microbial community dynamics (strongly correlated to salinity, temperature, and dissolved oxygen) to nitrification performance. Particularly, ammonia-oxidizing bacteria are severely affected by drastic changes in operational conditions, with direct consequences on WWTP performance.