In Vitro Assessment of Bio-Functional Properties from Lactiplantibacillus plantarum Strains.

In recent years, alongside the conventional screening procedures for the evaluation of probiotics for human usage, the pharmaceutical and food industries have encouraged scientific research towards the selection of new probiotic bacterial strains with particular functional features. Therefore, this study intended to explore novel functional properties of five Lactiplantibacillus plantarum strains isolated from bee bread. Specifically, antioxidant, antimicrobial and ß-glucosidase activities, exopolysaccharides (EPS) production and the ability to synthesize γ-aminobutyric acid (GABA) were evaluated. The results demonstrated that the investigated L. plantarum strains were effective in inhibiting the growth of some human opportunistic pathogens in vitro (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus). Moreover, the evaluation of antioxidant and ß-glucosidase activity and of EPS and GABA production, revealed a different behavior among the strains, testifying how these properties are strongly strain-dependent. This suggests that a careful selection within a given species is important in order to identify appropriate strains for specific biotechnological applications. The results highlighted that the five strains of L. plantarum are promising candidates for application as dietary supplements in the human diet and as microbial cultures in specific food productions.

The Lepidoptera Galleria mellonella "in vivo" model: a preliminary pilot study on oral administration of Lactobacillus plantarum (now Lactiplantibacillus plantarum).

This work aims to assess the in vitro adhesion of two type strains of Lactobacillus plantarum (ATCC 14917 and ATCC BAA-793) (now Lactiplantibacillus plantarum). The experiments were conducted both in vitro on colon cells lines (Caco-2 and HT-29) and in vivo by adopting Galleria mellonella, a well-known alternative preclinical model. Data comparison obtained from in vitro and in vivo assays showed that adhesion performance is comparable in both models. Moreover, the type strain BAA-793, originally isolated from human saliva, showed enhanced adhesion performance, either in vitro to the low mucus-producing cell line (HT-29) or in vivo into the G mellonella gut. These results suggest a possible adaptation of this strain to its ecological niche compared to ATCC 14917. This preliminary pilot study, once again, showed the reliability of G. mellonella oral administration model as a first-line screening tool for in vitro to in vivo translation. Also, for the first time, the permanence of Lactobacillus strains into G. mellonella gut has been reported, reinforcing the claim that this preclinical model can be used, together with standardised in vitro and in vivo procedures already accepted across the scientific community, for the evaluation and investigation of new potential probiotic strains.

Sequential inoculum of Hanseniaspora guilliermondii and Saccharomyces cerevisiae for winemaking Campanino on an industrial scale.

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.

Evolution of free amino acids during ripening of Caciocavallo cheeses made with different milks.

The evolution of free amino acids (FAA) in Caciocavallo cheeses, made with cow milk (CC) and cow milk mixed with ewe (CE) and goat (CG) milk, was studied throughout ripening. In all Caciocavallo cheeses produced, the total free amino acid (TFAA) content increased during ripening. In general, the highest TFAA content was found in cow cheeses, and the lowest in CG cheeses, whereas CE cheeses ranged over an intermediate level. In all the analyzed samples, during ripening, the content of the individual FAA increased with the exception of arginine. Tyrosine and histidine were found only in CE samples from the middle to the end of ripening. The major FAA found throughout the whole ripening period, in all types of cheese, were leucine, phenylalanine, lysine, valine, asparagine, γ-aminobutyric acid, and ornithine. The TFAA and several AA showed significant differences in ripening time, whereas tyrosine and histidine showed significant differences in kinds of milk.

Variability in chemical and microbiological profiles of long-ripened Caciocavallo cheeses.

