Massimo Iorizzo

Honey bee gut microbiota play important roles in host nutrition, immune regulation, and health, and their composition can vary in response to environmental conditions, diet, seasonality, and beekeeping management. In this two-year field study, we investigated seasonal variation in the gut bacterial communities of Apis mellifera ligustica colonies maintained under stationary or short-distance migratory beekeeping management. Six colonies originating from the same experimental apiary were assigned to stationary or migratory management, with three migratory colonies seasonally transferred to Mount Taburno during summer and three stationary colonies remaining at Castelpoto. Bacterial communities were characterized by high-throughput sequencing of the V1–V3 region of the 16S rRNA gene. The gut microbiota was consistently dominated by characteristic honey bee-associated taxa, particularly Gilliamella and Snodgrassella , although their relative abundances varied substantially across seasons and years. Alpha-diversity analyses based on harmonized genus-level relative abundance profiles revealed significant seasonal variation in both years, whereas no significant overall effect of management system was detected. Similarly, PERMANOVA of Bray–Curtis dissimilarities revealed significant seasonal structuring of bacterial community composition in 2022 and 2023, while stationary versus migratory management did not significantly explain overall community variation. The persistence of dominant gut-associated taxa across sampling periods indicates compositional continuity of major bacterial community members despite temporal variation, but does not by itself demonstrate functional stability or microbial resilience. Colony-performance measurements showed significantly higher annual honey production in migratory than in stationary colonies in 2022, whereas no significant differences in the investigated performance parameters were detected between management systems in 2023. Thus, the productive response was not consistent between years. Although temporal variation in gut bacterial communities occurred alongside differences in colony performance, the present study does not establish a causal or functional relationship between microbiota composition and productivity. Overall, these findings identify seasonality as the predominant factor associated with variation in the gut bacterial communities of the investigated colonies and provide no evidence for a consistent overall effect of short-distance migratory management on bacterial community diversity or structure. Further studies involving larger numbers of independent colonies and apiaries, together with direct functional analyses of the microbiome and characterization of dietary and environmental factors, are required to clarify the consequences of migratory beekeeping for honey bee microbiota and colony performance.

Background/Objectives: Propolis is a chemically complex product whose antimicrobial activity varies with geographical origin and composition. This study evaluated the chemical, antioxidant, and antimicrobial properties of five Romanian propolis ethanolic extracts (PEEs) and their combinations, focusing on antimicrobial interactions and chemical–biological relationships. Methods: Five PEEs were evaluated individually, as ten binary combinations, and as one five-extract mixture. Antioxidant activity and 20 phenolic compounds were determined. Antimicrobial activity against 16 strains was assessed using agar diffusion and broth microdilution assays. Extract interactions were evaluated using the fractional inhibitory concentration index (FICI). Principal Component Analysis (PCA), Spearman correlation, and Partial Least Squares (PLS) regression investigated chemical–biological relationships. Results: Significant differences were observed among preparations in phenolic composition, antioxidant activity, and antimicrobial responses. Total phenolic content ranged from 88.8 to 149.8 mg GAE/g, while A + S showed the highest antioxidant activity (3213.74 mg TE/g). C1 showed strong activity against several Gram-positive bacteria and Candida albicans, whereas S and S-containing combinations showed activity against several Gram-negative strains. FICI analysis showed predominantly indifferent interactions (60.8%), followed by additive (25.0%), synergistic (9.09%), and antagonistic (5.11%). The strongest synergy was observed for A + S against Pseudomonas aeruginosa (FICI = 0.195). Chemical PC1 was inversely associated with biological PC2 (ρ = −0.809, FDR-adjusted p = 0.0013). Conclusions: Antimicrobial activity depends on extract composition and the target microorganism. Combining extracts did not consistently improve activity; however, selected combinations produced additive or synergistic interactions, whereas antagonism was uncommon. Multivariate analyses further supported an association between phenolic composition and antimicrobial variability.

