Management of Listeria monocytogenes in fermented sausages using the Food Safety Objective concept underpinned by stochastic modeling and meta-analysis.

In the present work, a demonstration is made on how the risk from the presence of Listeria monocytogenes in fermented sausages can be managed using the concept of Food Safety Objective (FSO) aided by stochastic modeling (Bayesian analysis and Monte Carlo simulation) and meta-analysis. For this purpose, the ICMSF equation was used, which combines the initial level (H0) of the hazard and its subsequent reduction (ΣR) and/or increase (ΣI) along the production chain. Each element of the equation was described by a distribution to investigate the effect not only of the level of the hazard, but also the effect of the accompanying variability. The distribution of each element was determined by Bayesian modeling (H0) and meta-analysis (ΣR and ΣI). The output was a normal distribution N(-5.36, 2.56) (log cfu/g) from which the percentage of the non-conforming products, i.e. the fraction above the FSO of 2 log cfu/g, was estimated at 0.202%. Different control measures were examined such as lowering initial L. monocytogenes level and inclusion of an additional killing step along the process resulting in reduction of the non-conforming products from 0.195% to 0.003% based on the mean and/or square-root change of the normal distribution, and 0.001%, respectively.

Estimating the non-thermal inactivation of Listeria monocytogenes in fermented sausages relative to temperature, pH and water activity.

Data relative to in situ Listeria monocytogenes inactivation in fermented sausages were collected from 13 individual studies found in the literature. Inactivation rates were extrapolated and used to develop a predictive model to evaluate the relative effects of pH, water activity (a(w)) and temperature on L. monocytogenes fate during fermentation and ripening. Temperature explained ca. 60% of the data variability, while pH and a(w) only a small part. Temperature alone may not be sufficient to cause pathogen's inactivation, but inactivation rate is dominated by temperature when pH and a(w) are in the range which prevent L. monocytogenes growth. A predictive model based on two Arrhenius equations (ln[inactivation rate]=-25.71-[-0.6829 / (8.314 × T)] for fermentation; and ln[inactivation rate]=-44.86-[-1.219/(8.314 × T)] for ripening)was developed. The model can be used to quantify the effect of temperature and/or time changes on fermented sausage safety. The advantages and limitations of the model are discussed.

Differential gene expression profiling of Listeria monocytogenes in Cacciatore and Felino salami to reveal potential stress resistance biomarkers.

The current study reports a) the in situ transcriptional profiles of Listeria monocytogenes in response to fermented sausage stress and b) an approach in which in situ RT-qPCR data have been combined with advanced statistical techniques to discover potential stress resistance or cell viability biomarkers. Gene expression profiling of the pathogen has been investigated using RT-qPCR to understand how L. monocytogenes responds to the conditions encountered during the fermentation and ripening of sausages. A cocktail of five L. monocytogenes strains was inoculated into the batter of Cacciatore and Felino sausages. The RT-qPCR data showed that the acidic and osmotic stress-related genes were up-regulated. The transcripts of the lmo0669 gene increased during the fermentation and ripening of Cacciatore, whereas gbuA and lmo1421 were up-regulated during the ripening of Felino and Cacciatore, respectively. sigB expression was induced in both sausages throughout the whole process. Finally, the virulence-related gene prfA was down-regulated during the fermentation of Cacciatore. The multivariate gene expression profiling analysis suggested that sigB and lmo1421 or sigB and gbuA could be used as different types of stress resistance biomarkers to track, for example, stress resistance or cell viability in fermented sausages with short (Cacciatore) or long (Felino) maturation times, respectively.

Diversity and functional characterization of Lactobacillus spp. isolated throughout the ripening of a hard cheese.

The aim of this work was to study the Lactobacillus spp. intra- and inter- species diversity in a Piedmont hard cheese made of raw milk without thermal treatment and without addition of industrial starter, and to perform a first screening for potential functional properties. A total of 586 isolates were collected during the cheese production and identified by means of molecular methods: three hundred and four were identified as Lactobacillus rhamnosus, two hundred and forty as Lactobacillus helveticus, twenty six as Lactobacillus fermentum, eleven as Lactobacillus delbrueckii, three as Lactobacillus pontis, and two as Lactobacillus gasseri and Lactobacillus reuteri, respectively. A high genetic heterogeneity was detected by using the repetitive bacterial DNA element fingerprinting (rep-PCR) with the use of (GTG)5 primer resulting in eight clusters of L. helveticus and sixteen clusters in the case of L. rhamnosus. Most of isolates showed a high auto-aggregation property, low hydrophobicity values, and a general low survival to simulated digestion process. However, sixteen isolates showed promising functional characteristics.

High inhibition of Paenibacillus larvae and Listeria monocytogenes by Enterococcus isolated from different sources in Tunisia and identification of their bacteriocin genes.

UNLABELLED: A total of 300 isolates of Enterococcus, from different sources including faeces of poultry, cow and sheep, raw milk, ricotta cheese and water, in Tunisia, were screened for their antibacterial activity. Amongst them, 59 bacteriocin-producing strains were detected and identified by molecular methods. Genes encoding for entA, entP, entB, entL50A/B, AS-48 and bac31 bacteriocins were targeted by PCR. The bacteriocin-producing strains were assigned to the species Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, Enterococcus mundtii and Enterococcus durans, respectively, 34, 19, 3, 2 and 1 isolates. Antimicrobial activity was specifically observed against different spoilage and pathogenic micro-organisms, such as Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Escherichia coli, Ent. faecalis, Staphylococcus aureus, Salmonella enterica serovar Enteritidis and Paenibacillus larvae. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing the proteinaceous nature of the antimicrobial compound. Only three bacteriocin genes, namely entP, entA and entL50A/B were detected in the isolates included in this study. Enterocins A and P were the most frequent genes and they were found in 55 (93.2%) and 39 isolates (66.1%), respectively, followed by enterocin L50A/B present in 27 isolates (45.7%). These newly identified bacteriocin-producing enterococci have the potential to be used in bio-preservation of food as well as biological control of foulbrood disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci possess interesting properties not only for the food industry, but also for animal and human health. The antimicrobial potential of these bacteria includes principally bacteriocin-like molecules. With the aim of identifying bacteriocinogenic strains, a collection of 300 enterococci isolated from different origins were screened and their spectrum of action, as well as the gene encoding the bacteriocin, was determined. Fifty-nine bacteriocin-producing Enterococcus showed high activity against Listeria monocytogenes and Paenibacillus larvae, the causative agent of American foulbrood. Enterocins A, P and L50A/B were found in various combinations. The most important finding of this study is the growth inhibition of P. larvae due to bacteriocin-producing Enterococcus, which opens up the possibility to use these strains to control the disease in honeybees.

Microbial diversity, dynamics and activity throughout manufacturing and ripening of Castelmagno PDO cheese.

The diversity, dynamics and activity of Castelmagno PDO cheese microbiota were studied in three batches produced in a floor valley farm, in the Grana Valley (northwest Italy), during the wintertime. Samples of milk, curd and cheese (core and subsurface) at different ripening time were submitted to both culture-dependent and -independent analysis. In particular, DNA and RNA directly extracted from the matrices were studied by PCR-Denaturing gradient gel electrophoresis (DGGE) and reverse transcription (RT)-PCR-DGGE. Culture-dependent methods highlighted the initial dominance of a thermophilic streptococcal population with the species Streptococcus thermophilus and S. agalactiae. Then, mesophilic lactococci occurred among isolates during manufacturing, with Lactococcus lactis which was also well represented in the first month of Castelmagno PDO ripening. At this point and throughout the ripening, lactobacilli prevailed in cheese samples, represented from Lactobacillus plantarum and Lb. casei. Culture-independent analysis underlined the undoubted role of L. lactis, actively involved in both Castelmagno PDO manufacturing and ripening. Despite Lb. helveticus was never isolated on selective media, a DGGE band referred to this microorganism was detected, at RNA level, in samples from ripened cheeses. On the other hand, Lb. plantarum was widely isolated from the plates, among lactobacilli, but never detected by direct analysis. Due to the importance of microbiota in the sensory richness and properties of traditional cheeses, new information have been added, in this work, on microbial diversity of Castelmagno PDO cheese.

Maturing dynamics of surface microflora in Fontina PDO cheese studied by culture-dependent and -independent methods.

AIMS: To study the evolution of rind microbial communities in Fontina PDO cheese. METHODS AND RESULTS: Four batches were examined for their surface microflora during ripening, carried out in two different maturing caves, at Ollomont and Pré-Saint-Didier, Aosta Valley region, Northwest of Italy. Culture-dependent methodologies were combined with culture-independent analysis (PCR-DGGE). Yeasts were found to increase from 10(3) to 10(6) CFU cm(-2) in 28 days, with consequent rise of surface pH, which allowed the growth of salt-tolerant bacteria, in particular coryneforms which reached 10(9) CFU cm(-2) at the end of 3 months. Coagulase-negative cocci and lactic acid bacteria reached 10(7) CFU cm(-2) in the same period. Debaryomyces hansenii and Candida sake were the species more constantly present throughout the whole maturing process. As early as after 1 day since manufacture, Lactococcus lactis subsp. lactis and Streptococcus thermophilus were detected on cheese rinds. Arthrobacter nicotianae, Brevibacterium casei and Corynebacterium glutamicum were found after 7-28 days. CONCLUSIONS: According to cluster analysis of DGGE profiles, the maturing environment seemed to influence the dynamics of microbial groups on Fontina surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: These results represent a first picture of micro-organisms colonizing Fontina PDO rinds. Further studies are in progress to better understand the origin of this surface microflora and to formulate surface starters.