Five protected designation of origin (PDO) Caciocavallo Silano and 6 non-PDO Caciocavallo cheeses, ripened for 6mo and collected in the 5 Italian regions of the PDO area (Apulia, Basilicata, Calabria, Campania, and Molise, Italy), were studied to assess their physico-chemical (pH, acidity, moisture, fat, ash, protein, and free amino acid composition) and microbiological profiles. Analyses evidenced a certain fluctuation of previous parameters among samples regardless of the kind of cheese evaluated (PDO and non-PDO). The PCR-denaturing gradient gel electrophoresis analysis performed on the DNA directly extracted from cheeses gave different results, but a low number of bands was always observed. Only one band, corresponding to the species Streptococcus thermophilus, was detectable in 1 PDO and in 2 non-PDO cheese samples, whose free amino acid content was the lowest. Analyses were repeated on experimental Caciocavallo cheeses. Specifically, 2 productions were made, one mimicking the industrial technology (pasteurized milk and selected starter culture) and one the artisanal technology (raw milk and natural whey starter). Results obtained on experimental cheeses at 6mo of ripening showed that industrial samples had lower amounts of total free amino acids then the artisanal ones (1,188.2 vs. 7,523.67mg/100 g of dry matter). Moreover, the PCR-denaturing gradient gel electrophoresis analysis evidenced the sole presence of S. thermophilus in the case of the industrial technology. These data sustain the hypothesis that, out of 11 cheeses analyzed previously, 1 PDO Caciocavallo Silano and 2 non-PDO Caciocavallo cheeses were obtained with the industrial technology. These results could be of help in the discrimination of PDO products, taking into account that the PDO production regulation does not allow the milk pasteurization, nor the use of selected starters.

Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality.

In Italy, the sale of raw milk from vending machines has been allowed since 2004. Boiling treatment before its use is mandatory for the consumer, because the raw milk could be an important source of foodborne pathogens. This study fits into this context with the aim to evaluate the microbiological quality of 30 raw milk samples periodically collected (March 2013 to July 2013) from 3 vending machines located in Molise, a region of southern Italy. Milk samples were stored for 72 h at 4 °C and then subjected to different treatments, such as boiling and microwaving, to simulate domestic handling. The results show that all the raw milk samples examined immediately after their collection were affected by high microbial loads, with values very close to or even greater than those acceptable by Italian law. The microbial populations increased during refrigeration, reaching after 72 h values of about 8.0 log cfu/mL for Pseudomonas spp., 6.5 log cfu/mL for yeasts, and up to 4.0 log cfu/mL for Enterobacteriaceae. Boiling treatment, applied after 72 h to refrigerated milk samples, caused complete decontamination, but negatively affected the nutritional quality of the milk, as demonstrated by a drastic reduction of whey proteins. The microwave treatment at 900 W for 75 s produced microbiological decontamination similar to that of boiling, preserving the content in whey proteins of milk. The microbiological characteristics of raw milk observed in this study fully justify the obligation to boil the raw milk from vending machines before consumption. However, this study also showed that domestic boiling causes a drastic reduction in the nutritional value of milk. Microwave treatment could represent a good alternative to boiling, on the condition that the process variables are standardized for safe domestic application.

Biodiversity of Lactobacillus plantarum from traditional Italian wines.

In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine.

Diversity of fungal communities on Cabernet and Aglianico grapes from vineyards located in Southern Italy.

Grape-associated microbial community is influenced by a combination of viticultural, climatic, pedological and anthropological factors, collectively known as terroir. Therefore, grapes of the same cultivar grown in different areas can be appreciated for their distinctive biogeographic characteristics. In our previous study, we showed that the phenotypic response of Aglianico and Cabernet grapevines from Molise and Sicily regions is significantly influenced by the prevailing pedoclimatic conditions, particularly soil physical properties. However, the scale at which microbial communities differ could be important in clarifying the concept of terroir, including whether it is linked to the grape variety present in a particular vineyard. To explore this further, in the research presented here, a comparative study on the fungal communities inhabiting the berry surfaces of Cabernet and Aglianico cultivars was conducted on different vineyards located in Southern Italy (Molise, Sicily and Campania regions, the first two of which had been involved in our previous study) by using high-throughput sequencing (HTS) and multivariate data analysis. The descriptive approach through relative abundance analysis showed the most abundant phyla (Ascomycota, Basidiomycota, and Chytridiomycota), families (Cladosporiaceae, Saccotheciaceae, Pleosporaceae, Saccharomycodaceae, Sporidiobolaceae, Didymellaceae, Filobasidiaceae, Bulleribasidiaceae, and Saccharomycetaceae) and genera (Cladosporium, Aureobasidium, Alternaria, Stemphylium and Filobasidium) detected on grape berries. The multivariate data analysis performed by using different packages (phyloseq, Vegan, mixOmics, microbiomeMarker and ggplot2) highlighted that the variable "vineyard location" significantly affect the fungal community, while the variable "grape variety" has no significant effect. Thus, some taxa are found to be part of specific vineyard ecosystems rather than specific grape varieties, giving additional information on the microbial contribution to wine quality, thanks to the presence of fermentative yeasts or, conversely, to the involvement in negative or detrimental roles, due to the presence of grape-deriving fungi implied in the spoilage of wine or in grapevine pathogenesis. In this connection, the main functions of core taxa fungi, whose role in the vineyard environment is still poorly understood, are also described.