The increasing demand for sustainable and phytochemically enriched foods has encouraged the valorization of agricultural by-products as ingredients for beverage formulation. This study investigated the enrichment of grape juice with Vitis vinifera leaf extract and characterized the resulting phytochemical, antioxidant, volatile, and sensory profiles. The fortified juice was compared with non-enriched grape juice and a commercial sample. Samples were analyzed at production (t₀) and after 30 days of refrigerated storage (4 °C) for polyphenols, flavonoids, anthocyanins, carotenoids, tocopherols, and antioxidant capacity (ABTS, DPPH, and CUPRAC assays). Volatile organic compounds (VOCs), sensory properties, and microbiological stability were also evaluated. The fortified preparation showed higher concentrations of polyphenols, flavonoids, and anthocyanins than the non-fortified preparation. α-Tocopherol, γ-tocopherol, β-carotene, and lutein were detected exclusively in fortified samples. ABTS and CUPRAC assays showed comparatively high antioxidant activity values in the fortified preparation. Lower concentrations of anthocyanins, carotenoids, and tocopherols were observed at t₃₀ than at t₀, although substantial levels remained detectable after 30 days. HS-SPME/GC-MS detected 63 VOCs, with the fortified beverage showing a distinct volatile profile after 30 days of refrigerated storage, including comparatively high analytical responses for several monoterpenes and C6 alcohols. Sensory analysis revealed a distinctive profile characterized by greater sourness, bitterness, astringency, density, and aromatic complexity, together with lower overall sensory quality. Microbial counts remained below the respective detection limits under the investigated conditions. Overall, the investigated fortified preparation was characterized by a phytochemically enriched profile and by differences in antioxidant capacity, volatile composition, and sensory characteristics compared with the non-fortified preparation. These findings provide preliminary evidence of the compositional potential of grapevine leaf extract as an ingredient for phytochemically enriched grape-based beverages, while also identifying sensory constraints requiring formulation optimization.

The growing demand for clean-label and low-sulphite wines has increased interest in alternative preservation strategies capable of reducing sulphur dioxide (SO₂) usage while maintaining wine quality and stability. In this study, the effectiveness of Vitis vinifera leaf extract as a natural alternative to sulphur dioxide was evaluated in High Hydrostatic Pressure (HHP)-treated Fiano wines. Three experimental wines were produced: CW (control wine), SW (sulphited wine), and LW (leaf-extract wine). Following alcoholic fermentation, all wines were subjected to HHP treatment (600 MPa for 5 min) and stored at 4 °C for 60 days. Physicochemical parameters, volatile organic compounds (VOCs), and sensory characteristics were evaluated immediately after alcoholic fermentation and after HHP treatment followed by refrigerated storage. Compared with the control wine, LW exhibited approximately 20% higher total polyphenol concentrations, whereas SW showed the greatest preservation of fermentation-derived esters. HHP treatment induced only moderate changes in the volatile fraction, confirming the suitability of this non-thermal technology for wine stabilization. LW wines were characterized by higher abundances of terpene-related compounds, C6 alcohols, medium-chain fatty acids, and phenolic-associated volatiles, resulting in more pronounced floral, balsamic, herbaceous, and vegetal sensory attributes. Principal Component Analysis (PCA) explained 79.0% of the total VOC variability and clearly differentiated LW wines from CW and SW according to their volatile profiles, while sensory analysis confirmed the development of a distinctive aromatic identity associated with grapevine leaf extract. Overall, the results indicate that the combined application of V. vinifera leaf extract and HHP represents a promising strategy for the partial replacement of sulphur dioxide in white winemaking. This integrated approach contributes to wine stabilization while promoting the valorization of grapevine leaves as a sustainable winery by-product within a circular economy framework.

The honey bee gut microbiota plays a crucial role in host nutrition, immunity, and colony health, yet the relative influence of seasonal and colony-specific factors on its long-term dynamics remains incompletely understood. This study investigated temporal variation in the gut bacterial community of three Apis mellifera ligustica colonies maintained in the same apiary and monitored over two consecutive years (2022–2023). Worker bees were sampled during eight seasonal periods, and gut microbiota composition was characterized using 16S rRNA gene amplicon sequencing. Across all sampling periods, the microbiome was consistently dominated by the characteristic honey bee-associated genera Gilliamella , Snodgrassella , Bartonella , Frischella , Commensalibacter , and Lactobacillus , indicating the persistence of a conserved core bacterial community. Seasonal variation was primarily associated with changes in the relative abundance of dominant taxa rather than with major changes in community composition. In particular, Gilliamella apicola and Snodgrassella alvi exhibited complementary seasonal patterns, with Gilliamella reaching its highest abundance during autumn, particularly in autumn 2023, whereas Snodgrassella predominated during spring and winter. Alpha-diversity metrics (Observed OTUs, Chao1, Shannon, and Simpson indices) showed limited seasonal variation, whereas beta-diversity analyses detected significant differences in community composition among seasons. Principal Coordinates Analysis and PERMANOVA identified season as the factor most strongly associated with microbiome variation, while colony identity did not significantly influence bacterial community composition under the standardized experimental conditions adopted in this study. Overall, these findings show that the gut microbiome of A. mellifera ligustica maintains a conserved core bacterial community while exhibiting reproducible seasonal variation in the relative abundance of its dominant members. This study provides a longitudinal baseline for future investigations aimed at understanding the ecological mechanisms underlying seasonal microbiome dynamics and their relationship with honey bee biology and environmental change.

