The microbiota-gut-brain axis (MGBA) is a complex bidirectional communication system that plays a critical role in neurodevelopment, particularly during pediatric development, when the gut microbiota and the central nervous system undergo rapid and coordinated maturation. Dysbiosis due to external or physiological conditions may predispose to long-term neurological consequences. While microbiota-targeted interventions are gaining interest, the mechanistic role of specific microbial metabolites is often overlooked. This review highlights the role of riboflavin (vitamin B2) within the framework of pediatric MGBA. As an essential cofactor in mitochondrial energy metabolism, redox homeostasis, and neurotransmitter synthesis, riboflavin represents a vital intersection between nutrition and host-microbe interactions. Dietary intake remains the primary determinant of riboflavin status. However, changes in dietary habits and physiological conditions may lead to deficiencies influencing local availability and host-microbe interactions. Furthermore, riboflavin-overproducing probiotics are discussed as an emerging, function-driven strategy to modulate the MGBA. By enabling in situ vitamin production, these strains offer a targeted approach to enhance local bioavailability and promote beneficial microbial cross-feeding. Finally, we address current limitations in the field, emphasizing the lack of specific biomarkers to quantify host-accessible microbial riboflavin and the critical need for standardized pediatric clinical trials to translate experimental findings into evidence-based therapies.

Postbiotics, composed of inanimate microorganisms or their components, are gaining attention for managing intestinal discomfort, being safer than probiotics for vulnerable patients. This study investigates the effects of short-term consumption of a heat-stabilized VSL#3 formulation (VSL#3-HS) in a mouse model of dextran sulphate sodium (DSS)-induced colitis. Male C57BL/6 mice were divided into four groups: (1) Placebo (PLA); (2) VSL#3-HS (1 × 109 cells/mouse/day for 7 days); (3) DSS + PLA (2% DSS in drinking water from days 1-7, PLA from days 4-10); (4) DSS + VSL#3-HS (2% DSS from days 1-7, VSL#3-HS from days 4-10). All mice were euthanized on day 11 for tissue collection. VSL#3-HS rapidly alleviated clinical signs of the disease, including improvements in disease activity index and colon length. This effect was accompanied by a reduction in colonic pro-inflammatory mediators (IL-6, TNF-α, IL-1β) and a partial restoration of occludin gene expression in the intestine. However, no improvement was observed in MPO activity or microscopic tissue damage. The experimental findings demonstrate the efficacy of postbiotics in a murine model of UC and support further investigation into the efficacy of the heat-stabilized VSL#3-HS formulation to assess its potential applicability in human studies.

The increasing use of probiotics in dietary supplements and functional foods highlights the need for robust analytical strategies to assess microbial viability, functional performance, and safety. Conventional culture-based methods may underestimate probiotic potential, particularly in multi-strain formulations. In this study, a multi-analytical framework was applied to evaluate four commercial formulations containing Lactobacillus acidophilus LA-5®, differing in composition and delivery technology. A combination of culture-dependent and culture-independent approaches was employed, including plate counting, flow cytometry (FC), whole-genome sequencing (WGS), digital droplet PCR (ddPCR), antimicrobial activity assays, in vitro gastrointestinal digestion, and metabolomic profiling. WGS confirmed the taxonomic identity of all isolates as L. acidophilus and the absence of genes of concern. FC revealed that viable cells accounted for 59–88

