Roberta Nuvoloni

Experimental infection of dogs with Borrelia burgdorferi

Four beagle dogs were inoculated subcutaneously with the BITs1 Italian strain of Borrelia burgdorferi. Only one dog became infected and B burgdorferi was isolated from its blood and urine three and four weeks after infection. B burgdorferi antibodies were detected by immunofluorescence from four to 11 weeks after infection. An uninoculated dog kept in the same run as the infected dog, developed a positive serological response, but none of the five dogs showed clinical signs.

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 Hashimotos 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 1.8×10(4)Map 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.

Occurrence of Vancomycin-Resistant Enterococci in Foods of Animal Origin Purchased in Tuscany

Enterococci are ubiquitous microorganisms, commonly found in plants, insects and wild animals, and are normal residents of the gastrointestinal tract of both animals and humans. They frequently occur in large numbers in certain foods, especially those of animal origin. Over the last 10 years, enterococci have emerged as major nosocomial pathogens, thus representing an increasingly important problem for public health, in relation to their ability to acquire resistance to some antimicrobial agents of relevance to human therapy, such as glycopeptides, particularly vancomycin. In this regard, the food chain represents a possible source of vancomycin-resistant enterococci for humans, mainly as a result of the use of avoparcin, a vancomycin-like glycopeptide, as a feed additive for growth promotion of farm animals, as undoubtedly established by previous studies (Bates, 1997; Mc Donald et al., 1997). Although the utilization of avoparcin in animal husbandry has been banned since 1997, VRE have recently been isolated from foods of different typology (Giraffa et al., 2000; Knijff et al., 2002), therefore investigation into the frequency of recovery of VRE at this level is still recommendable. Thus, the present survey was carried out with the aim of evaluating the occurrence of VRE in foods of animal origin purchased in Tuscany, considering that these microorganisms could play a relevant role both directly as potential pathogens for humans, as well as in relation to their capability to spread antibiotic resistance genes to other pathogenic bacteria, such as Staphylococcus aureus.

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.

Fatty acid and microbiological profile of the meat (longissimus dorsi muscle) of wild boar (Sus scropha scropha) hunted in Tuscany

Abstract The aim of this work is to study the influence of gender, age and hunting period on the fatty acid composition of the longissimus dorsi muscle excised from 42 wild boars hunted on a farm located in Tuscany. A sub-sample of 22 muscles was used for the study of the microbiological profile of wild boar meat. The results show that gender had an effect only on C16:1 n7, which was higher in females (p ≤ .05). Slaughtering age influenced more parameters, thus resulting in a higher content of C6:0, C18:2, C20:1 and n6/n3 ratio in meat from adults (p ≤ .05) and in a higher percentage of C16:0 (p ≤ .05) in meat from young animals. The hunting month greatly influenced the meat quality because, in addition to the single fatty acids, it significantly modified the MUFA, PUFA, PUFA/SFA and n6/n3 ratio, underlining the great difference between meat of wild boar shot in October – November vs December – January. Ether extract was higher in sub-adults than in young animals (p ≤ .01) but was not influenced by gender and hunting month. Regarding the microbiological analyses, Salmonella spp. was only detected in one sample, while Yersinia enterocolitica was not detected at all. Data on the presence of pathogenic bacteria confirmed health risk for the consumer comparable to that associated with meat obtained from farm animals, while data on microbial loads (total aerobic mesophilic and psychrotrophic counts, Enterobacteriaceae, Escherichia coli) did not highlight any specific criticality.

Biogenic Amines Content of Four Types of “Pecorino” Cheese Manufactured in Tuscany

Biogenic amines content of four types of Tuscan ewes’ milk “pecorino” cheese was evaluated using HPLC-UV analysis. All cheeses were manufactured in the same dairy factory with different combinations of milk (raw or pasteurized) and type of ripening. Total biogenic amines and tyramine levels of a raw milk “pecorino” ripened five months, partly in a traditional cave, were significantly higher than those of a pasteurized milk “pecorino” with a similar ripening; and of a two months raw milk “pecorino” ripened in the dairy plant. No statistical significant difference was found when comparing total biogenic amines and tyramine contents of the same five month ripened raw milk “pecorino” with a pasteurized milk “pecorino” ripened six months, partly in a traditional “fossa.” In raw milk cave-ripened and “fossa”-ripened cheeses, total biogenic amines exceeded 1000 mg/kg. In cheeses manufactured with raw milk and/or in particular ripening environments, specific hygienic cares are needed to limit biogenic amines formation.

