Microbiological challenge testing for Listeria monocytogenes in ready-to-eat food: a practical approach

Food business operators (FBOs) are the primary responsible for the safety of food they place on the market. The definition and validation of the product’s shelf-life is an essential part for ensuring microbiological safety of food and health of consumers. In the frame of the Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs, FBOs shall conduct shelf-life studies in order to assure that their food does not exceed the food safety criteria throughout the defined shelf-life. In particular this is required for ready-to-eat (RTE) food that supports the growth of Listeria monocytogenes. Among other studies, FBOs can rely on the conclusion drawn by microbiological challenge tests. A microbiological challenge test consists in the artificial contamination of a food with a pathogen microorganism and aims at simulating its behaviour during processing and distribution under the foreseen storage and handling conditions. A number of documents published by international health authorities and research institutions describes how to conduct challenge studies. The authors reviewed the existing literature and described the methodology for implementing such laboratory studies. All the main aspects for the conduction of L. monocytogenes microbiological challenge tests were considered, from the selection of the strains, preparation and choice of the inoculum level and method of contamination, to the experimental design and data interpretation. The objective of the present document is to provide an exhaustive and practical guideline for laboratories that want to implement L. monocytogenes challenge testing on RTE food

Antibiotic Resistance in Staphylococcus aureus and Coagulase Negative Staphylococci Isolated from Goats with Subclinical Mastitis

Antimicrobial resistance patterns and gene coding for methicillin resistance (mecA) were determined in 25 S. aureus and 75 Coagulase Negative Staphylococci (CNS) strains isolates from half-udder milk samples collected from goats with subclinical mastitis. Fourteen (56.0%) S. aureus and thirty-one (41.3%) CNS isolates were resistant to one or more antimicrobial agents. S. aureus showed the highest resistance rate against kanamycin (28.0%), oxytetracycline (16.0%), and ampicillin (12.0%). The CNS tested were more frequently resistant to ampicillin (36.0%) and kanamycin (6.7%). Multiple antimicrobial resistance was observed in eight isolates, and one Staphylococcus epidermidis was found to be resistant to six antibiotics. The mecA gene was not found in any of the tested isolates. Single resistance against β-lactamics or aminoglicosides is the most common trait observed while multiresistance is less frequent

A Survey on aflatoxin M₁ content in sheep and goat milk produced in Sardinia region, Italy (2005-2013)

In the present work the results of a survey conducted in Sardinia Region on Aflatoxin M1 (AFM1) contamination in milk of small ruminants from 2005 to 2013 are reported. A total of 517 sheep and 88 goat milk samples from bulk tank, tank trucks and silo tank milk were collected. Analyses were performed by the Regional Farmers Association laboratory using high-performance liquid chromatography following the ISO 14501:1998 standard. None of the sheep milk samples analysed during 2005- 2012 showed AFM1 contamination. In sheep milk samples collected in 2013, 8 out of 172 (4.6%) were contaminated by AFM1 with a concentration (mean±SD) of 12.59±14.05 ng/L. In one bulk tank milk sample 58.82 ng/L AFM1 was detected, exceeding the EU limit. In none of goat milk samples analysed from 2010 to 2012 AFM1 was detected. In 2013, 9 out of 66 goat milk samples (13.6%) showed an AFM1 concentration of 47.21±19.58 ng/L. Two of these samples exceeded the EU limit, with concentrations of 62.09 and 138.6 ng/L. Higher contamination frequency and concentration rates were detected in bulk tank milk samples collected at farm than in bulk milk truck or silo samples, showing a dilution effect on AFM1 milk content along small ruminants supply chain. The rate and levels of AFM1 contamination in sheep and goat milk samples were lower than other countries. However, the small number of milk samples analysed for AFM1 in Sardinia Region in 2005-2013 give evidence that food business operators check programmes should be improved to ensure an adequate monitoring of AFM1 contamination in small ruminant dairy chain