IntroductionSpontaneous wine fermentation is driven by the ecological succession of vineyard-derived microorganisms, yet little is known about how this process unfolds in Picolit, a grape variety characterized by acinellatura (berry millerandage) and elevated sugar concentration. This study aimed to characterize the microbial and metabolic dynamics of spontaneous Picolit fermentation and to identify the ecological and functional transitions occurring throughout the process.MethodsAn integrated multi-omic approach combining shotgun metagenomics and untargeted metabolomics was applied to spontaneous fermentations of Picolit grapes produced at Aquila del Torre, an organic and biodynamic winery located in Savorgnano del Torre (Friuli-Venezia Giulia, Italy), within the newly established “Friuli Colli Orientali Sottozona Savorgnano D.O.C.” Five fermentation stages were sampled and analyzed to investigate microbial succession, functional pathways and metabolomic changes.ResultsThe initial must displayed high microbial richness dominated by non-Saccharomyces yeasts, oxidative bacteria and Botrytis cinerea. An atypical persistence and increasing abundance of B. cinerea suggested a strong interaction between grape physiology and fungal activity. Early fermentation stages were characterized by diverse non-Saccharomyces taxa, including Lachancea, Pichia, Torulaspora and Schizosaccharomyces, which were associated with acid modulation, aromatic precursor release and phenolic turnover. From mid-fermentation onward, a multi-species Saccharomyces consortium established functional dominance, coinciding with a marked reduction in bacterial diversity and a transition from aroma-related metabolic pathways to stress adaptation functions. Multi-omic network analyses revealed a progressive loss of modularity as fermentation progressed and the system became more stable.DiscussionThese findings demonstrate that spontaneous Picolit fermentation follows a distinctive ecological trajectory shaped by grape physiology, terroir and native microbial diversity. The persistence of B. cinerea, together with the succession of non-Saccharomyces and Saccharomyces populations, highlights unique microbial interactions that may contribute to wine identity. Overall, the results support the enological value of spontaneous fermentation and provide a microbial and functional framework for understanding and valorizing wines produced under the Savorgnano Bianco D.O.C.

Proper maintenance of the cold chain is essential to ensure both food quality and safety. This laboratory activity was designed to help undergraduate students understand the microbiological implications of cold chain failure through direct microbiological analysis and data interpretation on commercial foodstuffs. We designed an inquiry-based laboratory activity for final-year undergraduate students at the University of Milan (Milan, Italy) to explore the effects of thermal abuse on microbial populations in turkey hamburgers and ready-to-eat salads. Students analyzed key microbial groups, including aerobic mesophilic bacteria, lactic acid bacteria, yeasts, fungi, Pseudomonas spp., Escherichia coli, Enterococcus spp., staphylococci, and presumptive Bacillus cereus, under controlled and temperature-abused storage conditions. They classified samples according to microbial load and compared their results to food microbiology guidelines to assess their quality. Survey feedback revealed student engagement, with over 85% recognizing the activity's relevance to their future careers and its impact on critical thinking. This inquiry-based laboratory promotes food quality learning by integrating microbiological techniques with real-world challenges, strengthening students' appreciation for the role of food scientists and technologists in preventing risks to public health.

Akkermansia muciniphila MucT was authorized by EFSA in 2021 to be commercialized as novel food in its pasteurized form, corresponding to what it is commonly referred to as a postbiotic. As for probiotics, which are commercialized as living cells, postbiotics also require their correct quantification during the production process. Here we describe the optimization of a flow cytometry-based protocol for the absolute counting of pasteurized A. muciniphila cells in high cell density and standardized industrial batches. Protocol optimization required a sample preparation set-up and the choice of the appropriate diluent for the efficient rehydration of freeze-dried cells to limit cell aggregates. The developed quantification protocol allowed a more efficient cell counting with a lower standard deviation and coefficient of variation (CV) as compared with microscope-based quantification. The optimized protocol was successfully applied in a mass balance calculation over the production process, a standard validation in every industrial process for microbial biomass production that starts from high cell density batches to achieve standardized batches with the target cell dosage. Finally, the inter-laboratory trial, carried out among six independent laboratories using a ring test design, revealed good accuracy and precision of the optimized quantification protocol with an average CV ranging between 12.3 and 24.1 % and a Z score max of 2.64. In conclusion, our study revealed that developed flow cytometry protocol is a fast, reliable, reproducible and accurate method for the enumeration of the pasteurized A. muciniphila MucT that could be applied with minor modifications to other postbiotics.

