Valutazione della sicurezza e del potenziale tecnologico di batteri lattici isolati da pecorini tradizionali prodotti in Toscana

Il presente lavoro ha riguardato 54 ceppi di batteri lattici isolati da pecorini tradizionali toscani, prodotti a latte crudo, senza l’impiego di colture starter commerciali. I ceppi in esame erano suddivisi in 28 isolati appartenenti al genere Lactobacillus e 26 isolati appartenenti al genere Lactococcus. Nello specifico i ceppi appartenevano alle seguenti specie: Lc. lactis subsp. lactis (n=16), Lc. lactis subsp. cremoris (n=8), Lc. raffinolactis (n=2), Lb. paracasei (n=12) e Lb. plantarum (n=16). I ceppi ATCC334, ATCC20174 e ATCC19435 sono stati utilizzati come ceppi di referenza. Lo scopo della tesi è stato quello di caratterizzare, nella maniera il più completa possibile, tali microrganismi in modo da poterli utilizzare per la realizzazione di prodotti lattiero-caseari tradizionali. I ceppi selezionati sono stati dapprima sottoposti a valutazione del profilo di antibiotico-resistenza mediante metodiche fenotipiche (diffusione su agar e microdiluizioni). Gli antibiotici presi in considerazione sono stati: ampicillina, vancomicina, gentamicina, kanamicina, streptomicina, eritromicina, clindamicina, tetraciclina e cloramfenicolo. I ceppi resistenti sono stati sottoposti a PCR per la ricerca dei geni codificanti antibiotico-resistenza. Gli isolati sono stati successivamente sottoposti a caratterizzazione tecnologica (valutazione dell’attività acidificante, valutazione dell’attività proteolitica, resistenza a cloruro di sodio, resistenza al calore) e valutazione del profilo metabolico mediante sistemi miniaturizzati API. Infine, alcuni ceppi sono stati selezionati per un’indagine relativa alla biodiversità genotipica attraverso la metodica del Multilocus Sequence Typing (MLST). In accordo con la recente bibliografia, il presente studio ha messo in evidenza che il fenomeno di antibiotico-resistenza risulta essere globalmente contenuto nell’ambito delle flore lattiche. Le maggiori percentuali di resistenza fenotipica sono state riscontrate per il genere Lactobacillus nei confronti di cloramfenicolo (66,66%), tetraciclina (46,66%), streptomicina (23,07%) e kanamicina (6,6%). Per quanto riguarda Lactococcus una bassa percentuale di ceppi resistenti è stata invece riscontrata per tetraciclina (11,11%). E’ stato possibile osservare un fenotipo resistente accompagnato da un riscontro positivo anche per la presenza di geni codificanti la resistenza nel 5,5% della popolazione studiata e relativamente a tetraciclina (3 Lc. lactis subsp. cremoris positivi per il gene tet(M)). Per quanto riguarda Lactobacillus spp è stata evidenziata una discrepanza tra i risultati ottenuti tramite metodo di Kirby-Bauer e metodo delle microdiluizioni, probabilmente attribuibile alla mancanza di corretti parametri per l’interpretazione dei risultati ottenuti mediante metodo di Kirby-Bauer e alla mancanza di valori di cut off uniformati per i due metodi fenotipici utilizzati. Le attività acidificanti e proteolitiche hanno presentato trend simili nell’ambito delle specie considerate: Lb. paracasei e Lc. lactis subsp. lactis hanno mostrato attività maggiori, mentre Lb. plantarum e Lc. lactis subsp. cremoris si sono mostrate globalmente meno acidificanti e meno proteolitiche, seppur con alcune eccezioni. Per quanto riguarda la resistenza a temperatura, tutti i ceppi sono stati in grado di tollerare 40°C e 43°C, solamente 2 Lc. lactis subsp.cremoris e 1 Lb. parcacasei tolleravano 45°C. Relativamente alla resistenza al sale tutti lattobacilli tolleravano 4,5% e 6% NaCl, mentre solo 8 ceppi erano in grado di sviluppare in presenza del 6,5% di NaCl. I lattococchi sono risultati essere invece sensibili già al 4,5% di NaCl, solo 8 ceppi sono stati infatti in grado di crescere, seppur debolmente al 6% NaCl. Sono stati, inoltre, osservati diversi profili API: per quanto riguarda Lb. plantarum tra i 16 ceppi studiati sono stati evidenziati 5 profili diversi; per i 12 ceppi Lb. paracasei sono stati osservati 7 profili; per i 16 ceppi di Lc. lactis subsp lactis 7 profili; per gli 8 ceppi di Lc. lactis subsp cremoris 5 profili ed infine i due ceppi di Lc. raffinolactis presentavano 2 diversi profili biochimici. L’analisi attraverso MLST ha rappresentato un valido strumento per raggiungere un’identificazione a livello di ceppo. Sono stati osservati ceppi con fenotipo diverso, ma al contempo dotati di uno stesso profilo ST. Al contrario ceppi che avevano caratteristiche metaboliche e tecnologiche sovrapponibili hanno presentato poi un genotipo diverso. La maggiore biodiversità genotipica è stata osservata tra i ceppi appartenenti alla specie Lb. paracasei (9 ceppi, 7 profili ST), mentre per Lc. lactis è stata osservata una minore variabilità (18 ceppi, 10 profili ST). Questa metodica consente quindi di poter eliminare eventuali ridondanze a livello di ceppoteca e poter offrire ai produttori dei ceppi diversi da utilizzare in programmi di rotazione in caseificio, anche in previsione di un’eventuale infezione fagica

