M.J. Fraqueza

Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: Microbial ecosystems of processing environments

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.

Genetic diversity, antibiotic resistance and biofilm-forming ability of Arcobacter butzleri isolated from poultry and environment from a Portuguese slaughterhouse

The genus Arcobacter is an emerging pathogen associated with several clinical symptoms. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, where poultry is considered the major source. In this study, forty three Arcobacter butzleri strains isolated from poultry and environment of a Portuguese slaughterhouse, were characterized by pulsed field gel electrophoresis (PFGE) and assessed for antimicrobial susceptibility and ability to form biofilms. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed high genetic diversity, with 32 distinct PFGE patterns. Most of A. butzleri isolates presented multiple antimicrobial resistance, exhibiting four different resistance profiles. All 43 isolates were susceptible to gentamicin and 2.3% were resistant to chloramphenicol, in contrast to twenty four (55.8%) that were resistant to ciprofloxacin. Among 36 selected isolates, 26 strains presented biofilm-forming ability, which was dependent on the atmosphere and initial inoculum density. Overall, the results showed that A. butzleri displays a high genetic diversity, and presents resistance to several antibiotics, which together with its biofilm formation ability may represent a potential hazard for foodborne infections and a considerable risk for human health.

The effect on turkey meat shelf life of modified-atmosphere packaging with an argon mixture

There is a lack of knowledge related to the action of Ar on microbial development and prevention of oxidation when applied to raw meat under modified-atmosphere package (MAP). The aim of this study was to evaluate the effect of an anaerobic gas mixture with Ar on spoilage flora growth, color, and lipid oxidation stability of turkey meat under MAP stored at 0 degrees C. Breast muscles samples were collected on different working days from turkey carcasses (BUT9 and BIG6), fast-cooled in a tunnel (-2 degrees C, 2 m.s(-1), 90% RH) for 2 h and selected to be deboned according current practices in industrial slaughterhouses. The breasts were cut into slices that were individually packaged under aerobiosis (P0) and in 4 different modified atmospheres containing different gas mixtures as (P1) 100% N2, (P2) 50% Ar-50% N2, (P3) 50% Ar-50% CO2, and (P4) 50% N2-50% CO2. All samples were stored at 0+/-1 degrees C in the dark for between 12 and 25 d. Meat samples packaged in P0 were analyzed for their microbial and physicochemical characteristics on d 0, 5, and 12 of storage and then extended to 19 and 25 d when samples were under MAP. The microbial shelf life period extension of MAP sliced turkey meat compared with aerobic packaging (5-d shelf life) is 1 wk more for P1 and P2 mixtures, 2 wk for P4, and 3 wk for P3. The Ar-CO2 mixture was more efficient in delaying flora development than CO2-N2 with 1 log difference on the 25th day of storage, for total psychrotrophic counts, total anaerobic counts, and Brochothrix thermosphacta. The presence of Ar on gas mixtures did not seem to have any additional protective effect on lipid turkey meat oxidation.

Human, food and animal Campylobacter spp. isolated in Portugal: High genetic diversity and antibiotic resistance rates

Infections by Campylobacter jejuni and Campylobacter coli are considered the major cause of bacterial gastroenteritis in humans, with food being the main source of infection. In this study, a total of 196 Campylobacter strains (125 isolates from humans, 39 from retail food and 32 from food animal sources) isolated in Portugal between 2009 and 2012 were characterised by multilocus sequence typing (MLST) and flaA short variable region (SVR) typing. Susceptibility to six antibiotics as well as the mechanisms underlying antibiotic resistance phenotypes was also studied. Based on MLST typing, C. coli strains were genetically more conserved, with a predominant clonal complex (CC828), than C. jejuni strains. In contrast, C. coli isolates were genetically more variable than C. jejuni with regard to flaA-SVR typing. A high rate of resistance was observed for quinolones (100% to nalidixic acid, >90% to ciprofloxacin) and, in general, resistance was more common among C. coli, especially for erythromycin (40.2% vs. 6.7%). In addition, most isolates (86%) were resistant to multiple antimicrobial families. Besides the expected point mutations associated with antibiotic resistance, detected polymorphisms in the cmeABC locus likely play a role in the multiresistant phenotype. This study provides for the first time an overview of the genetic diversity of Campylobacter strains from Portugal. It also shows a worrying antibiotic multiresistance rate and the emergence of Campylobacter strains resistant to antibiotics of human use.