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.

Improving pork burgers quality using Zingiber officinale Roscoe powder (ginger)

Pork burgers were evaluated for physical-chemical characteristics, fatty acids profile, lipid oxidation, antioxidant capacity, microbiological growth and sensory evaluation during storage time of seven days at 4°C as function of three formulations as only meat (control, B) and meat added with ginger powder at the percentage of 1 and 2% (BG1 and BG2). BG1 and BG2 were less redness than control ones with incremented yellow hue. These modifications in color parameters did not modify sensory characteristics of burgers. PUFA were incremented (both PUFAω3 and PUFAω6) by the addition of ginger. Furthermore, BG1 and BG2 burgers showed to be less sensitive to lipid oxidation and to possess an increase in antioxidant capacity. Microbial growth evaluation of total aerobic count and Pseudomonas spp. showed that ginger powder delayed in time the bacterial contamination. Results highlighted that the presence of ginger led to an enhanced shelf life and health characteristics of burgers (increasing peroxidisability, ratio hypocholesterolemic/hypercholesterolemic and ratio ω3/ω6; reducing atherogenicity and thrombogenicity).

Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage. Lysozyme resistance and the ability to acidify donkey milk were chosen as main selection parameters. Different fermented beverages (C1-C9) were produced, each with a specific combination of isolates, and stored at refrigerated conditions for 35days. The pH values and viability of the isolates were weekly assessed. In addition, sensory analysis was performed. Both S. thermophilus and L.plantarum showed a high degree of resistance to lysozyme with a Minimum Bactericidal Concentration>6.4mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24h at 37°C, with an average ΔpH value of 2.91±0.16 and 1.78±0.66, respectively. Four L. plantarum and two S. thermophilus were chosen for the production of fermented milks. Those containing the association S. thermophilus/L. plantarum (C1-C4) reached a pH lower than 4.5 after 18h of fermentation and showed microbial loads higher than 7.00logcfu/mL until the end of the storage period. Moreover, comparing the microbial loads of samples containing both species and those containing S. thermophilus alone (C5), we highlighted the ability of L. plantarum to stimulate S. thermophilus replication. This boosted replication of S. thermophilus allowed to reach an appropriate pH in a time frame fitting the production schedule. This was not observed for samples containing a single species (C5-C9). Thus, L. plantarum strains seem to be good candidates in the production of a novel type of fermented milk, not only for their probiotic potential, but also for the enhancing effect on S. thermophilus growth.

Antimicrobial activity of four essential oils against pigmenting Pseudomonas fluorescens and biofilmproducing Staphylococcus aureus of dairy origin

Essential oils (EOs) are mixtures of secondary metabolites of plant origin with many useful properties, among which the antimicrobial activity is also of interest for the food industry. EOs can exert their antimicrobial potential both directly, in food products and active packaging, and indirectly, as sanitizing and anti-biofilm agents of food facility surfaces. Aim of this research was to evaluate the antimicrobial activity of four EOs (bergamot, cinnamon, manuka and thyme) against Pseudomonas fluorescens and Staphylococcus aureus isolated from milk and dairy products. The chemical composition of EOs was evaluated by Gas Chromatography-Mass Spectrometry analysis. Minimum Inhibitory Concentration values were determined by a microplate method against 9 Ps. fluorescens from marketed mozzarella with blue discoloration defect, and 3 biofilm-producing S. aureus from milk. Reference ATCC strains were included. Pigment production activity by Ps. fluorescens was assessed both in culture and in cheese. EOs of manuka (leptospermone 23%) and thyme (carvacrol 30%, pcymene 20%, thymol 15%) showed the highest antimicrobial activity against S. aureus, MIC values were 0.012%-0.024% and 0.024% v/v, respectively; meanwhile EOs from thyme and cinnamon (cinnamaldehyde 55%) exhibited the best activity against Ps. fluorescens with MIC values of 0.098%-0.195% and 0.195%-0.391% v/v, respectively. The antimicrobial activity of these EOs is promising and they could be exploited in the dairy production chain.