Beeswax: A minireview of its antimicrobial activity and its application in medicine

Abstract Beeswax is the substance that forms the structure of a honeycomb; the bees secrete wax to build the honeycombs where to store honey. Thanks to its rich hydrophobic protective properties, the beeswax is in fact present within cosmetics and body products. Also, beeswax is used in the food industry: as a film to wrap cheese for maturing or as a food additive (E901) to give shine to the products. Exactly as the honey which it contains, beeswax is also characterized by several therapeutic properties of great interest to us; it is thought to be particularly effective in healing bruises, inflammation and burns. Recently, the interest of researchers has moved even on antimicrobial properties of beeswax although there are still few studies in the literature focused only on the action of beeswax. The few studies showed an antimicrobic effectiveness of beeswax against overall Staphylococcus aureus , Salmonella enterica , Candida albicans and Aspergillus niger ; these inhibitory effects are enhanced synergistically with other natural products such as honey or olive oil. This minireview aims to be a collection of major scientific works that have considered the antimicrobial activity of beeswax alone or in combination with other natural products in recent years

Qualitative and quantitative evaluation of biogenic amines in vitro production by bacteria isolated from ewes' milk cheeses

Studying the production of biogenic amines (BA) by cheese microbiota is important, because high BA concentrations in food represent a health risk for consumers. Qualitative screening with differential media and HPLC quantification were used to investigate the production of 8 BA (2-phenylethylamine, cadaverine, histamine, putrescine, spermidine, spermine, tryptamine, and tyramine) by 72 isolates from curd and cheese samples manufactured with raw and pasteurised ewes' milk. Enterobacteriaceae showed good putrescine and cadaverine production, both for number of positive isolates and for concentrations produced (average: 341 and 785 micrograms/ml, respectively). Among Enterobacteriaceae data are provided on BA formation by Pantoea conspicua, previously not isolated from food samples. All enterococci formed tyramine, often in high amounts (average: 1608 micrograms/ml), and many produced notable 2-phenylethylamine, putrescine, and cadaverine concentrations (average: 184, 121, and 146 micrograms/ml, respectively). BA formation by lactobacilli was overall extremely limited, with the notable exception of high tyramine concentrations produced by 1 Lactobacillus paracasei (800 micrograms/ml) and 2 Lactobacillus curvatus (> 1700 micrograms/ml), all isolated from pasteurised milk samples. Thus, undesired and technologically useful microorganisms both play a role in BA accumulation in cheeses

Molecular survey of tick-borne pathogens in Ixodid ticks collected from hunted wild animals in Tuscany, Italy

Objective: The aim of the present study was to determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. Methods: PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. Results: A total of 432 ticks were collected: 30 (6.94%) Haemaphysalis punctata, 72 (16.7%) Dermacentor marginatus and 330 (76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58 (75.32%) resulted infected and among them 14 (18.18%) showed co-infections. In particular, 29 (37.66%) pools were positive for Bartonella spp., 23 (29.87%) for A. phagocytophilum, 16 (20.78%) for Rickettsia spp., and 5 (6.49%) for B. burgdorferi s.l. All samples were negative for C. burnetii. Conclusion: The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks

Caciotta della Garfagnana cheese: selection and evaluation of autochthonous mesophilic lactic acid bacteria as starter cultures

The aim of this study was to isolate, identify and select, with respect to acidification and proteolytic activities, the autochthonous mesophilic lactic acid bacteria (LAB) present in milk and Caciotta della Garfagnana, a cheese produced either with raw or thermised cow's milk in small dairies and family plants of Garfagnana (Tuscany), to obtain LAB strains with attributes suitable to be employed as starter cultures in this type of cheese, particularly when thermised milk is used to control spoilage microflora. Samples of raw milk, curd and cheese were collected from three representative farmers of the production area and used to isolate autochthonous LAB. Phenotypic and genotypic (species-specific PCR assay) identification of isolated LAB was done. Twenty-eight strains of LAB isolated from milk, curd and cheese were screened for acidifying and proteolytic activities. LAB strains with the better attributes were used as mesophilic starter cultures in technological trials: experimental cheeses manufactured with the addition of autochthonous LAB and control cheeses were compared for LAB and pH evolution. Experimental cheeses presented a significant increase in the mesophilic lactic acid microflora up to 14 days of ripening and significantly lower pH values up to seven days of ripening. The use of wild selected mesophilic lactic acid bacteria, together with thermisation of milk, for the Caciotta della Garfagnana looks very promising and could help to both standardise the production and improve quality and traditional characteristics of this type of cheese

Nalaz leptospira u nutrije (Myocastor coypus) i štakora selca (Rattus norvegicus) koji nastanjuju zaštićeno močvarno područje u Toskani u Italiji.

From September 2009 to February 2011, 122 coypus (Myocastor coypus) and 74 rats (Rattus norvegicus) were captured employing cage traps in a protected wetland in Tuscany (central Italy). Blood serum samples were collected from the animals and successively examined by the microagglutination test for several serovars of Leptospira: Bratislava, Ballum, Bataviae, Grippothyphosa, Icterohaemorrhagiae, Copenhageni, Mini, Pomona, Zanoni, Sejroe, Hardjo and Tarassovi. Kidney samples were collected from each animal and tested by bacteriological methods and submitted to Polymerase Chain Reaction. Thirty-four (27.87 %) coypu sera were positive to Leptospira interrogans serovar Bratislava, with antibody titers ranging from 1:100 to 1:400; no strain was isolated from coypu by bacteriological examination, while 12 (9.83 %) subjects were positive to PCR. All rats resulted seronegative; thirty-seven (50 %) Leptospira strains were isolated from rat kidneys; 30 were classified as Leptospira interrogans serovar Icterohaemorrhagiae and 7 as Leptospira interrogans serovar Ballum by the cross-agglutination test. Forty-five (60.81 %) rats resulted positive to PCR: 37 subjects positive to bacteriological examination and there were eight from which no strain were isolated from kidneys. These results would seem to suggest the minor zooepidemiological role of coypu in leptospirosis, which is widespread according to literature.Od rujna 2009. do veljače 2011. u zamke su bile uhvaćene 122 nutrije (Myocastor coypus) i 74 štakora selca (Rattus norvegicus) u zaštićenoj močvari u Toskani u središnjoj Italiji. Prikupljeni uzorci krvnog seruma bili su pretraženi mikroaglutinacijskim testom na nekoliko serovarova leptospira: Bratislava, Ballum, Bataviae, Grippothyphosa, Icterohaemorrhagiae, Copenhageni, Mini, Pomona, Zanoni, Sejroe, Hardjo i Tarassovi. Uzorci tkiva bubrega, uzeti od svake životinje, bili su pretraženi bakteriološki i lančanom reakcijom polimerazom. Tridesetčetiri (27,87 %) uzorka seruma nutrije bila su pozitivna na vrstu Leptospira interrogans serovar Bratislava s titrom protutijela od 1:100 do 1:400. Bakteriološkom pretragom iz nutrija nije bio izdvojen nijedan izolat, dok je 12 uzoraka (9,83 %) bilo pozitivno PCR-om. Svi štakori bili su serološki negativni, ali je 37 (50 %) izolata leptospira bilo izdvojeno iz tkiva njihovih bubrega. Od toga je 30 izolata pripadalo vrsti Leptospira interrogans serovaru Icterohaemorrhagiae, a 7 serovaru Ballum pretragom križnim aglutinacijskim testom. Ukupno su 45 štakora (60,81 %) bila pozitivna pretragom PCR-om, a 37 bakteriološkom pretragom. Od osam štakora nije bio izdvojen nijedan izolat leptospira iz bubrega. Ti rezultati upućuju na neznatnu zooepidemiološku ulogu nutrija u širenju leptospiroze

Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms.Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×104Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR

Short communication: Phenotypic and genetic diversity of wild Lactococcus lactis isolated from traditional Pecorino cheeses of Tuscany

Wild Lactococcus lactis isolates from traditional Pecorino cheeses in 4 regions of Tuscany were isolated and characterized to evaluate the diversity of autochthonous lactococci. Sixty strains of Lactococcus were clustered by the results of carbohydrate utilization and diagnostic enzyme activity. Twenty-one unique strains were then chosen for characterization of salt and temperature tolerance, as well as acidification and proteolytic activity in milk. Genetic analysis of these strains was performed via 16S ribosomal DNA sequencing and multilocus sequence typing (MLST) to elucidate diversity relative to their location of origin. Phylogenetic analysis showed distinct clustering by region within organism subspecies, and phenotypic properties demonstrated concomitant trends. Multilocus sequence typing thus allowed for the regional distinction of isolates separate from those of previous works, supporting the concept that distinctive regional qualities of cheeses are strongly influenced by microbial ecology

Staphylococcus aureus growth and enterotoxin production in Italian caciotta cheese

To evaluate growth and enterotoxin production of Staphylococcus aureus in raw milk caciotta, an Italian soft cheese, experimental cheesemaking trials were carried out with and without a commercial starter. Two different inocula of selected enterotoxinproducing S. aureus strains were tested: high (mean value 5.03 log CFU/mL of milk) and low (mean value 3.22 log CFU/mL of milk). Uninoculated cheeses with milk containing 2.15 log CFU/mL mean value of coagulase-positive staphylococci were also examined. With higher inocul a coagulase-positive staphylococci counts reached 7.57 log CFU/g without starter and had significantly higher values after the acidification phase than those with starter (below 6.5 log CFU/g). Using an enzyme immunoassay, enterotoxins were detected in cheeses both without and with starter, but the latter showed significantly lower photometrical absorbance values than the former. In low inoculum and uninoculated cheeses without starter, coagulase-positive staphylococci maximum values were between 5 and 6 log CFU/g, without enterotoxin production. To enhance the safety of this cheese it is fundamental to strictly prevent coagulase-positive staphylococci contamination and multiplication, particularly at the primary production level, and to correctly acidify the curd by starter cultures, which can substantially limit total coagulase-positive staphylococci increase

Microbiological and physicochemical profile of traditional <i>Salsicda toscana</i> during storage

The aim of this study was to evaluate the evolution during refrigerated storage of the main microbiological and physicochemical parameters of a traditional Italian fresh sausage, salsiccia toscana. The following parameters were analyzed at Days 0, 6 and 9 of storage: total aerobic mesophilic and psychrotrophic counts, Brochothrix thermosphacta, lactic acid bacteria, Pseudomonadaceae, Enterobacteriaceae, Escherichia coli, Micrococcaceae, yeasts and moulds, Salmonella spp., Listeria monocytogenes; pH, Aw, moisture, protein, total lipids, ash, fatty acid composition, Thiobarbituric Acid Reactive Substances, colour measurement. The results revealed a statistically significant increase in the main microbiological parameters between Days 0 and 9 of refrigerated storage, with total bacterial counts exceeding 107 CFU/g after nine days and Brochothrix thermosphacta representing the main spoilage microorganism. Among physicochemical parameters, discolouration was noted during the time of storage, with a significantly paler colour on the surface of sausages with casings after nine days of storage. Considering that the initial bacterial counts were higher than 105 CFU/g for Brochothrix thermosphacta, Pseudomonadaceae, lactic acid bacteria and Enterobacteriaceae, and that the product is traditionally purchased loose without any packaging, it is fundamental to improve the good manufacturing practices, particularly measures to control processing temperatures