The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

Amino acid decarboxylase activity of the Enterococcus faecalis strain EF37 was monitored during fermentation and ripening of a traditional dry fermented sausage from Northern Italy (Salame Veronese) by means of microbiological, chemical, and molecular approaches in relation to three technological factors: fermentation temperature, sodium chloride concentration, and amount of glucose added to the meat mixture. Besides the analytical determination of tyramine and phenylethylamine accumulation and the counts of enterococci, the presence and quantification of the tyrosine decarboxylase gene (tdc) and its mRNA transcript were also investigated by using real-time PCR. According to the mathematical models obtained, all of the three factors studied were statistically significant and microbiologically relevant for the early development of enterococci, although the fermentation temperature had a more relevant influence on the enterococcal viable cells of the ripened product. Sodium chloride concentration was the most determinant factor of the final tyramine and 2-phenylethylamine accumulation and also of the levels of tdc present in the final product. In contrast, an effect of glucose concentration on tdc expression was observed in the last period of ripening. Moreover, increasing amounts of sodium chloride and decreasing fermentation temperature resulted in a reduced tdc expression. This is the first time that bacterial tyrosine decarboxylase potential is directly examined through a molecular approach in a fermented meat. The quantification of tdc and its transcript can help to elucidate the critical steps and factors during food manufacturing at which bacterial aminogenesis is possible, thus allowing researchers to propose technological measures to control decarboxylase activities.

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In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

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The aim of this study was to investigate the diversity of tyramine production capability of four Enterococcus strains in buffered systems in relation to their genetic characteristics and environmental conditions. Cells of the strains Enterococcus faecalis EF37 and ATCC 29212, and E. faecium FC12 and FC643 were re-suspended in phosphate/citrate buffers with different pH, NaCl concentration and incubation temperature. At intervals, cell viability and tyramine production were assessed by plate counting and HPLC analysis, respectively. The activity of a purified tyrosine decarboxylase (TDC) was determined under the same conditions, as a reference. Reduced loss in cell viability was observed in all the tested conditions, except for pH 4 after 24 h. The TDC activity was greatly heterogeneous within the enterococci: EF37 and FC12 produced the higher tyramine concentrations, ATCC 29212 showed a reduced decarboxylase activity, while EF643 did not accumulate detectable amounts of tyramine in all the conditions assayed. Among the considerate variables, temperature was the most influencing factor on tyramine accumulation for enterococcal cells. To further correlate the phenotypic and genetic characteristics of the enterococci, the TDC operon region carrying the genes tyrosine decarboxylase (tyrDC), tyrosine/tyramine permease (tyrP), and Na+/H+ antiporter (nhaC-2) was amplified and sequenced. The genetic organization and nucleotide sequence of this operon region were highly conserved in the enterococcal strains of the same species. The heterogeneity in tyramine production found between the two E. faecalis strains could be ascribed to different regulation mechanisms not yet elucidated. On the contrary, a codon stop was identified in the translated tyrDC sequence of E. faecium FC643, supporting its inability to accumulate tyramine in the tested conditions. In addition, the presence of an additional putative tyrosine decarboxylase with different substrate specificity and genetic organization was noticed for the first time. Concluding, the high TDC activity heterogeneity within enterococci determined different accumulation of tyramine, depending on different genetic determinants, regulation mechanisms, and environmental factors. The present research contributes to elucidate the genetic characteristics of enterococcal strains and correlate specific mutations to their different strain-dependent tyraminogenic activity.

Aims:  To evaluate the concomitant effects of three technological variables (fermentation temperature, NaCl and glucose added to the meat batter) on diamines (cadaverine, putrescine and histamine) accumulation and microbial changes during ripening of dry fermented sausages. Methods and Results:  The variables were modulated according to an experimental design and predictive mathematical models were obtained. The models indicated that the sausages were characterized by low histamine amount independently on the applied conditions. In contrast, putrescine and cadaverine accumulation was considerable and significantly affected by the three variables. The microbial population dynamics suggest that lactic acid bacteria (LAB) and microstaphylococci are favoured by increasing glucose concentration until 0·7 g kg−1, while Enterobacteriaceae are negatively influenced by NaCl concentration and, to a lesser extent, by fermentation temperature. Conclusions:  Data obtained showed a relationship between Enterobacteriaceae growth and cadaverine and putrescine accumulation in sausages during ripening. The conditions more favourable for LAB and microstaphylococci induced a reduced growth of Enterobacteriaceae with a consequent reduced accumulation of putrescine and cadaverine. Significance and Impact of the Study:  The use of systematic experimental designs allows to individuate the technological conditions suitable to keep the aminogenic microflora under control, thus reducing the risk of diamines production during traditional fermented food manufacture.

In this study, microbiological aspects of Grana Trentino, a variant of Grana Padano cheese, were defined by plate counts, random amplified polymorphic DNA (RAPD) PCR genotying, 16S rRNA gene sequencing of bacterial isolates and PCR–denaturing gradient gel electrophoresis (PCR–DGGE). Results showed variability in monthly fluctuations of whey culture counts, differences in the diffusion of bacterial genotypes among producers and dairy plant‐specific microbial associations. Moreover, the presence of bacteria not previously reported in this cheese type was highlighted, including coagulase‐negative staphylococci and Lactobacillus sanfranciscensis‐like micro‐organisms.