The two probiotic bacteria Lactobacillus helveticus MIMLh5 and L. acidophilus NCFM exhibit homology, are both equipped with an S-layer made up of highly homologous proteins and are capable of stimulating Th1-inducing signals in dendritic cells. In this study, we aimed to compare the two strains as regards the thickness of the S-layer and their capacity to induce the production of the two Th1-inducing cytokines IL-12 and IFN-β. For both bacteria, stimulation with an increasing number of bacteria led to the higher and prompter production of IL-12 and IFN-β, but at all MOIs tested, the IL-12 response induced by NCFM was always the strongest. For both bacteria, the induction of IL-12 peaked at a multiplicity of infection (MOI) of 2–5, while IL-10, known to inhibit the induction of IL-12 cytokines, was induced more slowly and continued to increase at a higher MOI. By employing specific inhibitors, MIMLh5 and NCFM were also shown to activate different MAP kinase pathways. Endocytosed MIMLh5 showed higher survival in the DCs compared to NCFM. In the presence of mannan, previously shown to accelerate endosomal killing of Gram-positive bacteria, the survival of MIMLh5 was strongly decreased, and IL-12 increased to a level close to that induced by NCFM without the addition of mannan, indicating the importance of rapid endosomal degradation for a strong IL-12 response. When measuring the S-layer thickness, MIMLh5’s S-layer appeared to be more than twice the thickness of NCFM and exhibited an elastic modulus approximately twice as high, which is a measure of a cell’s resistance to an applied mechanic stress. When the two strains were depleted of S-layer protein, the elastic modulus was comparable. Together, our data suggests that the thicker S-layer of MIMLh5 compared to NCFM may contribute to its endosomal survival, thus reducing its capacity to induce IL-12. This may constitute an important parameter in the selection of probiotic bacteria for specific purposes.

Probiotic survival is crucial for their beneficial effect, but only a few studies have assessed the impact of delivery format on probiotic viability after passage through the gastrointestinal tract.Our study aimed to investigate the survival of Lacticaseibacillus rhamnosus (L. rhamnosus) CRL1505 through the gastrointestinal transit in healthy volunteers after a daily consumption of at least 1 billion CFUs per day administered as freeze-dried cells or incorporated into oat- and milk-fermented drinks. The probiotic's survival was evaluated by combining culture-based and molecular techniques. Our findings revealed that L. rhamnosus CRL1505 survival was significantly higher when the probiotic was administered as an oat- and as a milk-fermented drink. The study highlighted the persistence of CRL1505 in a subset of volunteers 1 week after the last administration. The metataxonomic analysis of fecal samples did not reveal significant changes in microbiota diversity among treatment groups, as expected after probiotic administration in healthy subjects.The survival of CRL1505 through the human gastrointestinal tract was demonstrated with and without the protection of food matrices, highlighting the strain's remarkable ability to withstand harsh conditions. Our findings emphasize the importance of food matrix selection for improving probiotic viability and its potential efficacy in functional foods.

Attenuation technologies applied to probiotics aim to modulate specific metabolic pathways, particularly acidification, while maintaining cell viability. Although ultrasound is an emerging tool in this context, its precise mechanism of action on probiotic cells remains poorly understood. The study aimed to establish a suitable method to investigate the effects of ultrasound attenuation on probiotics. Lacticaseibacillus casei ATCC 393 was exposed to sonication for 6 and 8 min in a water suspension. Morphological changes, cultivability, acidification capacity, and growth recovery were assessed using culture-dependent methods. Flow cytometry (FCM) combined with fluorescent staining was used to evaluate membrane integrity (as a marker of viability) and esterase activity (as a marker of metabolic activity). Moreover, plate count and FCM data were compared to estimate the overall effect of ultrasound. A reduction in cell size was observed, which was confirmed by decreases in forward and side scatter signals. Acidification capacity was dependent on the intensity applied, and only the 8-min treatment induced prolonged modulation over 24 h. Esterase activity was similarly affected by both sonicating times, whereas membrane integrity reduction was dependent on the treatment intensity. The probiotic demonstrated the ability to restore growth, with recovery time proportionally increasing with the duration of ultrasound treatment. Direct comparisons of the viable, culturable, and metabolically active subpopulations indicate that they are similarly affected by 6 min of sonication. On the contrary, 8 min of sonication increased sample heterogeneity, generating three different subpopulations. The lack of overlap between viable and culturable clusters suggested that the cells that were sonicated for 8 min entered the viable but non-culturable state. These results provide insight into the intensity-dependent effects of ultrasound on probiotic functionality and demonstrate the value of integrative analytical approaches (FCM combined with traditional methods) for characterizing bacterial responses to attenuation strategies.