Time-resolved image analysis for turbulent flows Conference paper

International audienceClassical Particle Image Velocimetry (PIV) uses two representations of the particle image distribution to determine the displacement of the particle image pattern by spatial cross-correlation. The accuracy and the robustness are however limited by the fact that only two representations at t and t +Δt are present. Thus, only a first order approximation of the velocity can be estimated. To enhance the precision in estimating the flow velocity, multi-pulse or multi-frame techniques were already investigated in the early days of PIV as summarized by Adrian (1991) and Hain and Kähler (2007). Today with the increasing power of high repetition rate lasers and enhanced sensitivity of the digital cameras it is possible to have a time-resolved sampling of even aerodynamically relevant flows, were the particles are much smaller than in water flows. The easiest sampling scheme is the equidistant temporal sampling of the particle distribution such that a robust displacement estimation between successive frames (1+2, 2+3, 3+4, ...) is possible. This so called TR-PIV does not only provide the possibility to follow the evolution of flow structures, but offers the ability to strengthen the data processing by using information from more than two frames (e.g. Hain and Kähler, 2007). Within the AFDAR-project (Advanced Flow Diagnostics for Aeronautical Research funded by the European Union) different approaches to evaluat time-resolved image series were developed by the different groups. The current contribution focuses on the comparison of the algorithms that were developed within the AFDAR project by the partners of the consortium. To verify and validate the performance of the different algorithms a short image sequence of an experiment on the flow over periodic hills (ERCOFTAC test case 81) was provided to all partners and evaluated with the current version of the algorithms

Hygienic and sensory quality factors affecting the shelf-life of Fruhe (Casu axedu) traditional Sardinian fresh cheese

A study was conducted to evaluate the dura- bility of the traditional fresh soft cheese Fruhe manufactured in Sardinia either from goats’ or sheep’s milk. Four farmstead cheese-making plants were visited three times during the Fruhe cheese-making season. During each visit environmental samples were collected from food contact and non-food contact sur- faces in order to evaluate the presence of Enterobacteriaceae, Escherichia coli, Pseudomonas spp. and Listeria spp. In a total of 60 environmental samples, Escherichia coli and Listeria spp. were never detected, while contamination with Enterobacteriaceae and Pseudomonas spp. was observed respectively in 48% and 43% of samples. The microbiological profile of 48 Fruhe cheese samples was assessed at different time points during the product shelf-life. Aerobic mesophilic bacteria, Enterobacteriaceae, E. coli, Pseudomonas spp., Bacillus cereus and Listeria monocytogenes were investigated at 0, 7, 14 and 21 days after production. E. coli, L. monocytogenes and B. cereus were never detected in the product. Enterobacteriaceae contamination was observed, showing decreasing levels over time. Pseudomonas spp. was recovered in only two Fruhe samples (3.3%) at day 0. Sensory analysis was also conducted using a triangle test to determine whether a difference between Fruhe samples at 14 and 21 days of shelf-life exists. Based on the evolution of the microbiological profile and the sensory attributes observed in the present study, it is reasonable to assume that the product shelf-life can be feasibly extended up to 21 days

Survey on the fatty acids profile of fluid goat milk

Fluid goat milk submitted to thermal treatment has interesting nutritional properties and a potential expanding market. The present study was aimed to conduct fatty acids profile characterisation of goat milk placed on market. Forty-nine fluid milk samples were collected: 12 pasteurised, 12 pasteurised at high temperature, 11 ultrahigh temperature (UHT) whole milk and 14 UHT semi-skimmed milk. Milk samples were collected at retail level from 7 different companies and from different production batches. After extraction and methilation, fatty acids (FAs) profile was determined on each sample using a gas chromatograph with flame ionisation detector (GC-FID) with high-polarity capillary column. The concentration (g/100mL) of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), trans fatty acids (t-FAs), and isomers of conjugated linoleic acid (CLA) was determined. N-6/n-3 ratio, atherogenic index (AI) and thrombogenic index (TI) were also assessed. Fluid goat milk lipid profile was characterised by SFAs (68.4% of total FAs), PUFAs (5.3%), MUFAs (21.3%), t-FAs (3.6%) and CLA (0.8%). The most represented fatty acids were: 16:0 (24.5%), 9cis-18:1 (18.2%), 18:0 (9.6%), 14:0 (9.5%), 10:0 (9.3%) and 12:0 (4.5%). Nutritional indices were 2.8-6.8 for n-6/n-3 ratio; 2.3-2.9 for AI; and 2.7-3.2 for TI. Milk produced by small scale plants, with no milk fat standardisation, showed greater differences in fatty acid profile as compared to industrial plants milk. Large scale production is characterised by commingled bulk tank milk of different origins and then is more homogeneous. The whole goat milk supply chain should be controlled to obtain milk with fatty acids of high nutritional value

Seroepidemiological and biomolecular survey on

Toxoplasma gondii is a zoonotic parasite able of infecting all warm-blooded animals. Toxoplasmosis is one of the major foodborne diseases globally. The consumption of wild boar (Sus scrofa) meat from recreational hunting has been linked to outbreaks of human toxoplasmosis. The island of Sardinia (Italy) contains a large wild boar population, thus providing an opportunity to assess the distribution of Toxoplasma in this species and the associated risks of transmission to humans. A total of 562 wild boars were screened: heart and meat juice samples were tested for T. gondii DNA via nested-PCR and IgG anti-Toxoplasma by commercial ELISA. Anti-Toxoplasma IgG were detected in 24.6% (138/562) of animals, while 37.2% (209/562) of the heart samples were PCR positive. The prevalence of T. gondii antibodies and DNA highlights the potential role of wild boar as an important reservoir for this parasite. The study suggests that wild boar could play a significant role in spreading the parasite to humans. As wild boar numbers are increasing throughout their range, their potential role in transmitting toxoplasmosis should be communicated to stakeholders, and the impact of different population control methods on disease transmission should be thoroughly assessed to mitigate potential threats effectively

Occurrence and traceability of Salmonella spp. in five Sardinian fermented sausage facilities

The aims of the present study were to evaluate the presence of Salmonella in five fermented sausage processing plants and their products during the production process, and to trace the possible sources of contamination. A total of 270 samples were collected: mixture of ground pork meat and fat, products at the end of acidification, sausages at the end of ripening and, during production stages, surfaces in contact with meat and surfaces not in contact with meat. For samples of ground meat, product at the end of acidification and sausages at the end of ripening, the pH and water activity (aw), were determined. All the samples were tested for the presence of Salmonella. Thirtytwo Salmonella isolates were obtained, subjected to serotyping and PFGE. The sausages at the end of ripening pH and aw mean values were 5.39±0.24 and 0.91±0.03, respectively. Salmonella was detected in three processing plants with an overall prevalence of 16.7% in food samples and 5.8% in environmental samples. Salmonella prevalence was 24% in ground meat and products at the end of acidification and was also detected in a sample of sausage at the end of ripening (2%). In environmental samples, Salmonella was detected in 6.6% of surfaces in contact with meat and 5% of surfaces not in contact with meat. Five serotypes were identified among 32 isolates: S. Derby (37.5%), S. Typhimurium and S. Rissen (both 25%), S. Give and monophasic S. Typhimurium (both 6.25%). Six different pulsotypes were obtained with PFGE. The serotypes and the PFGE pattern of the strains were specific for each facility with no overlapping between different processing plants. The same observation can be pointed out considering different sampling days for the same processing plants, thus presumably indicating the raw material (ground pork meat and fat) as the source of contamination. The detection of Salmonella in a sample of sausage at the end of ripening highlights the ability of the pathogen to survive during manufacturing process

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

Evaluation of vacuum packaging for extending the shelf life of Sardinian fermented sausage

Salsiccia sarda or Sardinian fermented sausage is a traditional dry-fermented sausage included in the list of traditional food products of Sardinia (Italy). At the request of some producing plants, the possibility of extending the shelf life of the vacuum-packed product up to 120 days was evaluated. Manufacturing of 90 samples, representing 3 different batches of Sardinian fermented sausage was carried out in two producing plants (A and B). In the packaged product and subsequently every 30 days for four months (T0, T30, T60, T120), the following analyses were conducted on all samples: physicochemical characteristics, total aerobic mesophilic count, Enterobacteriaceae count, detection of Listeria monocytogenes, Salmonella spp., mesophilic lactic acid bacteria, and coagulase-positive Staphylococci. Moreover, surfaces in contact and surfaces not in contact with food were sampled in both producing plants. Sensory profile analysis was also performed for every analysis time. At the end of the extended shelf life, pH values were equal to 5.90±0.11 (producing plant A) and 5.61±0.29 (producing plant B). Water activity mean values at T120 were 0.894±0.02 (producing plant A) and 0.875±0.01 (producing plant B). L. monocytogenes was detected in 73.3% (33/45) of the samples from producing plant A, with mean levels of 1.12±0.76 log10 CFU/g. In producing plant B, L. monocytogenes was never detected. Enterobacteriaceae were detected in 91.1% (41/45) of samples in producing plant A with mean values of 3.15±1.21 log10 CFU/g, and in 35.5% (16/45) samples in producing plant B samples with mean values of 0.72±0.86 log10 CFU/g. Salmonella and Staphylococcus aureus were never detected. Regarding environmental samples, the sites that were most contaminated by L. monocytogenes were the bagging table (contact surface) and processing room floor drains (non-contact surface) with a prevalence of 50% each (8/16 positive samples for both sampling sites). Sensory analysis results showed that at T30 the overall sensory quality was at its highest; moreover, the visual-tactile aspect, the olfactory characteristics, the gustatory aspects, and the texture showed significant differences in samples throughout the shelf life, with a decreased intensity at 120 days of storage. Overall, the quality and sensory acceptance of the vacuum-packed Sardinian fermented sausage were not affected until 120 days of shelf-life. However, the possible contamination by L. monocytogenes calls attention to the hygienic management of the entire technological process. The environmental sampling was confirmed as a useful verification tool during control