Plant-Based Ingredients Utilized as Fat Replacers and Natural Antimicrobial Agents in Beef Burgers.

The present study aimed to find solutions based on the use of plant-based ingredients that would improve the nutritional quality of meat products as well as ensure sensory and microbiological quality. Two fat replacers, lemon albedo (Citrus lemon) and carob seed gum (Ceratonia siliqua), were investigated by chemical analysis and panel testing to evaluate their effect on the nutritional and sensory quality of beef burgers. The antimicrobial activity of two plant extracts, from nettle (Urtica dioica) leaves and medlar (Eriobotrya japonica) seeds, was studied, evaluating the intensity of inhibitory action and the minimum inhibitory concentration against Pseudomonas spp. and Listeria innocua strains by plate test. In addition, the antioxidant activity of both extracts was evaluated. Based on the results, lemon albedo and medlar seed extracts were validated in a food model (beef burger) by a storage test and a challenge test. The storage test results highlight that medlar seed extract prevents the formation of thiobarbituric acid reactive substances (TBARSs) and ensures microbiological quality, inhibiting Enterobacteriaceae and Pseudomonas spp. Anti-Listeria efficacy was confirmed in situ by challenge test results. In conclusion, although fat replacers ensure nutritional and sensory quality, they do not satisfy microbiological quality. This study clearly demonstrates that the safety of low-fat burgers can only be achieved through the combination of appropriate fat replacers with well-selected natural antimicrobial extracts.

Investigating the impact of pedoclimatic conditions on the oenological performance of two red cultivars grown throughout southern Italy.

The cultivated grapevine, Vitis vinifera subsp. vinifera, possesses a rich biodiversity with numerous varieties. Each variety adapts differently to varying pedoclimatic conditions, which greatly influence the terroir expression of wine regions. These conditions impact vine growth, physiology, and berry composition, ultimately shaping the unique characteristics and typicity of the wines produced. Nowadays, the potential of the different adaptation capacities of grape varieties has not yet been thoroughly investigated. We addressed this issue by studying two grape varieties, Aglianico and Cabernet Sauvignon, in two different pedoclimatic conditions of Southern Italy. We evaluated and compared the effect of different pedoclimatic conditions on plant physiology, the microbial quality of grapes using Next-Generation Sequencing (NGS) technology, the expression trends of key genes in ripe berries and the concentration of phenolic compounds in grapes and wines by HPLC-MS, HPLC-DAD, NMR and spectrophotometric analyses. Metabolomic and microbiome data were integrated with quantitative gene expression analyses to examine varietal differences and plasticity of genes involved in important oenological pathways. The data collected showed that the phenotypic response of studied grapes in terms of vigor, production, and fruit quality is strongly influenced by the pedoclimatic conditions and, in particular, by soil physical properties. Furthermore, Aglianico grape variety was more influenced than the Cabernet Sauvignon by environmental conditions. In conclusion, the obtained findings not only reinforce the terroir concept and our comprehension of grape's ability to adapt to climate variations but can also have implications for the future usage of grape genetic resources.

Fungi Occurrence in Ready-to-Eat Hazelnuts (Corylus avellana) From Different Boreal Hemisphere Areas.