The increasing demand for sustainable and health-oriented foods has encouraged the valorization of agricultural by-products as sources of bioactive compounds for functional beverages. This study enriched grape juice with Vitis vinifera leaf extract to enhance its phytochemical composition, antioxidant capacity, and aromatic profile. The fortified juice was compared with non-enriched grape juice and a commercial sample. Samples were analyzed at production (t₀) and after 30 days of refrigerated storage (4 °C) for polyphenols, flavonoids, anthocyanins, carotenoids, tocopherols, and antioxidant capacity (ABTS, DPPH, and CUPRAC assays). Volatile organic compounds (VOCs), sensory properties, and microbiological stability were also evaluated. Enriched beverages showed significantly higher concentrations of polyphenols, flavonoids, and anthocyanins. α-Tocopherol, γ-tocopherol, β-carotene, and lutein were detected exclusively in fortified samples. ABTS and CUPRAC assays revealed the highest antioxidant activity in enriched juices. Although storage reduced anthocyanin, carotenoids, and tocopherols, substantial levels persisted after 30 days. HS-SPME/GC-MS identified 63 VOCs, with leaf fortification increasing monoterpenes and C6 alcohols linked to floral, citrusy, herbaceous, and green notes. Sensory analysis indicated greater sourness, astringency, density, and aromatic complexity, while maintaining satisfactory overall quality. All samples remained microbiologically stable. These findings support grapevine leaf extract as a promising sustainable ingredient for functional grape-based beverages.

Aligning with the circular economy principles and the valorization of agri-food wastes, this study investigated the major bioactive compounds in leaves of two representative grapevine cultivars from Campania region in Italy: Aglianico and Falanghina. Key phytochemicals, such as carotenoids, tocols, total and single polyphenols, were analysed through chromatographic and advanced mass spectrometry techniques (HPLC-DAD-FLD and HPLC-ESI-Q-TOF MS). Antioxidant capacity was evaluated using the DPPH, CUPRAC, ABTS assays. The most representative tocols were α-tocopherol (maximum values of about 200 mg/100 g f.w.) and γ-tocopherol (highest levels of 80 mg/100 g f.w.), while β-carotene and lutein were the predominant carotenoids (highest amounts about 15 and 25 mg/100 g, respectively). Caftaric acid and quercetin-3- O -glucuronide were the major phenolic compounds. Considering the Recommended Daily Allowance (RDA) established by EU Regulation, 100 g of the analysed leaves provide significant amounts of vitamins A and E. Seasonal monitoring (from June to November) revealed that the concentrations of the investigated bioactive compounds varied significantly across leaf developmental stages, with carotenoids and total phenols decreasing and tocols increasing during the phenological stages. Results showed that a good period of harvesting could be in September, after the grape harvest.

