Lactobacilli Cell-Free Supernatants Modulate Inflammation and Oxidative Stress in Human Microglia via NRF2-SOD1 Signaling.

Microglia are macrophage cells residing in the brain, where they exert a key role in neuronal protection. Through the gut-brain axis, metabolites produced by gut commensal microbes can influence brain functions, including microglial activity. The nuclear factor erythroid 2-related factor 2 (NRF2) is a key regulator of the oxidative stress response in microglia, controlling the expression of cytoprotective genes. Lactobacilli-derived cell-free supernatants (CFSs) are postbiotics that have shown antioxidant and immunomodulatory effects in several in vitro and in vivo studies. This study aimed to explore the effects of lactobacilli CFSs on modulating microglial responses against oxidative stress and inflammation. HMC3 microglia were exposed to lipopolysaccaride (LPS), as an inflammatory trigger, before and after administration of CFSs from three human gut probiotic species. The NRF2 nuclear protein activation and the expression of NRF2-controlled antioxidant genes were investigated by immunoassay and quantitative RT-PCR, respectively. Furthermore, the level of pro- and anti-inflammatory cytokines was evaluated by immunoassay. All CFSs induced a significant increase of NRF2 nuclear activity in basal conditions and upon inflammation. The transcription of antioxidant genes, namely heme oxygenase 1, superoxide dismutase (SOD), glutathione-S transferase, glutathione peroxidase, and catalase also increased, especially after inflammatory stimulus. Besides, higher SOD1 activity was detected relative to inflamed microglia. In addition, CFSs pre-treatment of microglia attenuated pro-inflammatory TNF-α levels while increasing anti-inflammatory IL-10 levels. These findings confirmed that gut microorganisms' metabolites can play a relevant role in adjuvating the microglia cellular response against neuroinflammation and oxidative stress, which are known to cause neurodegenerative diseases.

Potential of Non-Saccharomyces Yeasts as Probiotics and Alternatives to Antibiotics in Animal Production.

Probiotics are live yeast or bacterial organisms that have beneficial effects on the host. Several microorganisms exhibit probiotic properties, the most common types being lactic acid bacteria, Bifidobacteria, spore-forming bacteria, and some yeast strains. Saccharomyces cerevisiae var. boulardii is the most important probiotic yeast species. However, another group of foodborne microorganisms, the so-called non-Saccharomyces yeasts (NSYs), has recently been re-evaluated and shown to have enormous potential in various fields of application, ranging from food fermentation to human and animal applications. NSYs are able to produce a range of bioactive compounds such as antimicrobials, mannoproteins, enzymes, polyunsaturated fatty acids, essential amino acids, vitamins, and ß-glucans, which increases their potential applications as a new class of probiotics and/or alternatives to antibiotics in animal husbandry. In this review, we aim to highlight the potential and benefits of NSYs as probiotics and natural antimicrobials to improve animal health. Furthermore, the use of NSYs as biological alternatives to antibiotics to control foodborne pathogens in animal production is discussed.

Psychobiotic Properties of Lactiplantibacillus plantarum in Neurodegenerative Diseases.

Neurodegenerative disorders are the main cause of cognitive and physical disabilities, affect millions of people worldwide, and their incidence is on the rise. Emerging evidence pinpoints a disturbance of the communication of the gut-brain axis, and in particular to gut microbial dysbiosis, as one of the contributors to the pathogenesis of these diseases. In fact, dysbiosis has been associated with neuro-inflammatory processes, hyperactivation of the neuronal immune system, impaired cognitive functions, aging, depression, sleeping disorders, and anxiety. With the rapid advance in metagenomics, metabolomics, and big data analysis, together with a multidisciplinary approach, a new horizon has just emerged in the fields of translational neurodegenerative disease. In fact, recent studies focusing on taxonomic profiling and leaky gut in the pathogenesis of neurodegenerative disorders are not only shedding light on an overlooked field but are also creating opportunities for biomarker discovery and development of new therapeutic and adjuvant strategies to treat these disorders. Lactiplantibacillus plantarum (LBP) strains are emerging as promising psychobiotics for the treatment of these diseases. In fact, LBP strains are able to promote eubiosis, increase the enrichment of bacteria producing beneficial metabolites such as short-chain fatty acids, boost the production of neurotransmitters, and support the homeostasis of the gut-brain axis. In this review, we summarize the current knowledge on the role of the gut microbiota in the pathogenesis of neurodegenerative disorders with a particular focus on the benefits of LBP strains in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autism, anxiety, and depression.

Knock out of sHSP genes determines some modifications in the probiotic attitude of Lactiplantibacillus plantarum.

