Use of commercial protective cultures in portioned sheep milk cheeses to control Listeria monocytogenes.

The main objective of this study was to innovate soft and semi-cooked sheep milk cheese production processes with the use of a commercial protective culture able to control Listeria monocytogenes growth. A freeze-dried commercial culture of Lactobacillus plantarum was tested in DS cheese and PS cheese, two types of pasteurized sheep milk, raw-paste cheeses aged for no less than 20 and 30 days respectively. In the first step, in vitro tests were conducted to identify the most suitable matrix for the growth of L. plantarum in order to create a subculture that could be used at industrial cheese-making plants. During the second phase of the study, L. plantarum culture was introduced in the manufacturing process of the cheeses in a production plant. Finally, a challenge test was conducted on portioned DS and PS cheeses in order to evaluate the activity of the protective culture against L. monocytogenes: the cheeses were portioned, experimentally contaminated with L. monocytogenes strains, vacuum packed and stored at +4°C (correct storage conditions) and at +10°C (thermal abuse). Cheeses were analysed at the end of the shelf-life to evaluate the presence and growth of L. monocytogenes, to enumerate lactic acid bacteria and determine chemicalphysical features. The results confirmed that protective cultures are a useful technological innovation to control L. monocytogenes growth during cheese storage without altering composition, microflora and chemical- physical characteristics of the product. However, the use of protective cultures should be applied as an integration of risk control measures and not as a substitute for preventive actions.

Preliminary data on the microbial profile of dry and wet aged bovine meat obtained from different breeds in Sardinia.

This study aimed to evaluate the influence of dry and wet aging on microbial profile and physicochemical characteristics of bovine loins obtained from four animals of two different breeds, namely two Friesian cull cows and two Sardo-Bruna bovines. During dry and wet aging aerobic colony count, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas, molds and yeasts, Salmonella enterica, Listeria monocytogenes and Yersinia enterocolitica, pH and water activity (aw) were determined in meat samples collected from the internal part of the loins. Moreover, the microbial profile was determined with sponge samples taken from the surface of the meat cuts. Samples obtained from Friesian cows were analyzed starting from the first day of the aging period and after 7, 14, and 21 days. Samples obtained from the Sardo Bruna bovines were also analyzed after 28 and 35 days. Wet aging allowed better control of Pseudomonas spp. during storage that showed statistically lower levels (P>0.05) in wet-aged meats with respect to dry-aged meats during aging and particularly at the end of the period (P>0.01) in both cattle breeds. At the end of the experiment (21 days), aerobic colony count and Pseudomonas in Fresian cows' dry-aged meats showed mean levels >8 log, while lactic acid bacteria mean counts >7 log were detected in wet-aged meats of both cattle breeds. In meats submitted to dry aging, pH was significantly higher (P<0.01) with respect to wet-aged meats at all analysis times and in both cattle breeds. Aw showed a stable trend during both dry and wet aging without significant differences. These preliminary results highlight the critical importance of the strict application of good hygiene practices during all stages of production of these particular cuts of meat intended for aging.

Traceability, virulence and antimicrobial resistance of Yersinia enterocolitica in two industrial cheese-making plants.

Between 2018 and 2019, 309 environmental and food samples were collected from two industrial cheese-making plants located in Sardinia, in order to investigate Y. enterocolitica presence and to characterize the isolates. Y. enterocolitica isolates were further compared with isolates detected during a previous investigation from sheep and goat raw milk samples. Y. enterocolitica was detected in 7.4 % of the samples and the prevalence was higher, even if not significantly (P > 0.05) higher in non-food contact surface samples (10.2 %) than in food contact surface samples (3.8 %). The highest prevalence was detected in floor samples (13.5 %), followed by drain samples (7.2 %), which might serve as main harborage sites for further contamination. Y. enterocolitica was also detected in food contact surfaces, namely shelves of the Ricotta cooling room and packaging room, one cheese cutting machine surface and one raw milk filter sample. The biotype 1A isolates identified in this study were classified into six different serotypes. Additionally, a bioserotype 2/O:5,27 isolate was identified in one goat milk sample. All 1A isolates possessed the virulence genes invA and ystB while the 2/O:5,27 isolate showed the presence of ail, ystA, invA and yadA genes, thus confirming a pathogenic potential. The isolates showed intrinsic resistance to amoxicillin-clavulanic acid, ticarcillin and cefoxitin due to the presence of the blaA gene. Whole genome sequencing allowed to identify seven different sequence types among the 1A isolates, thus showing a high genetic diversity. The same Y. enterocolitica sequence type (ST3) was detected from three different areas of the same cheese-making plant, indicating a possible transfer of the microorganism along the processing lines. Y. enterocolitica contamination in cheese-making plants can pose a risk to human health. Preventive measures include the hygienic design of the plant layout and equipment, in association with proper cleaning and disinfection programmes.

Selection of commercial protective cultures to be added in Sardinian fermented sausage to control Listeria monocytogenes.

Sardinian fermented sausage "Salsiccia Sarda" is a Mediterranean-style, semi-dry, fermented, RTE product, representing the main pork meat product in Sardinia (Italy). The high variability that characterizes the technological processes applied in different production plants results in sausages with different chemico-physical features sometimes permissive for the growth of Listeria monocytogenes. In order to guarantee the hygienic-sanitary quality of the final product and to innovate the manufacturing process, the main objective of this study was to evaluate the use of different commercial protective cultures to control L. monocytogenes growth in the Sardinian fermented sausage. In the first step, in vitro tests were carried out to evaluate the effectiveness of five freeze-dried bioprotective cultures availabe on the market in limiting the growth of L. monocytogenes. The two protective cultures that showed the best in vitro results were selected for a challenge test on artificially contaminated Sardinian fermented sausages. Moreover, the protective culture that showed the best results in inhibiting the growth of L. monocytogenes according to in vitro and challenge test experiments, was included into real production settings and validated in three producing plants. As a result, it was observed that protective cultures represent an important technological innovation for the Sardinian fermented sausage processing plants as they allow to control L. monocytogenes growth without altering the composition, the microflora and the chemical-physical characteristics of the product, thus ensuring safety and quality. Protective cultures also showed to reduce Enterobacteriaceae mean levels at the end of ripening and not to affect the natural concentration of lactic acid bacteria and coagulase-negative staphylococci.

Trend of Salmonella enterica occurrence and serotypes in Sardinian pig slaughterhouses.

The aim of this study was to evaluate Salmonella prevalence and serotypes in four Sardinian pig slaughterhouses. Moreover, a population study was conducted with pulsed field gel electrophoresis (PFGE). The results were compared with previous investigations carried out during years 2008 and 2014. A total of 147 samples were collected, 117 from slaughtered pigs (lymph nodes, colon content and carcass surface) and 30 from the slaughterhouse environment (surfaces in contact and not in contact with meat). Salmonella was isolated from 3.4% pig samples and was not detected from environmental samples. Comparing the results with those of previous investigations, occurrence showed a sharp decrease through the years in both animals (18.8% in 2008, 10% in 2014 and 3.4% in 2020) and environmental samples (34.1% in 2008, 3.7 in 2014, and 0% in 2020). At the same time, prevalence of carriers (pigs positive at lymph nodes and/or colon content level) showed a reduction through the years and was always lower in animals coming from local farms rather than those coming from other European Member States, probably indicating the role of stressful factors as transport in increasing Salmonella susceptibility and shedding. Salmonella serotypes were monophasic Typhimurium, Rissen and Muenchen. Overall, 13 different Salmonella serotypes were identified during the three surveys with the most prevalent being serotypes often isolated from slaughtered pigs and during human salmonellosis cases: S. Derby and S. Typhimurium in 2008, S. Anatum and S. Rissen in 2014, monophasic S. Typhimurium in 2020. Population study with pulsed field gel electrophoresis showed a high similarity between Salmonella strains belonging to the same serotype. The results of the investigations showed a decrease of Salmonella occurrence during twelve years in Sardinia, probably due to the improvement in the application of correct GMPs and GHPs at slaughterhouse and also to a reduction of the rate of carrier pigs at farm level.