The present study evaluated the fungal contamination of ready-to-eat dried hazelnuts considering for the first time the application of the same condition drying process of several hazelnut cultivars from different boreal hemisphere areas. Fifty lots of hazelnuts (Corylus avellana), belonging to eight cultivars from seven regions in four countries, were analyzed for fungal microbiota, describing both load levels and species diversity. For this purpose, a polyphasic approach consisting of morphological examination (optical and scanning electron microscope observation) and molecular characterization [PCR-DGGE analysis and sequence analyses of the internal transcribed spacer (ITS)] was performed. The results show that different fungal populations occur in dried hazelnuts regardless of their geographical area of production. Although some varieties appear to be relatively less susceptible, species related to Aspergillus, such as A. commune and A. ochraceus, Penicillium, including P. commune, P. solitum, and P. expansum, and Rhizopus, for instance, R. stolonifer and R. oryzae, have generally been found. A related character "hazelnut cultivar-fungi" was found for species related to the genera Trichoderma and Fusarium, including F. oxyxporum, F. solani, and F. falciforme. All 14 species found are known to host pathogenic strains. Therefore, their presence in a ready-to-eat product, such as dried hazelnuts, can pose a real danger to the consumer. Based on these considerations, the development of new protective strategies seems highly desirable. The species-level description of the contaminating fungal community acquired through this study is the starting point for the development of tailor-made protective biotechnologies.

Probiotic Potentiality from Versatile Lactiplantibacillus plantarum Strains as Resource to Enhance Freshwater Fish Health.

Dietary probiotic supplementation has the potential to enhance the health of fish and their disease resistance. In this study, some properties of ten Lactiplantibacillus plantarum strains have been evaluated, for their potential use as probiotics in freshwater fish diet. In particular, antimicrobial activity, antioxidant activity, the potentiality to survive the gastrointestinal transit and persist in the intestine, were evaluated in vitro. The experimental tests were carried out at 15 °C and 30 °C to determine the suitability of these lactic acid bacteria to be used as probiotics in the diet of fish grown at different temperatures. The results demonstrated that the evaluated Lp. plantarum strains, which often have significant differences among themselves, are characterized by important functional characteristics such as cell surface properties (auto-aggregation and hydrophobicity), ability to produce antioxidant substances, capacity to survive in the presence of 0.3% bile salts and acidic environment (2.5 pH), antagonistic activity against some fish opportunistic pathogens (A. salmonicida, Ps. aeruginosa, E. coli and C. freundii) and other unwanted bacteria present in fish products (S. aureus and L. innocua). The outcomes suggest that these Lp. plantarum strains may be candidates as probiotics in warm- and cold-water aquaculture.

Presence of Lactic Acid Bacteria in the Intestinal Tract of the Mediterranean Trout (Salmo macrostigma) in Its Natural Environment.

Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. Few reports have been produced on the composition of the gut microbiota and on the presence of LAB in the intestines of freshwater fish that inhabit river environments. In this study, we investigated the LAB community that was present in the gastrointestinal tract (GIT) of Mediterranean trout (Salmo macrostigma) that colonized the Biferno and Volturno rivers of the Molise region (Italy). The partial 16S rRNA gene sequences of these strains were determined for the species-level taxonomic placement. The phylogenetic analysis revealed that the isolated LABs belonged to seven genera (Carnobacterium, Enterococcus, Lactobacillus, Lactiplantibacillus, Vagococcus, Lactococcus, and Weissella). The study of the enzymatic activities showed that these LABs could contribute to the breakdown of polysaccharides, proteins, and lipids. In future studies, a greater understanding of how the LABs act against pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome and enhance disease control and fish health.

Probiotic Properties and Potentiality of Lactiplantibacillus plantarum Strains for the Biological Control of Chalkbrood Disease.

