Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages.

The aim of this study was to determine the technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase and enterotoxigenic activities) of 38 strains of Staphylococcaceae (11 of Staphylococcus epidermidis, 15 of Staphylococcus equorum, 5 of Staphylococcus pasteuri and 7 of Staphylococcus saprophyticus) isolated from Androlla and Botillo, two Spanish traditional sausages, in order to evaluate their suitability as potential starter cultures in the manufacture of these sausages. Most strains were able to grow at 10 °C, in the presence of 10% and 15% NaCl and at pH values of 5.5 and 5.0, except for S. equorum strains, growth of which was reduced at these pH values. The proteolytic activity assessed by the agar plate method showed that 89.5% and 52.6% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were not confirmed by electrophoretic assays as only 47.2% of the strains changed the SDS-PAGE profile of actin, myosin and/or sarcoplasmic protein extracts. The assessment of the lipolytic activity by titration showed that only 21.0% of the strains can hydrolyze pork fat to any extent; whereas the profiles of the freed fatty acids were different in the different strains. Most of the strains showed decarboxylase activity against histidine, lysine, ornithine and tyrosine, but the quantities of biogenic amines produced were in most cases <25 ppm and <5 ppm for putrescine and cadaverine, respectively. Only four strains (10.5%), of S. epidermidis, produced enterotoxin C.

Monitoring the bacterial population dynamics during the ripening of Galician chorizo, a traditional dry fermented Spanish sausage.

The dynamics of the bacterial population throughout the ripening of Galician chorizo, a traditional dry fermented sausage produced in the north-west of Spain, were investigated by using classical and molecular approaches. Fermented sausages are certainly complex matrices with PCR inhibitors and background microbiota. Therefore, two different DNA preparation methods were performed to elaborate each standard curve and no significant differences were found, showing a very good correlation between both methods (R(2)>0.994). The combination of the results obtained from microbial counts, species and genus-specific PCR as well as real-time quantitative PCR (qPCR) allowed the identification for the dominant bacterial species and the study of the variation in the community composition over the ripening period. According to the data obtained both by identification of plate isolates and by real-time PCR, the dominant species among staphylococci and lactobacilli were Staphylococcus equorum and Lactobacillus sakei, respectively. However, only real-time PCR assay showed enough sensitivity to detect and quantify staphylococci in meat batter before stuffing, showing values of 5.28logCFU/g when quantifying Staphylococcus spp. and 2.87logCFU/g when quantifying S. equorum. In conclusion, real-time PCR was shown to be an efficient tool for the study of the complex associations developed in meat fermentations and for the characterization of dominant populations.

Study of the lactic acid bacteria throughout the manufacture of dry-cured lacón (a Spanish traditional meat product). Effect of some additives.

Total aerobic mesophilic microflora (on SPC agar), lactic acid bacteria (on MRS agar) and lactobacilli (on Rogosa agar) were enumerated in samples from the surface and the interior of the pieces throughout the manufacture of six batches of lacón. Three of the batches were made without additives and three with additives (glucose (2 g/kg), sodium nitrite (E(250)) (125 mg/kg), sodium nitrate (E(251)) (175 mg/kg), sodium ascorbate (E(301)) (500 mg/kg), and sodium citrate (E(331)) (100 mg/kg)). The counts decreased throughout the manufacturing process, particularly after the salting stage. The use of additives did not affect the counts or the evolution of the microbial groups, except for the lactobacilli, which were present in higher numbers in the batches with additives. In four batches (two without and two with additives), from MRS agar and from Rogosa agar plates, 10 colonies were randomly taken from each sampling point of each batch (five from the surface sample and five from the interior sample) and from each culture medium; a total of 224 strains from MRS agar, and 176 strains from Rogosa agar that were identified by classical methods. The MRS agar displayed moderate selectivity for the isolation of lactic acid bacteria, and only 59% of the isolated strains belonged to this microbial group. Homofermentative and facultative heterofermentative lactobacilli (particularly Lactobacillus curvatus and Lactobacillus sakei) were the most abundant species isolated on this medium. The selectivity of the Rogosa agar for lactobacilli was extremely high. The species of lactobacilli isolated on this medium at different stages of manufacture of the four batches of lacón were consistent with those isolated from MRS agar. The use of additives in the lacón did not appreciably affect the kinds and proportions of species isolated on either MRS agar or Rogosa agar.

