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

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 form..

Prevalence, phenotypic and genetic diversity of Campylobacter in poultry fresh meat and poultry products on retail sale in Tuscany (Italy)

In this study, the prevalence of Campylobacter spp. in poultry fresh meat and ready‑to‑cook products was evaluated. Seventy‑three samples were collected at retail level from supermarkets and discount stores, obtaining 61.6% positivity. Of 133 Campylobacter isolates, 86 strains (Campylobacter coli, 58.1% and Campylobacter jejuni, 41.9%) were selected for characterisation on the basis of their SmaI and kpnI pulsed field gel‑electrophoresis (PFGE) profiles, to exclude clonal replicates. Campylobacters resulted highly resistant to tetracycline, ciprofloxacin, and nalidixic acid (79.1%, 72.1% and 65.1%, respectively); 50% of C. coli and 13.9% of C. jejuni were resistant to ciprofloxacin and erythromycin, the most important antimicrobials for human campylobacteriosis therapy. Five C. coli were resistant to 5/7 of the tested antimicrobials. HS4c was the prevailing C. jejuni serotype group (22.3%), whereas 8 other serotypes were identified in low percentages. SmaI and kpnI profiles showed a wide variability. The survey showed a high Campylobacter contamination of poultry meat and poultry products at retail level in Tuscany, Italy. A wide strains’ heterogeneity and a remarkable level of strains’ antimicrobial resistance have been reported, confirming the need for an improvement of specific preventive measures along the production chain

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

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

In Vitro Antibacterial Activity of Manuka (Leptospermum scoparium J.R. et G. Forst) and winter Savory (Satureja montana L.) Essential Oils and Their Blends against Pathogenic E. coli Isolates from Pigs

Neonatal diarrhoea (ND), post-weaning diarrhoea (PWD) and oedema disease (OD) are among the most important diseases affecting pig farming due to economic losses. Among the main aetiological agents, strains of Escherichia coli are identified as the major responsible pathogens involved. Several strategies have been put in place to prevent these infections and, today, research is increasingly studying alternative methods to antibiotics to reduce the antibiotic resistance phenomenon. Essential oils (EOs) are among the alternative tools that are being investigated. In this study, the in vitro effectiveness of winter savory and manuka essential oils and their mixtures in different proportions against strains of E. coli isolated from episodes of disease in pigs was evaluated. The EOs alone demonstrated slight antibacterial effectiveness, whereas the blends, by virtue of their synergistic action, showed remarkable activity, especially the 70%–30% winter savory–manuka blend, showing itself as a potential tool for prevention and therapy

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 35 days. 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 &gt; 6.4 mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24 h 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 18 h of fermentation and showed microbial loads higher than 7.00 log cfu/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 biofilm-producing 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 mik 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%, p-cymene 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 (cinnamaldheyde 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 coul be exploited in the dairy production chain

BIOGENIC AMINE CONTENT IN "PECORINO DEL PARCO DI MIGLIARINO - SAN ROSSORE"

Biogenic amines (BAs) can be naturally present in several foods. They are mainly produced in large amounts by amino acid decarboxylases activity of bacteria. The BAs content has been associated to the quality of raw material and to fermentation or spoilage processes. The aim of the present study was to asses the content of BAs (single and total value) in the core and in the external part of a Tuscan traditional pecorino cheese. Sixteen "Pecorino del Parco di Migliarino-San Rossore" cheeses belonging to same batch were tested during ripening time, up to 5 months. BAs content was analyzed by an HPLC-UV method. The BAs content was significantly higher in the core than in the external part. Tyramine was the amine most frequently detected and largely quantized, followed by putrescine, histamine and cadaverine