ABSTRACT This study aimed to assess the potential of Lentilactobacillus hilgardii as a novel candidate for malolactic fermentation (MLF) in winemaking, through comparative genomics and experimental validation, in direct comparison with Oenococcus oeni. We performed a pangenome analysis on 16 L. hilgardii and 7 O. oeni strains to explore their genetic diversity, focusing on wine‐related traits. Functional predictions were generated using genome‐scale metabolic models (ModelSEED/KBase), including in silico co‐inoculation with Saccharomyces cerevisiae EC1118 and post‐alcoholic fermentation simulations. The reference strains L. hilgardii DSM 20176 and O. oeni DSM 20252 were experimentally tested for MLF performance in a synthetic wine‐like medium at 25°C and 10°C. Core‐genome comparison revealed that 67.9% of the malolactic enzyme sequence is conserved between the two species, with comparable docking affinity to L‐malic acid. L. hilgardii harboured unique enzymes with potential oenological interest (phenolic acid decarboxylase, mannitol dehydrogenase, glucosidase) and distinctive stress‐related proteins (YaaA, HrcA, ASP23), suggesting improved tolerance to oxidative, temperature, and alkaline stresses. Notably, L. hilgardii showed genomic potential to degrade putrescine, arginine, and ornithine, precursors of ethyl carbamate. Experimentally, L. hilgardii reduced L‐malic acid from 2.5 g/L to < 0.1 g/L within 12 days at 10°C, while O. oeni showed no MLF activity at this temperature. At 25°C, both strains completed MLF within 6–7 days. L. hilgardii also consumed > 80% of residual fructose at 10°C, whereas O. oeni showed minimal utilisation. Our results demonstrate that L. hilgardii combines a favourable genomic repertoire for wine adaptation with superior MLF performance at low temperature, suggesting its potential as an alternative to O. oeni in cool‐climate winemaking. This work provides the first genome‐scale comparative and functional evaluation of L. hilgardii in the winemaking context, highlighting its technological promise to improve fermentation reliability, reduce spoilage risk, and expand the biodiversity of malolactic starters.

Background: The disruption of the intestinal barrier and the imbalance of the gut microbiota (GM) seem to play a major role in the complex pathogenesis of irritable bowel syndrome (IBS). Specific microbial strains could improve the gut microenvironment, promoting anti-inflammatory pathways; similarly, vitamin D supplementation could play a role in enhancing the barrier integrity and modulating the immune response in the gut. This study aims to evaluate the efficacy of a new multistrain probiotic, combined with vitamin D, in improving gut barrier function in IBS without constipation. Methods: In this phase IIb double-blind randomized placebo-controlled, parallel-group, multicenter, clinical trial, 35 patients were treated for 12 weeks with OttaBac®, a high concentration multistrain probiotic plus cholecalciferol, or placebo and were followed up until week 16. Symptoms, quality of life, intestinal permeability, fecal biomarkers, and microbiota composition were evaluated at 0, 12, and 16 weeks. Results: Mean zonulin values showed a significant progressive reduction in the active group (−10.2 ng/mL at week 12, p = 0.0375; −19.5 ng/mL at week 16, p = 0.0002), with a significant difference between groups at week 16 in the per-protocol population (−19.01, p = 0.0053). The active group showed a more stable trend toward improvement in stool frequency and consistency at both week 12 and 16, with a significant improvement compared to the baseline and to the placebo group (−23.2, p = 0.0265, and 5.57 vs. −23.2, p = 0.0492, respectively). No differences were found in regards to the lactulose/mannitol ratio, Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) and Short Form Health Survey (SF-36) total scores, plasmalemmal vesicle associated protein-1 (PV-1), and citrulline levels. In the active group, Bifidobacterium animalis subsp. lactis and Streptococcus thermophilus levels were increased (p < 0.05), while those for Lachnospira were decreased (p < 0.05), and significant changes in Actinobacteria and Proteobacteria were observed (p < 0.05). Lactate (p < 0.01) and acetate (p < 0.05) levels increased post-treatment. Correlation analysis pointed out a significant association between the microbial biomarkers and the symptoms (p < 0.05). Conclusions: Probiotic plus vitamin D could improve IBS-associated symptoms through gut microbiota modulation and gut barrier enhancement, with persistent benefits after treatment discontinuation.

The microbial plate count continues to be an essential technique in food microbiology research and quality control. In food science education, this technique is often taught through traditional fact-based methods, requiring students to follow pre-established protocols. This approach may not fully engage students or enhance their problem-solving skills. Eighty-two final-year Food Science and Technology bachelor's students at the University of Milan were asked to combine their knowledge on general microbiology and microbial physiology for quality assessment of different probiotic products, including freeze-dried cells, fermented milk, and bacterial spore suspension. They were required to select the appropriate culture media, incubation conditions, and treatments based on flow cytometry cell quantification to optimize microbial plate counts and selectively count the different microbial species present in the probiotic formulations. The project aimed to bridge theoretical knowledge with practical applications, emphasizing the most appropriate procedure for viable and cultivable cell quantification, particularly relevant in probiotic formulation. Survey feedback indicated high satisfaction and relevance to future careers, with the majority finding the activity challenging yet engaging. The project demonstrated the effectiveness of combining different microbiological techniques in education, fostering a deeper understanding of the cultivability of probiotics and the role of selective agents.

