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Research has been conducted on lactic acid bacteria and gram-positive catalase-positive cocci from low-acid fermented sausages. The authors have investigated microbial hygienic quality of these sausages, and have identified the main bacterial species of technological interest via the use of species-specific techniques.

Any bacterial strain to be used as starter culture should have suitable characteristics, including a lack of amino acid decarboxylase activity. In this study, the decarboxylase activity of 76 bacterial strains, including lactic acid bacteria and grampositive, catalase-positive cocci, was investigated. These strains were previously isolated from European traditional fermented sausages to develop autochthonous starter cultures. Of all the strains tested, 48% of the lactic acid bacteria strains and 13% of gram-positive, catalase-positive cocci decarboxylated one or more amino acids. Aminogenic potential was strain dependent, although some species had a higher proportion of aminogenic strains than did others. Thus, all Lactobacillus curvatus strains and 70% of Lactobacillus brevis strains had the capacity to produce tyramine and β-phenylethylamine. Some strains also produced other aromatic amines, such as tryptamine and the diamines putrescine and cadaverine. All the enterococcal strains tested were decarboxylase positive, producing high amounts of tyramine and considerable amounts of β-phenylethylamine. None of the staphylococcal strains had tyrosine-decarboxylase activity, but some produced other amines. From the aminogenic point of view, Lactobacillus plantarum, Lactobacillus sakei, and Staphylococcus xylosus strains would be the most suitable for use as autochthonous starter cultures for traditional fermented sausages. [ABSTRACT FROM AUTHOR]

Aims: Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages. Methods and Results: Different points during the production of a traditional fermented sausage type ( fuet) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers’ hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source. Conclusions: The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin. Significance and Impact of the Study: This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment. [ABSTRACT FROM AUTHOR]

Abstract: Consumers demand high quality, natural, nutritious, fresh appearance and convenient meat products with natural flavour and taste and an extended shelf-life. To match all these demands without compromising safety, in the last decades alternative non-thermal preservation technologies such as HHP, irradiation, light pulses, natural biopreservatives together with active packaging have been proposed and further investigated. They are efficient to inactivate the vegetative microorganisms, most commonly related to food-borne diseases, but not spores. The combination of several non-thermal and thermal preservation technologies under the so-called hurdle concept has also been investigated in order to increase their efficiency. Quick thermal technologies such as microwave and radiofrequency tunnels or steam pasteurization bring new possibilities to the pasteurization of meat products especially in ready to eat meals. Their application after final packaging will prevent further cross-contamination during post-processing handling. The benefits of these new technologies and their limitations in an industrial application will be presented and discussed. [Copyright &y& Elsevier]

Sources and tracing of Staphylococcus aureus in alheira (garlic sausage) production were evaluated by multifactorial correspondence analysis (MCA) of occurrence data and a random amplified polymorphic DNA (RAPD) on S. aureus isolates. Samples from four production lines, four different production batches, and 14 different sampling sites (including raw material, different contact surfaces, and several stages of alheira manufacturing) were analyzed at four sampling times. From the 896 microbial analyses completed, a collection of 170 S. aureus isolates was obtained. Although analysis of the occurrence data alone was not elucidative enough, MCA and RAPD-PCR were able to assess the sources of contamination and to trace the spread of this microorganism along the production lines. MCA results indicated that the presence of S. aureus in alheira was related to its presence in the intermediate manufacturing stages after heat treatment but before stuffing in the casings. It was also possible to associate a cross-contamination path related to handler procedures. RAPD-PCR typing in accordance to MCA results confirmed the cross-contamination path between the raw material and casings and the role of handlers as an important cross-contamination vehicle. [ABSTRACT FROM AUTHOR]