OBJECTIVE: We investigated whether the knock out of small heat shock protein (sHSP) genes (hsp1, hsp2 and hsp3) impact on probiotic features of Lactiplantibacillus plantarum WCFS1, aiming to find specific microbial effectors involved in microbe-host interplay. RESULTS: The probiotic properties of L. plantarum WCFS1 wild type, hsp1, hsp2 and hsp3 mutant clones were evaluated and compared through in vitro trials. Oro-gastro-intestinal assays pointed to significantly lower survival for hsp1 and hsp2 mutants under stomach-like conditions, and for hsp3 mutant under intestinal stress. Adhesion to human enterocyte-like cells was similar for all clones, though the hsp2 mutant exhibited higher adhesiveness. L. plantarum cells attenuated the transcriptional induction of pro-inflammatory cytokines on lipopolysaccharide-treated human macrophages, with some exception for the hsp1 mutant. Intriguingly, this clone also induced a higher IL10/IL12 ratio, which is assumed to indicate the anti-inflammatory potential of probiotics. CONCLUSIONS: sHSP genes deletion determined some differences in gut stress resistance, cellular adhesion and immuno-modulation, also implying effects on in vivo interaction with the host. HSP1 might contribute to immunomodulatory mechanisms, though additional experiments are necessary to test this feature.

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action.

Lactiplantibacillus plantarum (L. plantarum) is a well-studied and versatile species of lactobacilli. It is found in several niches, including human mucosal surfaces, and it is largely employed in the food industry and boasts a millenary tradition of safe use, sharing a long-lasting relationship with humans. L. plantarum is generally recognised as safe and exhibits a strong probiotic character, so that several strains are commercialised as health-promoting supplements and functional food products. For these reasons, L. plantarum represents a valuable model to gain insight into the nature and mechanisms of antimicrobials as key factors underlying the probiotic action of health-promoting microbes. Probiotic antimicrobials can inhibit the growth of pathogens in the gut ensuring the intestinal homeostasis and contributing to the host health. Furthermore, they may be attractive alternatives to conventional antibiotics, holding potential in several biomedical applications. The aim of this review is to investigate the most relevant papers published in the last ten years, bioprospecting the antimicrobial activity of characterised probiotic L. plantarum strains. Specifically, it focuses on the different chemical nature, the action spectra and the mechanisms underlying the bioactivity of their antibacterial and antiviral agents. Emerging trends in postbiotics, some in vivo applications of L. plantarum antimicrobials, including strengths and limitations of their therapeutic potential, are addressed and discussed.

Lactobacillus fermentum: a bacterial species with potential for food preservation and biomedical applications.

Lactic acid-producing bacteria are the most commonly used probiotics that play an important role in protecting the host against harmful microorganisms, strengthening the host immune system, improving feed digestibility, and reducing metabolic disorders. Lactobacillus fermentum (Lb. fermentum) is a Gram-positive bacterium belonging to Lactobacillus genus, and many reportedly to enhance the immunologic response as well as prevent community-acquired gastrointestinal and upper respiratory infections. Additionally, Lb. fermentum strains produce diverse and potent antimicrobial peptides, which can be applied as food preservative agents or as alternatives to antibiotics. Further functions attributed to probiotic Lb. fermentum strains are their abilities to decrease the level of blood stream cholesterol (as cholesterol-lowering agents) and to potentially help prevent alcoholic liver disease and colorectal cancer among humans. Finally, Lb. fermentum is a key microorganism in sourdough technology, contributing to flavor, texture, or health-promoting dough ingredients, and has recently been used to develop new foods stuffs such as fortified and functional foods with beneficial attributes for human health. Development of such new foodstuffs are currently taking important proportions of the food industry market. Furthermore, an increasing awareness of the consumers prompts the food-makers to implement alternative environmental friendly solutions in the production processes and/or suitable biological alternative to limit the use of antibiotics in feed and food. Here, we give an account on the application of Lb. fermentum strains in the biomedical and food preservation fields, with a focus on probiotic features such as bacteriocin production. We also summarize the use of Lb. fermentum as cell factories with the aim to improve the efficacy and health value of functional food.

How probiotics face food stress: They get by with a little help.

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.

Riboflavin-overproducing lactobacilli for the enrichment of fermented soymilk: insights into improved nutritional and functional attributes.

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.

In situ riboflavin fortification of different kefir-like cereal-based beverages using selected Andean LAB strains.

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.

A Metagenomic-Based Approach for the Characterization of Bacterial Diversity Associated with Spontaneous Malolactic Fermentations in Wine.

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.

Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties.

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.

Correction to: Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties.

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.

Starter cultures as biocontrol strategy to prevent Brettanomyces bruxellensis proliferation in wine.

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.

The potential of lactic acid bacteria to colonize biotic and abiotic surfaces and the investigation of their interactions and mechanisms.

Lactic acid bacteria (LAB) are a heterogeneous group of Gram-positive bacteria that comprise several species which have evolved in close association with humans (food and lifestyle). While their use to ferment food dates back to very ancient times, in the last decades, LAB have attracted much attention for their documented beneficial properties and for potential biomedical applications. Some LAB are commensal that colonize, stably or transiently, host mucosal surfaces, inlcuding the gut, where they may contribute to host health. In this review, we present and discuss the main factors enabling LAB adaptation to such lifestyle, including the gene reprogramming accompanying gut colonization, the specific bacterial components involved in adhesion and interaction with host, and how the gut niche has shaped the genome of intestine-adapted species. Moreover, the capacity of LAB to colonize abiotic surfaces by forming structured communities, i.e., biofilms, is briefly discussed, taking into account the main bacterial and environmental factors involved, particularly in relation to food-related environments. The vast spread of LAB surface-associated communities and the ability to control their occurrence hold great potentials for human health and food safety biotechnologies.

In Situ ß-Glucan Fortification of Cereal-Based Matrices by Pediococcus parvulus 2.6: Technological Aspects and Prebiotic Potential.

Bacterial exopolysaccharides produced by lactic acid bacteria are of increasing interest in the food industry, since they might enhance the technological and functional properties of some edible matrices. In this work, Pediococcus parvulus 2.6, which produces an O2-substituted (1,3)-ß-d-glucan exopolysaccharide only synthesised by bacteria, was proposed as a starter culture for the production of three cereal-based fermented foods. The obtained fermented matrices were naturally bio-fortified in microbial ß-glucans, and used to investigate the prebiotic potential of the bacterial exopolysaccharide by analysing the impact on the survival of a probiotic Lactobacillus plantarum strain under starvation and gastrointestinal simulated conditions. All of the assays were performed by using as control of the P. parvulus 2.6's performance, the isogenic ß-glucan non-producing 2.6NR strain. Our results showed a differential capability of P. parvulus to ferment the cereal flours. During the fermentation step, the ß-glucans produced were specifically quantified and their concentration correlated with an increased viscosity of the products. The survival of the model probiotic L. plantarum WCFS1 was improved by the presence of the bacterial ß-glucans in oat and rice fermented foods under starvation conditions. The probiotic bacteria showed a significantly higher viability when submitted to a simulated intestinal stress in the oat matrix fermented by the 2.6 strain. Therefore, the cereal flours were a suitable substrate for in situ bio-fortification with the bacterial ß-glucan, and these matrices could be used as carriers to enhance the beneficial properties of probiotic bacteria.

Strain-Specific Features of Extracellular Polysaccharides and Their Impact on Lactobacillus plantarum-Host Interactions.

UNLABELLED: 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.

Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms.

A wide range of lactic acid bacteria (LAB) is able to produce capsular or extracellular polysaccharides, with various chemical compositions and properties. Polysaccharides produced by LAB alter the rheological properties of the matrix in which they are dispersed, leading to typically viscous and "ropy" products. Polysaccharides are involved in several mechanisms such as prebiosis and probiosis, tolerance to stress associated to food process, and technological properties of food. In this paper, we summarize the beneficial properties of exopolysaccharides (EPS) produced by LAB with particular attention to prebiotic properties and to the effect of exopolysaccharides on the LAB-host interaction mechanisms, such as bacterial tolerance to gastrointestinal tract conditions, ability of ESP-producing probiotics to adhere to intestinal epithelium, their immune-modulatory activity, and their role in biofilm formation. The pro-technological aspect of exopolysaccharides is discussed, focusing on advantageous applications of EPS in the food industry, i.e., yogurt and gluten-free bakery products, since it was found that these microbial biopolymers positively affect the texture of foods. Finally, the involvement of EPS in tolerance to stress conditions that are commonly encountered in fermented beverages such as wine is discussed.

Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study.

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.

Technological properties of Lactobacillus plantarum strains isolated from grape must fermentation.

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 L. plantarum in grape must improves 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.

Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach.

The spoilage potential of Brettanomyces bruxellensis in wine is strongly connected with the aptitude of this yeast to enter in a Viable But Non Culturable (VBNC) state when exposed to the harsh wine conditions. In this work, we characterized the VBNC behaviour of seven strains of B. bruxellensis representing a regional intraspecific biodiversity, reporting conclusive evidence for the assessment of VBNC as a strain-dependent character. The VBNC behaviour was monitored by fluorescein diacetate staining/flow cytometry for eleven days after addition of 0.4, 0.6, 0.8, 1 and 1.2 mg/L of molecular SO2 (entrance in the VBNC state) and after SO2 removal (exit from the VBNC state). Furthermore, one representative strain was selected and RNA-seq analysis performed after exposure to 1.2 mg/L SO2 and during the recovery phase. 30 and 1634 genes were identified as differentially expressed following VBNC entrance and 'resuscitation', respectively. The results reported strongly suggested that the entrance in the SO2-induced VBNC state in B. bruxellensis is associated with both, sulfite toxicity and oxidative stress response, confirming the crucial role of genes/proteins involved in redox cell homeostasis. Among the genes induced during recovery, the expression of genes involved in carbohydrate metabolism and encoding heat shock proteins, as well as enriched categories including amino acid transport and transporter activity was observed. The evidences of a general repression of genes involved in DNA replication suggest the occurrence of a true resuscitation of cell rather than a simple regrowth.