Ascosphaera apis is an entomopathogenic fungus that affects honeybees. In stressful conditions, this fungus (due not only to its presence, but also to the combination of other biotic and abiotic stressors) can cause chalkbrood disease. In recent years, there has been increasing attention paid towards the use of lactic acid bacteria (LAB) in the honeybees' diets to improve their health, productivity and ability to resist infections by pathogenic microorganisms. The screening of 22 strains of Lactiplantibacillus plantarum, isolated from the gastrointestinal tracts of honeybees and beebread, led to the selection of five strains possessing high antagonistic activity against A. apis. This study focused on the antifungal activity of these five strains against A. apis DSM 3116 and DSM 3117 using different matrices: cell lysate, broth culture, cell-free supernatant and cell pellet. In addition, some functional properties and the antioxidant activity of the five L. plantarum strains were evaluated. All five strains exhibited high antagonistic activity against A. apis, good surface cellular properties (extracellular polysaccharide (EPS) production and biofilm formation) and antioxidant activity. Although preliminary, these results are encouraging, and in future investigations, the effectiveness of these bacteria as probiotics in honeybee nutrition will be tested in vivo in the context of an eco-friendly strategy for the biological control of chalkbrood disease.

Effect of Biofilm Formation by Lactobacillus plantarum on the Malolactic Fermentation in Model Wine.

Biofilm life-style of Lactobacillus plantarum (L. plantarum) strains was evaluated in vitro as a new and suitable biotechnological strategy to assure L-malic acid conversion in wine stress conditions. Sixty-eight L. plantarum strains isolated from diverse sources were assessed for their ability to form biofilm in acid (pH 3.5 or 3.2) or in ethanol (12% or 14%) stress conditions. The effect of incubation times (24 and 72 h) on the biofilm formation was evaluated. The study highlighted that, regardless of isolation source and stress conditions, the ability to form biofilm was strain-dependent. Specifically, two clusters, formed by high and low biofilm producer strains, were identified. Among high producer strains, L. plantarum Lpls22 was chosen as the highest producer strain and cultivated in planktonic form or in biofilm using oak supports. Model wines at 12% of ethanol and pH 3.5 or 3.2 were used to assess planktonic and biofilm cells survival and to evaluate the effect of biofilm on L-malic acid conversion. For cells in planktonic form, a strong survival decay was detected. In contrast, cells in biofilm life-style showed high resistance, assuring a prompt and complete L-malic acid conversion.

Low-Fat and High-Quality Fermented Sausages.

The present study, considering for the first time microbiological concerns due to the use of lemon albedo as a fat replacer, aimed at the selection of an anti-Listeria strain to be used as protective culture in low-fat southern Italian fermented sausages. In fact, these kinds of products require appropriate bio-protective strategies to avoid risks due to Listeria monocytogenes. Sixty-seven Lactiplantibacillus plantarum strains isolated from diverse sources were screened for their antimicrobial activity and their interaction with starter strains (Latilactobacillus sakei 152 and Staphylococcus xylosus MVS9). Lactiplantibacillus plantarum Lpls100, highlighting both listericidal activity and the ability to promote Staphylococcus xylosus MVS9 growth, was used as a protective strain in low-fat fermented sausages prepared with lemon albedo as a fat replacer. The effect of the albedo and the protective strain on the fermentation process and the final quality was ascertained. Results highlighted that the use of the albedo did not affect the growth of starter strains and enhanced some quality features, such as fatty acid profiles and certain sensory attributes. However, the albedo also produced a slow decrease in water activity, compromising the microbial quality. The anti-Listeria strain, enhancing coagulase negative cocci growth and exerting antimicrobial activity, avoided the inconveniences caused by the use of the albedo. Moreover, the anti-Listeria effectiveness was assessed through a challenge test using a Listeria cocktail. The study revealed that Lactiplantibacillus plantarum Lpls100, regardless of the presence of the albedo, assures a prompt inhibition of Listeria spp. Therefore, its use could be an important contribution to the quality of low-fat fermented sausages.

Antimicrobial Activity against Paenibacillus larvae and Functional Properties of Lactiplantibacillus plantarum Strains: Potential Benefits for Honeybee Health.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty-one Lactiplantibacillus plantarum strains, against P. larvae ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against P. larvae ATCC 9545were selected for further physiological and biochemical characterizations. In  particular, the hydrophobicity, auto-aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five L. plantarum selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against P. larvae opens up interesting perspectives for new biocontrol strategies of diseases such as AFB.

Potential Application of Apilactobacillus kunkeei for Human Use: Evaluation of Probiotic and Functional Properties.