Water composition is a key factor influencing brewing performance and beer quality due to its impact on mash chemistry, fermentation kinetics, and fermentation-derived metabolites. This study evaluated the effect of four Albanian spring waters (Bogova, Germenji, Selita, and Lajthiza), each with distinct mineral compositions, on the fermentation behaviour and physicochemical characteristics of Pale Ale beer produced under standardised brewing conditions. All beers were brewed using the same malt formulation, hopping regime, yeast strain, and fermentation parameters, with water source as the sole experimental variable. The produced worts showed only moderate differences in pH, colour, extract, free amino nitrogen (FAN), bitterness, and density, whereas alcoholic fermentation proceeded efficiently in all treatments and was completed within seven days. Final alcohol contents ranged from 5.56 to 5.70% v/v, confirming comparable fermentation performance among treatments. More pronounced differences were observed in acidity-related parameters and fermentation-derived compounds. Volatile acidity ranged from 0.19 to 0.93 g/L, with the highest values in beers produced with Selita and Lajthiza waters. Glycerol concentrations varied from 0.88 to 1.24 g/L, with Germenji beer showing the highest value, whereas acetaldehyde ranged from 3.16 to 6.04 mg/L, with the lowest concentration in Germenji beer. Pearson correlation analysis and exploratory principal component analysis (PCA) identified associations between water mineralisation and selected physicochemical and fermentation-derived beer parameters. Calcium, magnesium, conductivity, and hardness were positively associated with glycerol concentration, whereas bicarbonate concentration was associated with beer pH and acidity-related parameters. The first two principal components explained 87.7% of the total variance. Overall, the results indicate that Albanian spring waters are suitable for Pale Ale production and show that differences in water mineral composition were associated with variations in the physicochemical and fermentation-derived characteristics of the final beers. These findings highlight that brewing water should not be regarded as a neutral processing medium but rather as an important technological factor associated with differences in the physicochemical characteristics of beer, while supporting the valorisation of Albanian spring waters for geographically distinctive craft brewing applications.

Lactiplantibacillus plantarum is a highly versatile lactic acid bacterium, widely distributed across diverse ecological niches. Although often described as a nomadic species, increasing evidence suggests that strains from different habitats may retain niche-associated functional traits. This study investigated the phenotypic biodiversity of forty L. plantarum strains isolated from four ecologically distinct environments: wine, honeybee gut, trout intestine, and pre-weaning infant feces. Growth performance at different temperatures and on various carbon sources, acidification capacity, and β-glucosidase activity were evaluated and integrated using multivariate statistical analyses. Significant differences in β-glucosidase activity were observed among ecological groups (Kruskal–Wallis, p = 0.001), with wine-associated strains exhibiting the highest enzymatic activities and trout-derived isolates the lowest. Growth and acidification traits showed more limited variation among habitats, indicating that these physiological characteristics are largely conserved within the species. Heatmap visualization, principal component analysis (PCA), and hierarchical clustering revealed substantial phenotypic heterogeneity among strains. PCA indicated that growth performance and acidification traits contributed primarily to the first principal component, whereas β-glucosidase activity and differential fructose utilization were major contributors to the second component. Permutational multivariate analysis of variance (PERMANOVA) confirmed a significant effect of ecological origin on the overall phenotypic structure (p = 0.006), although habitat explained only 15.3% of the total variance (R² = 0.153). Overall, the results show that ecological origin contributes to the phenotypic diversification of L. plantarum populations while preserving the extensive functional versatility characteristic of this species. β-Glucosidase activity emerged as the most discriminating phenotypic trait among ecological groups and represented the principal niche-associated functional signature identified in this study.

Water composition is a key factor influencing brewing performance and beer quality due to its impact on mash chemistry, fermentation kinetics, and yeast metabolism. This study evaluated the effect of four Albanian spring waters (Bogova, Germenji, Selita, and Lajthiza), each with distinct mineral compositions, on the fermentation behaviour and physicochemical characteristics of Pale Ale beer produced under standardised brewing conditions. All beers were brewed using the same malt formulation, hopping regime, yeast strain, and fermentation parameters, with water source as the sole experimental variable. The beer worts showed only moderate differences in pH, colour, extract, and density, while alcoholic fermentation proceeded efficiently in all treatments and was completed within seven days. The final beers exhibited alcohol contents within the expected range for Pale Ale beers, while more pronounced differences were observed in acidity-related parameters and secondary metabolites. In particular, volatile acidity, glycerol, acetaldehyde, and organic acid concentrations varied significantly among treatments. Beers produced with Selita and Lajthiza waters showed higher volatile acidity and acetaldehyde levels, whereas Germenji water promoted increased glycerol production and lower acetaldehyde concentration. Correlation analysis and principal component analysis confirmed significant associations between brewing water mineralisation and fermentation-derived compounds. Calcium, magnesium, conductivity, and hardness were positively associated with glycerol production, while bicarbonate concentration influenced final beer pH and acidity development. Overall, the results demonstrate that Albanian spring waters are suitable for Pale Ale production and confirm that water mineral composition significantly affects yeast metabolism and the chemical profile of the final beers. These findings highlight the importance of water chemistry as a technological factor in craft beer production and may contribute to the valorisation of Albanian spring waters for geographically distinctive brewing applications.