This work aimed to quantify the impact of a w and fat content of dry-cured ham on the Log reduction of Salmonella enterica by high pressure (HP). Dry-cured ham with adjusted a w (0.86–0.96) and fat content (10–50%) was inoculated with S. enterica and pressurised (347–852 MPa, 5 min/15 °C), following a Central Composite Design. Polynomial regression indicated a significant impact of pressure and a w on S. enterica HP-lethality. By lowering a w a clear piezoprotection was observed. At low a w (0.88) the S. enterica reduction was little affected by increasing pressure (e.g. 2.3 to 3.2 Logs at 450 to 750 MPa, respectively). At the highest a w the estimated inactivation ranged from 3.3 to 8.9 Logs at 450 to 750 MPa, respectively. No significant piezoprotective effect on S. enterica was recorded by the fat content. The relevance of food characteristics on the HP-lethality of S. enterica indicate the need to validate the HP effectiveness on the specific product. [ABSTRACT FROM AUTHOR]

High pressure processing (HPP) is a promising food preservation technology as an alternative to thermal processing for microbial inactivation. The technological parameters, the type of microorganism, and the food composition can greatly affect the microbicidal potential of HPP against spoilage and pathogenic microorganisms. Presently, the number of available models quantifying the influence of food characteristics on the pathogen inactivation is scarce. The aim of this study was to model the inactivation of Listeria monocytogenes CTC1034 in dry-cured ham, as a function of pressure (347–852 MPa, 5 min/15 °C), water activity (a w , 0.86–0.96) and fat content (10–50%) according to a Central Composite Design. The response surface methodology, based on the equation obtained with a stepwise multivariate linear regression, was used to describe the relationship between bacterial inactivation and the studied variables. According to the best fitting polynomial equation, besides pressure intensity, both a w and fat content exerted a significant influence on HP-inactivation of L. monocytogenes . A clear linear piezoprotection trend was found lowering the a w of the substrate within the whole range of tested pressure. Fat content was included in the model through the quadratic term and as interaction term with pressure, resulting in a particular behavior. A protective effect due to the presence of high fat content was identified for pressure treatments above ca. 700 MPa. At lower pressure, higher inactivation of L. monocytogenes occurred by increasing the fat content above 30%. The results emphasize the relevant influence of intrinsic factors on the L. monocytogenes inactivation by HPP, making necessary to assess and validate the effectiveness of HPP on specific food products and consequently set process criteria adjusted to each particular food product. [ABSTRACT FROM AUTHOR]

Abstract: To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347–852 MPa), time (2.3–15.8 min) and temperature (7.6–24.4 °C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process. [Copyright &y& Elsevier]

Abstract: The effect of high pressure processing parameters on the inactivation of a potentially virulent Enterococcus faecalis strain inoculated on sliced dry-cured ham was investigated. The response surface methodology was applied in combination with a central composite design of the technological parameters: including pressure (347–852MPa), length of the treatment (2.3–15.8min) and temperature (7.6–24.4°C). Bacterial inactivation, as logarithmic reductions of Enterococcus spp. counts, was modeled through the backward stepwise procedure. According to the best fitting and most significant polynomial equation, pressure and time were the factors determining the Enterococcus inactivation, while temperature was not statistically significant. E. faecalis showed a notable resistance to high pressure processing, similar to that reported for Staphylococcus aureus and higher than the baroresistance of pathogens such as Listeria monocytogenes or Salmonella. To achieve 4 log reduction of E. faecalis on dry-cured ham, pressures from 750MPa for at least 9.5min would be required. Industrial Relevance: The results of this work can help food processors to set optimum technological parameters of high pressure processing to inactivate undesired microorganisms in food. [Copyright &y& Elsevier]

Abstract: The application of high hydrostatic pressure (200MPa) to meat batter just before sausage fermentation and the inoculation of starter culture were studied to improve the safety and quality of traditional Spanish fermented sausages (fuet and chorizo). Higher amounts of biogenic amines were formed in chorizo than in fuet. Without interfering with the ripening performance in terms of acidification, drying and proteolysis, hydrostatic pressure prevented enterobacteria growth but did not affect Gram-positive bacteria significantly. Subsequently, a strong inhibition of diamine (putrescine and cadaverine) accumulation was observed, but that of tyramine was not affected. The inoculated decarboxylase-negative strains, selected from indigenous bacteria of traditional sausages, were resistant to the HHP treatment, being able to lead the fermentation process, prevent enterococci development and significantly reduce enterobacteria counts. In sausages manufactured with either non-pressurized or pressurized meat batter, starter culture was the most protective measure against the accumulation of tyramine and both diamines. [Copyright &y& Elsevier]

Various predictive models are available for high pressure inactivation of Listeria monocytogenes in food, but currently available models do not consider the growth kinetics of surviving cells during the subsequent storage of products. Therefore, we characterised the growth of L. monocytogenes in sliced cooked meat products after a pressurization treatment. Two inoculum levels (107 or 104 CFU/g) and two physiological states before pressurization (freeze-stressed or cold-adapted) were evaluated. Samples of cooked ham and mortadella were inoculated, high pressure processed (400 MPa, 5 min) and subsequently stored at 4, 8 and 12 °C. The Logistic model with delay was used to estimate lag phase (λ) and maximum specific growth rate (μmax) values from the obtained growth curves. The effect of storage temperature on μmax and λ was modelled using the Ratkowsky square root model and the relative lag time (RLT) concept. Compared with cold-adapted cells the freeze-stressed cells were more pressure-resistant and showed a much longer lag phase during growth after the pressure treatment. Interestingly, for high-pressure inactivation and subsequent growth, the time to achieve a concentration of L. monocytogenes 100-fold (2-log) higher than the cell concentration prior to the pressure treatment was similar for the two studied physiological states of the inoculum. Two secondary models were necessary to describe the different growth behaviour of L. monocytogenes on ready-to-eat cooked ham (lean product) and mortadella (fatty product). This supported the need of a product-oriented approach to assess growth after high pressure processing. The performance of the developed predictive models for the growth of L. monocytogenes in high-pressure processed cooked ham and mortadella was evaluated by comparison with available data from the literature and by using the Acceptable Simulation Zone approach. Overall, 91% of the relative errors fell into the Acceptable Simulation Zone. [ABSTRACT FROM AUTHOR]

Abstract: High pressure (HP) inactivation curves of Listeria monocytogenes CTC1034 (ca. 107 CFU/g) on sliced RTE cooked meat products (ham and mortadella) were obtained at pressures from 300 to 800MPa. A clear tail shape was observed at pressures above 450MPa and the log-linear with tail primary model provided the best fit to the HP-inactivation kinetics. The relationships between the primary kinetic parameters (log kmax and log Nres ) and pressure treatments were described by a polynomial secondary model. To estimate HP-inactivation of L. monocytogenes in log (N/N 0) over time, a one-step global fitting procedure was applied. The secondary model was integrated into the primary model and the combined equation was fitted to the entire data-set to readjust parameter values. Validation of the developed models both under dynamic conditions and using external independent data supported their suitability for predictive purposes, e.g., to set the process criteria required to meet food safety objectives. Industrial relevance: Quantitative mathematical models for predicting inactivation of pathogens by HPP provide useful tools for a process optimization and real time control of a unit operation. The developed models can assist food industries to define the process criteria compatible with the Food Safety Objectives for Listeria monocytogenes in RTE cooked meat products. [Copyright &y& Elsevier]

Abstract: The lethal effect of high pressure on microorganisms is influenced by a number of factors in relation to the technological variables, microorganisms and food matrix, which have to be considered when designing high pressure processes. The present work aimed to develop and validate a model of the inactivation of Salmonella enterica on dry-cured ham by high pressure processing (HPP), as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with S. enterica was treated at different HPP conditions (at 347 to 852MPa; for 2.3 to 15.75min; at 7.6 to 24.4°C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of Salmonella counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. Temperature showed significant influence through its interaction with both pressure and holding time. The model was validated with results obtained from further experiments within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, for instance to predict the process criteria to meet the lethality safety standards. The results of this work may help food processors to select optimum processing conditions of HPP to ensure the microbiological safety of pressure-treated foods. [Copyright &y& Elsevier]

Abstract: The aim of the work was to develop and validate a model of the inactivation of Listeria monocytogenes on dry-cured ham by high hydrostatic pressure (HHP) processing, as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with L. monocytogenes was treated at different HHP conditions (at 347–852 MPa; for 2.3 to 15.75 min; at 7.6 to 24.4 °C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of L. monocytogenes counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. The significance of the quadratic term of pressure and time indicated that little effect was observed below 450 MPa, whereas holding time longer than 10 min did not result in a meaningful reduction of L. monocytogenes counts. Temperature did not show significant influence at the range assayed. The model was validated with results obtained from further experiments and bibliographical data within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, such as prediction of the process criteria to meet the Food Safety Objectives. The results of this work may help food processors to select optimum processing conditions of HHP. [Copyright &y& Elsevier]

Abstract: The volatile profiles of slightly-fermented sausages inoculated with eight different enterococci strains were determined and compared with a fermented sausage manufactured without any inoculation. The volatile profile was determined by solid-phase microextraction (SPME) and gas chromatography coupled to mass spectrometry (GC/MS). A total of 121 volatile and semi-volatile compounds were identified in the samples of the final products (day 21). Terpenes from spices added during manufacture of dry-fermented sausages were the main group of compounds in the volatile profile. Other groups included alcohols (18% of total volatile compounds), aldehydes (16%), alkanes and alkenes (12%), and ketones (10%). These are derived from lipids, proteins or carbohydrates and formed during fermentation and ripening. In terms of global volatile composition, the batches inoculated with enterococci strains were similar to those obtained for non-inoculated batch. However, some quantitative differences in the volatile profiles of different batches were found, mainly in the compounds formed from amino acid catabolism (i.e.: propanol, isoamyl), from free fatty acid oxidation (i.e.: hexanal, hexanol), or from carbohydrate fermentation (i.e.: 2-butanone, acetoin). In comparison with non-inoculated sausages, some batches were more resistant to oxidation; they contained fewer volatile compounds arising from lipid oxidation. The use of different selected enterococci strains did not alter the volatile profile of slightly-fermented sausages. The enterococci strains provide similar volatile compounds to those usually reported in the literature. [ABSTRACT FROM AUTHOR]

Abstract: Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units. [Copyright &y& Elsevier]

Abstract: The population of Gram-positive catalase-positive cocci from slightly fermented sausages was characterized at species and strain level by molecular techniques and some technological and hygienic aspects were also considered. Staphylococcus xylosus was the predominant species (80.8%) followed by Staphylococcus warneri (8.3%), Staphylococcus epidermidis (5.8%) Staphylococcus carnosus (4.6%), and Kocuria varians (0.4%). Proteolytic activity was observed in 23% of the isolates. The species with the highest percentage of proteolytic strains was S. warneri. Lipolytic activity was found in 45.8% of the isolates and S. xylosus was the species with the highest percentage of lipolytic isolates. Biogenic amine production was not widely distributed (only 14.6% of the isolates). Tyramine was the most intense amine produced, although by only 4.6% of the isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. Some strains also produced putrescine (3.3%), cadaverine (2.9%), histamine (1.3%) and tryptamine (0.4%). All isolates were susceptible to linezolid and vancomicin and over 70% were resistant to penicillin G, ampicilin and sulphonamides. Most of the mecA + strains (only 4.6% of isolates) also displayed resistance to multiple antibiotics. A reduced enterotoxigenic potential was found. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene. The combination of RAPD-PCR and plasmid profiling allowed the discrimination of 208 different profiles among the 240 Gram-positive catalase-positive cocci characterized, indicating a great genetic variability. [Copyright &y& Elsevier]

A multiplex PCR (m-PCR) assay with an internal amplification control (IAC) was developed for the simultaneous detection of Salmonella spp. and Listeria monocytogenes through invA and prfA genes, respectively. To ensure the detection of the pathogens in cooked ham, samples were enriched in both buffered peptone-water and Half Fraser broth. Subsequently, equal volumes of enrichment broths were mixed and DNA purification was performed prior to m-PCR reaction, saving considerable time and effort. The m-PCR also proved to be very useful as a simple and ready-to-go method for simultaneous confirmation of presumptive L. monocytogenes and Salmonella spp. colonies directly from agar plates without any DNA extraction steps. [Copyright &y& Elsevier]

Databases that integrate information from primary and specialist care medical records offer an information source that may be useful for studies of epidemiology, therapeutic adaptation and health/non-healthcare resource use and associated costs. The objective of the study was to determine the validity and representativeness of the BIG-PAC database electronic medical records in usual clinical practice in Spain. [Extracted from the article]