Cultivable microorganisms associated with honeys of different geographical and botanical origin

In this study, the composition of the cultivable microbial populations of 38 nectar honey and honeydew honey samples of different botanical and geographical origin were assessed. After growth in specific media, various colonies with different appearance were isolated and purified before phenotypic (morphological, physiological and biochemical traits) and genotypic [randomly amplified polymorphic DNA (RAPD), repetitive DNA elements-PCR (rep-PCR) and restriction fragment length polymorphism (RFLP)] differentiation. The identification was carried out by 16S rRNA gene sequencing for bacteria and, in addition to RFLP, by sequencing the D1/D2 region of the 26S rRNA gene for yeasts and the 5.8S-ITS rRNA region for filamentous fungi. The results showed the presence of 13 species of bacteria, 5 of yeasts and 17 of filamentous fungi; the species most frequently isolated were Bacillus amyloliquefaciens, Zygosaccharomyces mellis and Aspergillus niger for the three microbial groups, respectively. The highest microbial diversity was found in multifloral honeys. No correlation among the microbial species and the botanical/geographical origin was found, but some strains were highly adapted to these matrices since they were found in several samples of different origin

Formation and Characterization of Early Bacterial Biofilms on Different Wood Typologies Applied in Dairy Production

The main hypothesis of this work was that Sicilian forestry resources are suitable for the production of equipment to be used in cheese making and indigenous milk lactic acid bacteria (LAB) are able to develop stable biofilms providing starter and nonstarter cultures necessary for curd fermentation and cheese ripening, respectively. Hence, the present work was carried out with deproteinized whey to evaluate LAB biofilm formation on different woods derived from tree species grown in Sicily. Microbiological and scanning electron microscopy analyses showed minimal differences in microbial levels and compositions for the neoformed biofilms. The specific investigation of Salmonella spp., Listeria monocytogenes, Escherichia coli, coagulase-positive staphylococci (CPS), and sulfite-reducing anaerobes did not generate any colony for all vats before and after bacterial adhesion. LAB populations dominated all vat surfaces. The highest levels (7.63 log CFU/cm2) were registered for thermophilic cocci. Different colonies were characterized physiologically, biochemically, and genetically (at strain and species levels). Six species within the genera Enterococcus, Lactobacillus, Lactococcus, and Streptococcus were identified. The species most frequently present were Lactobacillus fermentum and Lactococcus lactis. LAB found on the surfaces of the wooden vats in this study showed interesting characteristics important for dairy manufacture. To thoroughly investigate the safety of the wooden vat, a test of artificial contamination on new Calabrian chestnut (control wood) vats was carried out. The results showed that LAB represent efficient barriers to the adhesion of the main dairy pathogens, probably due to their acidity and bacteriocin generation. IMPORTANCE This study highlights the importance of using wooden vats for traditional cheese production and provides evidence for the valorization the Sicilian forest wood resources via the production of dairy equipment

Evolution of the lactic acid bacterial biofilms on different wood typologies applied for the production of vats intended for cheese making

The present work was carried out to evaluate the lactic acid bacterial biofilms formation on wooden vat produced with seven different woods, derived from trees species grown in Sicily. The biofilm formation on the wooden surfaces was performed as reported by Gaglio et al. Microbiological and scanning electron microscopy analyses did not show differences in terms of microbial levels and composition within the neoformed biofilms (Fig.1). The specific investigation of Salmonella spp., Listeria monocytogenes, Escherichia coli and positive-coagulase staphylococci did not generate any colony for all vats before and after microbial activation. Lactic acid bacteria (LAB) populations dominated the surfaces of all vats and the highest concentration of approximately 7.63 Log CFU/cm2 was registered for thermophilic cocci. All colonies of different morphologies were isolated and characterized for their physiological and biochemical characteristics before being investigated genetically at strain level by RAPD-PCR and species level by a polyphasic approach consisting of 16S rRNA gene sequencing. As reported in a preview investigation conducted on wooden vat surfaces. we found species within the genera Enterococcus, Lactobacillus, Lactococcus, and Streptococcus. The species most frequently present were Lactobacillus fermentum and Lactococcus lactis. The technological characterization of the LAB found at high numbers on to the surfaces of the wooden vats showed interesting dairy properties. In order to deeply investigate on the safety of the wooden vat, a test of artificial contamination on new Calabrian chestnut vats was carried out showing that the acidity and the bacteriocin generated by LAB represent efficient barriers to their adhesion. This study highlights the importance to use the wooden vats for traditional cheese production and provides evidences for their safety in terms of bacterial community evolution

Microbial activation of wooden vats used for traditional cheese production and evolution of the neoformed biofilms

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese

Valorisation of Dairy Wastes Through Kefir Grain Production

The main aim of this work was to investigate on kefir grains (KGs) increase using different dairy by-products. To this purpose, whey and deproteinized whey from cow, goat and sheep milk were compared to the pasteurized milk of the corresponding animal species. Each substrate was inoculated with 3% (w/v) of KGs cultivated in ultra-high temperature cow milk and evalu- ated for pH decrease, total titratable acidity increase and development of lactic acid bacteria (LAB) and yeasts released in the matrices after 24 h incubation at three different temperatures (20, 25 and 30 \ub0C). The genotypic characterization of the dominating microbial populations resulted in the identification of two LAB belonging to the species Lactococcus lactis and Leuconostoc mesenteroides and one yeast strain within the species Kluyveromyces marxianus. Among whey based fermenta- tion media deproteinized sheep whey allowed the highest KGs increase (almost 50%). Results of the present investigations reveal the potential of the dairy wastes for KGs production at industrial level

Development of an ad hoc natural whey starter culture for the production of Vastedda della valle del Belìce cheese

This work was aimed to characterize the autochthonous lactic acid bacteria (LAB) of PDO Vastedda della valle del Belìce cheeses, produced in several dairy factories, for the development of an ad hoc starter culture preparation for the production of this cheese. To this purpose, winter and spring productions were analysed, in order to isolate LAB adapted to perform the fermentation at low temperatures. Plate counts showed the total microbial counts (TMC) till levels of 9 -1almost 10 CFU g and all cheese samples were dominated by coccus LAB. Not all samples were positive for the presence of enterobacteria, but when they were found their concentrations were at similar levels in both seasons. All colonies with different morphological appearance were isolated and differentiated on the basis of phenotypic characteristics and by randomly amplified polymorphic DNA (RAPD)- PCR analysis. A total of 65 strains were considered and subjected to the genotypic analysis by means of 16S rRNA gene sequencing, which identified 13 LAB species belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). The species most frequently found were Streptococcus macedonicus, Streptococcus thermophilus, Lactococcus lactis and Leuconostoc mesenteroides. The 65 strains were investigated in vitro for their general dairy aptitudes and some strains of the above four species showed technological traits relevant to act as starter strains for this cheese production. Among those strains, 12 LAB (Lactobacillus delbrueckii PON79, PON256 and PON405, Lactococcus lactis PON36, PON46 and PON203, Leuconostoc mesenteroides PON169, PON259 and PON559, Streptococcus thermophilus PON3, PON120 and PON261) were used in different combinations [all strains belonging to each species in triple combinations (Lb, lactobacilli; Lc, lactococci; Ln, leunostocs; St, streptococci), all thermophilic strains (Lb-St, lactobacilli and streptococci) and all mesophilic strains (Lc-Ln, lactococci and leuconostocs)] to produce experimental cheese by means of a dairy pilot plant. The different 7bacterial combinations (final concentration of approximately 10 CFU/mL) were tested in different conditions: 1, after growth in the optimal synthetic media, re- suspended in Ringer’s solution and inoculated in pasteurised ewes’ milk; 2, after growth in whey-based medium (WBM) and inoculated in pasteurised ewes’ milk; 3, after growth in WBM and inoculated in raw ewes’ milk. Plate counts and RAPD analysis were applied to monitor the bacterial evolution during the different trials, while pH was measured to follow their acidifying activities. All lactococci and leuconostocs were able to perform the rapid acidification of the curd in winter conditions; the experimentation was carried in February with 10°C as minimum ambient temperature registered in the room where the curd were left to acidify. A sensory evaluation of the resulting cheeses, obtained after stretching of the acidified curds, indicated the cheeses processed with lactococci in single and multiple combinations as those well appreciated by the judges. These cheeses were then subjected to the analysis of the volatile organic compounds carried out by gas chromatography coupled with mass spectrometry (GC/MS) which clearly showed substantial differences with the control cheese

Influence of the early bacterial biofilms developed on vats made with seven wood types on PDO Vastedda della valle del Belìce cheese characteristics

Early vat bacterial biofilms developed spontaneously through contact with whey have been characterized on seven wood types (Castanea sativa Miller, Cedrus libani, A. Rich., Prunus avium L., Fraxinus ornus L., Juglans regia L., Pinus nigra J.F. Arnold and Populus nigra L.). The present study aimed to evaluate the influence of these biofilms on the microbiological, chemical, physical and sensory characteristics of PDO Vastedda della valle del Belìce (VdB) cheese, processed traditionally from raw ewe's milk using wooden tools. To this purpose, the experimental cheeses after 15 d of refrigerated storage were examined. Lactic acid bacteria (LAB) populations dominated the microbial community of all samples. The species more frequently identified were Lactococcus lactis among starter LAB and Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum and Pediococcus pentosaceus among non starter LAB. Culture-independent analysis of microbiota diversity was performed by MiSeq Illumina that identified Streptococcus as major group followed by members of Enterobacteriaceae family, Lactococcus and Lactobacillus. Generally, the seven tree species did not negatively affect the physicochemical composition of VdB cheeses. Chestnut (both Sicilian and Calabrian) vats produced cheeses with significant lower hue angle (a*/b*) than other wood types. Among chemical parameters, significant variations were registered for a w , primary and secondary lipid oxidation state (significantly lower for the VdB cheeses produced with poplar wood), and volatile organic compounds (VOCs). The significant differences detected among the VOCs emitted from cheeses were not perceived by the panelists who recognized all cheeses from the different trials as similar. This study confirmed the suitability of cedar, cherry, ash, walnut, black pine and poplar as alternative woods to chestnut for the production of the wooden vats employed in cheese making for the Sicilian traditional dairy productions