Global increasing awareness about the health benefits of probiotics resulted to explorational growth in probiotic food supplement market. However, in some countries such as Montenegro, specific probiotic supplement regulation and comprehensive market analysis are absent, hampering the understanding of consumer preferences, market trends, and potential economic impacts of this industry. This article aims to delve into the Montenegrin market of probiotic food supplements, thoroughly examining various product types and their key characteristics. Using the case study of a pharmacy chain, as an example of organizational level, the sales, sale patterns, and trends are examined. Furthermore, we developed and employed a machine learning model for forecasting future sales. The market analysis highlighted the importance of setting national probiotic supplement regulations to enhance Montenegrin consumer understanding and trust, ensuring product efficacy and safety. Our study clearly showed increased interest in probiotic supplements as well as a constant positive trend in probiotic supplement sales. Furthermore, we found the correlation between foreign tourist visits in Montenegro and the yearly seasonality of probiotic supplement sales. Developed support vector regression machine learning model on time series data showed a good forecasting accuracy, clearly indicating that the same could be used for national sales forecasting. The insights from this study could promote the establishment of national probiotic supplement regulations, enhancing consumer protection and market credibility. Additionally, developed machine learning model provides the industry with valuable predictive tool, enabling companies to optimize their supply chains, effectively meet demand, and make data-driven decisions that could support sustainable market growth.

Intestinal microbial composition not only affects the health of the gut but also influences centrally mediated systems involved in mood, through the “gut-brain” axis, a bidirectional communication between gut microbiota and the brain. In this context, the modulation of intestinal microbiota and its metabolites through the administration of probiotics seems to represent a very promising approach in the treatment of the central nervous system alterations. Early postnatal life is a critical period during which the brain undergoes profound and essential modulations in terms of maturation and plasticity. Maternal separation (MS), i.e., the disruption of the mother–pup interaction, represents a pivotal paradigm in the study of stress-related mood disorders, by inducing persistent changes in the immune system, inflammatory processes, and emotional behavior in adult mammals. We conducted experiments to investigate whether sustained consumption of a multi-strain probiotic formulation by adult male mice could mitigate the effects of maternal separation. Our data demonstrated that the treatment with probiotics was able to totally reverse the anxiety- and depressive-like behavior; normalize the neuro-inflammatory state, by restoring the resting state of microglia; and finally induce a proneurogenic effect. Mice subjected to maternal separation showed changes in microbiota composition compared to the control group that resulted in permissive colonization by the administered multi-strain probiotic product. As a consequence, the probiotic treatment also significantly affected the production of SCFA and in particular the level of butyrate. Gut microbiota and its metabolites mediate the therapeutic action of the probiotic mix on MS-induced brain dysfunctions. Our findings extend the knowledge on the use of probiotics as a therapeutic tool in the presence of alterations of the emotional sphere that significantly impact on gut microbiota composition.