Distribution of aminogenic activity among potential autochthonous starter cultures for dry fermented sausages.

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 gram-positive, 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 beta-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 beta-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.

Effects of lairage temperature and holding time on pig behaviour and on carcass and meat quality

Abstract Twenty three groups of about 30 pigs each were kept in an environmentally controlled room in lairage, at temperatures of 20 or 35°C for periods of 0.5 or 3 h, to establish the effects of these parameters on animal behaviour and meat quality. Following initial exploration, 50% of pigs kept at 35°C but only 5% kept at 20°C lay down before the end of the 0.5 h period. Seven percent of pigs fought in both temperature conditions. When held in lairage for 0.5 h there was no difference in meat quality or skin damage at either temperature. When held for 3 h, about 95% of pigs were lying down after 2 h in the pens. Again, the percentage of animals fighting was similar for both temperatures, the number of encounters increasing during the first 30–40 min. The more intense initial fighting within the group held at 35°C resulted in the pigs lying down slightly earlier. Irrespective of the lairage time, the frequency of sexual activity decreased with temperature. The proportion of carcasses with skin damage increased with lairage time due, perhaps, to the longer duration of aggressive encounters. Increased lairage time at 20°C reduced the incidence of PSE meat, but at 35°C the longer lairage time showed no benefit to animal welfare or meat quality.

Antibiotic resistance of lactic acid bacteria isolated from dry-fermented sausages

Dry-fermented sausages are meat products highly valued by many consumers. Manufacturing process involves fermentation driven by natural microbiota or intentionally added starter cultures and further drying. The most relevant fermentative microbiota is lactic acid bacteria (LAB) such as Lactobacillus, Pediococcus and Enterococcus, producing mainly lactate and contributing to product preservation. The great diversity of LAB in dry-fermented sausages is linked to manufacturing practices. Indigenous starters development is considered to be a very promising field, because it allows for high sanitary and sensorial quality of sausage production. LAB have a long history of safe use in fermented food, however, since they are present in human gastrointestinal tract, and are also intentionally added to the diet, concerns have been raised about the antimicrobial resistance in these beneficial bacteria. In fact, the food chain has been recognized as one of the key routes of antimicrobial resistance transmission from animal to human bacterial populations. The World Health Organization 2014 report on global surveillance of antimicrobial resistance reveals that this issue is no longer a future prediction, since evidences establish a link between the antimicrobial drugs use in food-producing animals and the emergence of resistance among common pathogens. This poses a risk to the treatment of nosocomial and community-acquired infections. This review describes the possible sources and transmission routes of antibiotic resistant LAB of dry-fermented sausages, presenting LAB antibiotic resistance profile and related genetic determinants. Whenever LAB are used as starters in dry-fermented sausages processing, safety concerns regarding antimicrobial resistance should be addressed since antibiotic resistant genes could be mobilized and transferred to other bacteria.

Effect of gamma radiation on microbial population of natural casings

Abstract The high microbial load of fresh and dry natural casings increases the risk of meat product contamination with pathogenic microorganisms, agents of foodborn diseases. The aim of this work is to evaluate the killing effect of gamma radiation of the resident microbial population of pork and beef casings, to improve their hygiene and safety. Portions of fresh pork (small intestines and colon) and dry beef casings were irradiated in a Cobalt 60 source with with absorbed doses of 1,2,5 and 10 kGy. The D 10 values of total aerobic microorganisms in the pork casings were 1.65 kGy for colon and 1.54 kGy for small intestine. The D 10 value found in beef dry casings (small intestine) was 10.17 kGy. Radurization with 5 kGy was able to reduce, at least, 6 logs the coliform bacteria in pork casings. The killing effect over faecal Streptococci was 4 logs for pork fresh casings and 2 logs for beef dry casings. Gamma radiation with 5 kGy proved to be a convenient method to reduce substantially the microbial population of pork fresh casings. Otherwise, the microbial population of beef dry casings still resisted to 10 kGy.

Antimicrobial resistance among Campylobacter spp. strains isolated from different poultry production systems at slaughterhouse level

The aim of the current work was to evaluate the prevalence and antimicrobial susceptibility of Campylobacter spp. isolated from different chicken production systems at the slaughterhouse level. Chicken sampling at slaughterhouse was performed for cecum, carcass, and breast meat from flocks of organic (n = 6), extensive indoor (n = 14), and intensive production (n = 14), totaling 34 ceca pools, 64 neck skin pools, and 132 breasts, representing 96,386 chickens. A collection of 167 strains were identified as Campylobacter coli (n = 85) and Campylobacter jejuni (n = 82) and were tested for susceptibility to 11 antimicrobial agents by the disk diffusion method. The frequency of Campylobacter in chicken samples from different production systems was between 79 and 100%. Campylobacter isolated from all origins were resistant to the fluoroquinolones studied (80-98%). However, for ciprofloxacin and ofloxacin, the Campylobacter isolates from extensive indoor chicken were significantly (P < 0.05) less resistant (77 and 58%) than that from organic (97 and 91%) and intensive production (96 and 95%). A high probability of tetracycline resistance occurrence was also found for the Campylobacter spp. tested (58% for C. jejuni and 76% for C. coli). A more frequent profile of multidrug resistance was noticed for isolates from intensive and organic production than for extensive indoor production. These results reinforce the need of efficient strategy implementation to control and reduce Campylobacter in chickens at production and slaughter levels, and the necessity to reduce the use of antimicrobials in poultry sector.

Biogenic amine formation in turkey meat under modified atmosphere packaging with extended shelf life: Index of freshness

The aim of this study was to evaluate the effect of modified atmosphere packaging (MAP) on biogenic amine production in turkey meat according to its shelf life period, determining an index of freshness. Sliced meat samples of different meat quality categories (according to color and pH₂₄) were individually packaged under aerobiosis (aerobic package) and in 6 different modified atmospheres containing different gas mixtures: MAP1, 50% N₂/50% CO₂; MAP2, 0.5% CO/50% CO₂/49.5% N₂; MAP3, 50% Ar/50% N₂; MAP4, 0.5% CO/80% CO₂/19.5% N₂; MAP5, 100% N₂; and MAP6, 50% Ar/50% CO₂. All samples were stored at 0 ± 1°C in the dark for between 12 and 25 d. Meat samples packaged in aerobic packaging were analyzed for their microbial and physicochemical characteristics on d 0, 5, and 12 of storage, and then extended to 19 and 25 d when samples were under MAP. The production of biogenic amines analyzed in turkey meat increased over time. The values of putrescine, cadaverine, and tyramine increased significantly (P < 0.05) during storage time in samples packaged under aerobiosis, MAP3, and MAP5. Histamine was not detected in turkey meat packaged under study conditions, or when present, the levels were below the limit of quantification (1.03 mg/kg). Tyramine in turkey meat under MAP was not the best amine indicator of meat deterioration, with cadaverine being suggested instead, or the sum of the amines putrescine, cadaverine, and tyramine, to characterize and quantify meat freshness. After 25 d of storage, the meat packaged under MAP with a mixture containing a higher concentration of CO₂ and with CO was the one with a lower index value (11.36 mg/kg), although not significantly different from the indices provided by the meat packaged with MAP1, 2, and 6.