Metabolic characterization of Bacillus subtilis and Bacillus amyloliquefaciens strains isolated from traditional dry-cured sausages.

The aim of this study was to investigate the effect of pH, temperature, and NaCl on growth, proteolytic and lipolytic activities, and the ability to produce biogenic amines of 19 strains of Bacillus isolated from Androlla and Botillo (two Spanish traditional sausages) to elucidate the role of these bacteria in sausage manufacture. All strains grew in the presence of 10% salt and at pH values of 5.0 and 5.5, whereas only 9 strains grew at 10°C. Proteolytic activity was assessed by the agar plate method, which revealed that 100 and 94.7% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were confirmed by electrophoretic assays. The titration method revealed that only two strains hydrolyzed pork fat to any extent, and the profiles of the fatty acids freed were different. Most strains produced biogenic amines, but the quantities were generally low.

Microbiological and physicochemical characterization of dry-cured Halal goat meat. Effect of salting time and addition of olive oil and paprika covering.

The objective of this work was to define a simple technological process for dry-cured Halal goat meat elaboration. The aims of this study were to analyze physicochemical parameters and to enumerate the microbial population at the end of the different manufacturing processes (two salting times and the addition of olive oil and paprika covering) on 36 units of meat product. A total of 532 strains were isolated from several selective culture media and then identified using classical and molecular methods. In general, salt effect and the addition of olive oil and paprika were significant for all the studied microbial groups as well as on NaCl content and water activity. Molecular analysis proves that staphylococci, especially Staphylococcus xylosus and Staphylococcus equorum, were the most common naturally occurring microbiota. The best manufacturing process would be obtained with a longer salting time and the addition of the olive oil and paprika covering.

Effect of modified atmosphere and vacuum packaging on some quality characteristics and the shelf-life of "morcilla", a typical cooked blood sausage.

The effect of modified atmosphere and vacuum packaging on the shelf-life of "morcilla", a traditional cooked blood sausage, was investigated. A total of 99 "morcillas" were packaged under vacuum and in modified atmosphere using three different gas mixtures: 15:35:50/O(2):N(2):CO(2) (atmosphere 1), 60:40/N(2):CO(2) (atmosphere 2) and 40:60/N(2):CO(2) (atmosphere 3), and stored during 2, 4, 6 and 8 weeks at 4 °C. Shelf life evaluation was based on pH, water activity (a(w)), colour (CIE L*, a*, b*, C* and h*), TBARS formation and microbial counts. The results indicated that, in general, storage time affected (P<0.05) all parameters whereas no significant differences were observed (P>0.05) among packaging conditions. Based on the microbial counts, the shelf-life of "morcilla" would be greater than 8 weeks for all packaging conditions. Samples packaged with high CO(2) concentrations (40:60/N(2):CO(2)) showed the lowest values of TBARS at the end of storage.

Production of biogenic amines "in vitro" in relation to the growth phase by Enterobacteriaceae species isolated from traditional sausages.

Histidine, lysine, ornithine and tyrosine decarboxylase activities were tested in 79 strains of Enterobacteriaceae (41 of Hafnia alvei, 17 of Serratia liquefaciens, 5 of Enterobacter cloacae, 4 of Citrobacter braakii, 2 of Proteus vulgaris, 2 of Proteus mirabilis, 2 of Providencia stuartii, 2 of Klebsiella terrigena, 1 of Rahnella aquatilis, 1 of Salmonella arizonae, 1 of Citrobacter youngae and 1 of Escherichia coli) isolated from Botillo, a Spanish traditional sausage. In general, the strains were positive for all four activities, with the exception of two strains of H. alvei and the E. coli strain, which did not display histidine decarboxylase activity. The strains of P. mirabilis and P. stuartii did not exhibit any of the four activities tested. Accumulation of putrescine and cadaverine was studied throughout growth of the 75 strains that displayed ornithine and lysine decarboxylase activities. Biogenic amines were produced particularly in the exponential phase, with maximum accumulation occurring after between 12 to 72 h, depending on the biogenic amine and microbial species considered. Maximum accumulation of putrescine varied greatly between species and within the same species, and ranged from 18 mg/l in the R. aquatilis strain to 7325 mg/l in a H. alvei strain. Maximum accumulation of cadaverine varied less than that of putrescine, and ranged from 30 mg/l in the R. aquatilis strain to 1935 mg/l in a S. liquefaciens strain.