Efficient grapevine downy mildew control necessitates the implementation of anti-resistance strategies to ensure the ongoing efficacy of available substances and optimal disease control. With the gradual disappearance of multi-site fungicides from the market, reliance on single-site fungicides poses a long-term risk of selecting strains resistant to multiple modes of action. Challenges in disease management encompass selecting optimal spray programs and monitoring field population sensitivity. This study evaluated the efficacy of anti-resistance strategies, including two single-site fungicides (mandipropamid and oxathiapiprolin), on disease control and fungicide sensitivity through a combination of field trials and laboratory tests for the biological and molecular characterization of the pathogen populations over a three-year period (2019–2021). Mandipropamid, a cellulose synthase inhibitor, is used since a long time for downy mildew control, while oxathiapiprolin, an OxySterol Binding Protein homologue Inhibitor, was introduced recently. Field trials demonstrated effective disease control, even in the presence of mandipropamid-resistant strains (with G1105S/V mutations in PvCesA3 ) and revealed a pronounced selection and spread of resistance to both fungicides in the vineyard where disease pressure was higher. Characterizing pathogen strains remained a significant obstacle in sensitivity monitoring, hindering precise determination of resistance frequencies related to fungicide programs. Traditional techniques, in fact, lack the resolution required for high-throughput isolation and characterization of resistant individuals. To address this challenge, we propose utilizing flow cytometry and fluorescence-activated cell sorting on field sporangia populations, a method able to determine both the number of resistant isolates and isolate pathogen strains in a single assay.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29 vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Micronutrient deficiency is a form of malnutrition responsible for different metabolic diseases, widely shared among developing low-and middle-income African countries. While deficiencies of calcium, iron, vitamin A, zinc, and selenium have been counteracted mainly by implementing mandatory food fortification programs, little attention was given so far on strategies to decrease inadequate intake of water-soluble B-group vitamins. In this review, we summarize the physiological role of B-group vitamins, and discuss the approaches commonly used to tackle their deficiencies in Africa, namely (i) dietary diversification, (ii) supplementation, and (iii) fortification, with the main focus being here the microbial-based biofortification of food. We report the increasing evidence of plant-based African fermented foods as important sources of these vitamins and how microbial-based biofortification strategies may enhance their content and bioavailability during plant-based fermentation, especially seen for folate (vitamin B9), riboflavin (vitamin B2), and cobalamin (vitamin B12). The selection of pro-technological functional microbial strains from spontaneous fermentation and/or unconventional food matrices, the employment of vitamin overproducing lactic acid bacteria, as well as the implementation of adequate food processes are promising tools that could be implemented in the production of staple home-made fermented foods to counteract B-group vitamins deficiencies. Further research is needed to explore the biotechnological potential of underexploited indigenous microbial strains and the impact of fortified foods on gut host health.

Using culture enrichment methods, 100 strains of bacilli of lactic acid bacteria (LAB) were isolated from honeybee Apis mellifera intermissa and fresh honey, collected from apiaries located in the north-east of Algeria. Amongst all of the isolated LAB, 19 selected strains were closely affiliated to four species-Fructobacillus fructosus (10), Apilactobacillus kunkeei (5), Lactobacillus kimbladii and/or Lactobacillus kullabergensis (4)-using phylogenetic and phenotypic approaches. The in vitro probiotic characteristics (simulated gastrointestinal fluids tolerance, autoaggregation and hydrophobicity abilities, antimicrobial activity and cholesterol reduction) and safety properties (hemolytic activity, antibiotic resistance and absence of biogenic amines) were evaluated. The results indicated that some strains showed promising potential probiotic properties. In addition, neither hemolytic activity nor biogenic amines were produced. The carbohydrate fermentation test (API 50 CHL) revealed that the strains could efficiently use a broad range of carbohydrates; additionally, four strains belonging to Apilactobacillus kunkeei and Fructobacillus fructosus were found to be exopolysaccharides (EPS) producers. This study demonstrates the honeybee Apis mellifera intermissa and one of her products as a reservoir for novel LAB with potential probiotic features, suggesting suitability for promoting host health.

SCOPE:This study evaluates how manufacturing conditions of probiotic biomass production, using two different cryoprotectants, Cryo-A and Cryo-B, can affect Streptococcus thermophilus BT01 in vivo gastrointestinal tract survival and its ability to modulate the level of urease activity in fecal samples of healthy subjects.METHODS AND RESULTS:A randomized controlled cross-over study is carried out on 20 adult healthy subjects to evaluate total and viable loads, persistence of S. thermophilus BT01, and urease activity in fecal samples. Strain-specific quantification by using developed culture-based method and molecular qPCR tool allows to quantify viable S. thermophilus BT01 strain in 90% of the subjects. The quantification of both total DNA and recovered viable S. thermophilus BT01 in fecal samples does not reveal significant differences between Cryo-A or Cryo-B treated biomass. However, the administration of S. thermophilus BT01 produced with Cryo-A results in a decreased urease activity in fecal samples compared to Cryo-B protected cells.CONCLUSION:This study i) highlights how the manufacturing conditions can play a role in influencing the probiotic functionality in vivo and ii) represents the first evidence that links S. thermophilus to a specific probiotic mechanism, the reduction of urease activity in fecal samples.

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E%max-E%40h) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature.