Margherita Cruciata

Animal rennets as sources of dairy lactic acid bacteria.

ABSTRACT The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri, were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus lactis, Lactobacillus delbrueckii, and Streptococcus thermophilus, while the other strains, all belonging to the genus Enterococcus, could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.

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.

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms

ABSTRACT 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.

Microbiological investigation of Raphanus sativus L. grown hydroponically in nutrient solutions contaminated with spoilage and pathogenic bacteria.

The survival of eight undesired (spoilage/pathogenic) food related bacteria (Citrobacter freundii PSS60, Enterobacter spp. PSS11, Escherichia coli PSS2, Klebsiella oxytoca PSS82, Serratia grimesii PSS72, Pseudomonas putida PSS21, Stenotrophomonas maltophilia PSS52 and Listeria monocytogenes ATCC 19114(T)) was investigated in mineral nutrient solution (MNS) during the crop cycle of radishes (Raphanus sativus L.) cultivated in hydroponics in a greenhouse. MNSs were microbiologically analyzed weekly by plate count. The evolution of the pure cultures was also evaluated in sterile MNS in test tubes. The inoculated trials contained an initial total mesophilic count (TMC) ranging between 6.69 and 7.78Log CFU/mL, while non-sterile and sterile control trials showed levels of 4.39 and 0.97Log CFU/mL, respectively. In general, all inoculated trials showed similar levels of TMC in MNS during the experimentation, even though the levels of the inoculated bacteria decreased. The presence of the inoculums was ascertained by randomly amplified polymorphic DNA (RAPD) analysis applied on the isolates collected at 7-day intervals. At harvest, MNSs were also analyzed by denaturing gradient gel electrophoresis (DGGE). The last analysis, except P. putida PSS21 in the corresponding trial, did not detect the other bacteria, but confirmed that pseudomonads were present in un-inoculated MNSs. Despite the high counts detected (6.44 and 7.24CFU/g), only C. freundii PSS60, Enterobacter spp. PSS11 and K. oxytoca PSS82 were detected in radishes in a living form, suggesting their internalization.

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

ABSTRACT 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 of the Sicilian forest wood resources via the production of dairy equipment.

Optimization of environmental conditions for kefiran production by kefir grain as scaffold for tissue engineering

Optimization of Environmental Conditions for Kefiran Production by Kefir Grain as Scaffold for Tissue Engineering Salvatore Montesanto*, Giuseppa Calò, Margherita Cruciata, Luca Settanni, Valerio B. Brucato, Vincenzo La Carrubba a Department of Civil, Environmental, Aerospace, Materials Engineering (DICAM)University of Palermo, Viale delle Sicenze Ed. 8, 90128 Palermo, Italy. b Department of Science and Biological Thecnologies, Chemicals and Farmaceutics (STEBICEF)University of Palermo, Viale delle Sicenze Ed. 16, 90128 Palermo, Italy. c Department of Agricultural and Forestry Science, University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy salvatore.montesanto1985@gmail.com

Ecology of Vastedda della valle del Belìce cheeses: a review and recent findings to stabilize the traditional production

ABSTRACT The increasing request for classic foods has led to a higher demand for cheeses enjoying a “recognition of quality” status. This phenomenon has also affected Sicilian dairy products, in particular protected designation of origin (PDO) “Vastedda della valle del Belìce” (VdB), a cheese traditionally available only during the summer season, but requested year round. The variations of the microbial populations of raw milk among the seasons influence the microbiological quality of the cheeses produced. This review article reports the historical importance of cheeses and the traditional equipment used for dairy productions in Sicily with a specific focus on VdB. The microbial ecology of this cheese is thoroughly discussed. The recent findings to stabilize the traditional VdB production protocol with regards to the microbial composition and the organoleptic characteristics of the final cheeses are reviewed. The selection of starter lactic acid bacteria from PDO cheeses, their in vitro and in vivo application, and, overall, the setup of their inoculation, have all generated a novel production protocol. The innovation was respectful of the traditional discipline, essentially based on the ad hoc biofilm formation on the traditional wooden equipment, and allowed the preservation of VdB typicality. Future prospects are summarized.

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

In order to help laboratories to face with their regulatory requirements, BIPEA (Bureau Interprofessionnel d’Études Analytiques) developed a production of external reference materials (ERM) for microbiology in food. These samples should allow laboratories to check the trueness of their results at any time, outside the regular proficiency-testing schemes (PTS). For this purpose, stable and homogeneous samples of minced meat spiked with various bacterial strains are produced by BIPEA: Escherichia coli, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes, at levels between 102 to 104 microorganisms per gram. Controls were performed by analysing samples all along the production process, at the beginning of the study and regularly during a 6-month period. The homogeneity was checked by calculating coefficients of variation, which were inferior to 25 % for all the analytical parameters. The stability was characterized by comparing means of three samples to the mean obtained at the beginning of the study. The samples produced were thus considered as being sufficiently homogeneous and stable to meet the ERM requirements: the results of enumeration of the different micro-organisms present in the minced meat after 6 months of storage at (-24 ± 6)°C showed a good stability, with a maximum deviation less than 0.5 log (CFU/g: colony forming unity per gram). ISSN 2576-0564 Research Article Citation: Marie de BORT, Elvire MESSINEO, Romain LE NEVE, et al. Production of External Reference Materials in Food Microbiology. Int J Microbiol Infect Dis. 2018; 2(1): 1-3.In this study, essential oil of Sahara Nigella sativa L. was extracted using a rapid extraction, the microwave steam distillation (MSD) and the cryogenic grinding (CG). Two procedures have been investigated, the MSD1 (seeds inside of oven apparatus) and MSD2 (seeds outside of oven apparatus). Forty-six compounds were identified and significant differences in quantities of the major constituents were observed, mainly were thymoquinone (CLG: 331.82-443.55 mg and CG: 272.95413.57 mg/100 g of seeds), p-cymene (CLG: 181.71-244.17 mg, CG: 369.80- 374.40 mg/100 g of seeds), dehydro-sabina ketone (CLG: 24.60- 25.83 mg, GC: 44.02-50.69 mg/100g of seeds), carvacrol (CLG: 10.32-10.96 mg, CG: 3.91-12.67 mg/100 g of seeds) and longifolene (CLG: 11.9016.43 mg, CG: 12.72-19.58 mg/100 g of seeds).Results showed that essential oils exhibit a good activity in each antioxidant system with a special attention for β-carotene bleaching test (IC50: 21 to 27 μg/ml) and reducing power (EC50: 9 to 14 μg/ml).The N. Sativa essential oils exhibited higher antibacterial and antifungal activities varying according to technique extraction and grinding mode used, with a high effectiveness against Gram-positive bacteria with a diameter of inhibition zones growth ranging from 9.5 to 35 mm and MIC and MBC values ranging from (0.042–0.10 mg/ml) to (0.20–0.75 mg/ml), respectively.T mold growth leading physical, chemical and sensory changing in food products and carrying health risks with toxin production is a main problem in the food industry. In our study, as an alternative solution, bioactive materials were developed by incorporating Williopsis saturnus var. saturnus at different concentrations (0; 3.2; 6.8 and 8.8 cfu/cm2) into whey protein concentrate (WPC) based edible films. Viability of W. saturnus var. saturnus in the films was investigated during the time of the storage. Moreover, the antifungal activity of the films incorporated with W. saturnus var. saturnus was also tested against Penicillium expansum and Aspergillus niger. The effect of the antagonist yeast occurrence in the film on physical properties and microstructural properties of the films were analyzed. The optical properties and the mechanical properties of the film samples were also observed by SEM and Instron, respectively. The results showed that bioactive WPC films containing 8.8 log cfu/cm2 W. saturnus var. saturnus were able to maintain more than 60% of the initial antagonist yeast population. In addition, the films incorporated with the antagonist yeasts reduced mycelium growth of P. expansum and A. niger on the medium by more than 29% and 19% at pH 4.5, respectively. Incorporation of more than 3.2 log cfu/cm2 antagonist yeasts significantly increased water vapor permeability of the films (P<0.05). Increased population of the yeasts also significantly increased percent water solubility of the films (P<0.05). Moreover, the distribution of the antagonistic yeasts in the films presented homogenous microstructure at SEM. Increasing concentration of the killer yeasts in the films presented a significant tendency to greenness and yellowness as well as lightness values (P<0.05). The concentrations more than 3.2 log cfu/cm2 of killer yeast population in the films significantly increased tensile strength but decreased percent elongation at break (P<0.05).L are rods belonging to the Lactic Acid Bacteria (LAB), and includes: Gram-positive, catalase negative, non-motile, non-sporulating, facultative anaerobes, growing under microaerophilic to strictly anaerobic conditions. Recently, some research has been focused for selected strains from goat’s milk with a technological potential and safety, susceptible to be used as adjunct cultures in dairy products strains intended for the use in food systems as starters or probiotics adjunct culture should therefore be carefully examined following important criteria. This study is a widespread of our research that has of objective to identify and study in vitro the technological traits heterogeneity and their safety aspect of lactobacilli strains isolated from goats milk samples collected in different regions of Algeria. Using API50 CHL system and 16S rDNA sequencing, 51 % of strains were assigned as L. plantarum, 34 % as L. pentosus, 7 % as L. rhamnosus and 8 % as L. fermentum. A large variability was noted for the acidifying capacity in skimmed milk after 6, 12 and 24 h of incubation. All strains expressed aminopeptidase activity against alanine-ρ-NA and leucine-ρ-NA at different levels. All strains were resistant to vancomycin and most of strains showed more susceptibility to β-lactam antibiotic. High susceptibility toward the inhibitors of protein synthesis was also observed. Minimum inhibitory concentrations data obtained revealed that isolates were susceptible to penicillin and chloramphenicol, and resistant to gentamicin and vancomycin. Minimum inhibitory concentration distribution of other antibiotics showed variability. The analysis of graphical representation of principal component analysis of technological properties of L. plantarum and L. pentosus strains showed diversity among the isolates. Finally, eight L. plantarum, four L. pentosus and two L. rhamnosus strains, could be good candidates as adjunct culture in dairy product in Algeria.Background: The realization that stress alters brain–gut interaction which is known to play a critical role in health and disease is rapidly advancing. In a stress model, we aimed at evaluating the effect of fermented maillard reaction product treatment on intestinal barrier and inflammation. Moreover, the fermentation of MRP by Lactobacillus rhamnosus 4B15 synergistically enhanced the above effect compared to those of unfermented MRP.Methodology & Theoretical Orientation: A total of 129 samples, consisting in 123 samples of mussels (Mytilus galloprovincialis) and 6 oysters (Crassostrea cornucopiae), were purchased from outlets in the city of Murcia (southeast Spain). The isolation of C. difficile strains was carried out using enrichment broth suplemented with sodium taurocholate followed by etanol shock, prior to the culture on a selective media. The identification was carried out detecting Tpi gene using molecular techniques. The isolation and enumeration of bacterial indicators were investigated according to the ISO norm.Background: The realization that stress alters brain-gut interaction which is known to play a critical role in health and disease, including neuropsychiatric disorders, is rapidly advancing. Here we tested whether fermented Maillard reaction product (f-MRP) treatment modifies behavior across domains relevant to anxiety, depression, cognition, stress response, and social behavior. Furthermore, the fermentation of MRP by Lactobacillus rhamnosus 4B15 synergistically enhanced the above activities compared to those of unfermented MRP.