Apilactobacillus kunkeei is an insect symbiont with documented beneficial effects on the health of honeybees. It belongs to fructophilic lactic acid bacteria (FLAB), a subgroup of lactic acid bacteria (LAB) notably recognized for their safe status. This fact, together with its recurrent isolation from hive products that are traditionally part of the human diet, suggests its possible safe use as human probiotic. Our data concerning three strains of A. kunkeei isolated from bee bread and honeybee gut highlighted several interesting features, such as the presence of beneficial enzymes (ß-glucosidase, ß-galactosidase and leucine arylamidase), the low antibiotic resistance, the ability to inhibit P. aeruginosa and, for one tested strain, E. faecalis, and an excellent viability in presence of high sugar concentrations, especially for one strain tested in sugar syrup stored at 4 °C for 30 d. This datum is particularly stimulating, since it demonstrates that selected strains of A. kunkeei can be used for the probiotication of fruit preparations, which are often used in the diet of hospitalized and immunocompromised patients. Finally, we tested for the first time the survival of strains belonging to the species A. kunkeei during simulated gastrointestinal transit, detecting a similar if not a better performance than that showed by Lacticaseibacillus rhamnosus GG, used as probiotic control in each trial.

Inter- and Intra-Species Diversity of Lactic Acid Bacteria in Apis mellifera ligustica Colonies.

Lactic acid bacteria could positively affect the health of honey bees, including nutritional supplementation, immune system development and pathogen colonization resistance. Based on these considerations the present study evaluated predominant Lactic Acid Bacteria (LAB) species from beebread as well as from the social stomach and midgut of Apis mellifera ligustica honey bee foragers. In detail, for each compartment, the diversity in species and biotypes was ascertained through multiple culture-dependent approaches, consisting of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE), 16S rRNA gene sequencing and Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The study of a lactic acid bacteria community, performed with PCR-DGGE and sequence analysis targeting the V1-V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Apilactobacillus kunkeei, Lactiplantibacillus plantarum, Fructobacillus fructosus, Levilactobacillus brevis and Lactobacillus delbrueckii subsp. lactis. Depending on the different compartments, diverse levels of biodiversity in species were found. Particularly, a very low inter-species biodiversity was detected in the midgut that was prevalently dominated by the presence of Apilactobacillus kunkeei. On the other hand, the beebread was characterized by a reasonable biodiversity showing the presence of five species and the predominance of Apilactobacillus kunkeei, Lactiplantibacillus plantarum and Fructobacillus fructosus. The RAPD-PCR analysis performed on the three predominant species allowed the differentiation into several biotypes for each species. Moreover, a relationship between biotypes and compartments has been detected and each biotype was able to express a specific biochemical profile. The biotypes that populated the social stomach and midgut were able to metabolize sugars considered toxic for bees while those isolated from beebread could contribute to release useful compounds with functional properties. Based on this knowledge, new biotechnological approaches could be developed to improve the health of honey bees and the quality of bee products.

Antagonistic Activity against Ascosphaera apis and Functional Properties of Lactobacillus kunkeei Strains.

: Lactic acid bacteria (LAB) are an important group of honeybee gut microbiota. These bacteria are involved in food digestion, stimulate the immune system, and may antagonize undesirable microorganisms in the gastrointestinal tract. Lactobacillus kunkeei is a fructophilic lactic acid bacterium (FLAB) most frequently found in the gastrointestinal tracts of honeybees. Ascosphaera apis is an important pathogenic fungus of honeybee larvae; it can colonize the intestine, especially in conditions of nutritional or environmental stress that cause microbial dysbiosis. In this work, some functional properties of nine selected L. kunkeei strains were evaluated. The study focused on the antifungal activity of these strains against A. apis DSM 3116, using different matrices: cell lysate, broth culture, cell-free supernatant, and cell pellet. The cell lysate showed the highest antifungal activity. Moreover, the strains were shown to possess good cell-surface properties (hydrophobicity, auto-aggregation, and biofilm production) and a good resistance to high sugar concentrations. These L. kunkeei strains were demonstrated to be functional for use in "probiotic syrup", useful to restore the symbiotic communities of the intestine in case of dysbiosis and to exert a prophylactic action against A. apis.