Eleonora Bargossi

Correlation between volatile profiles of Italian fermented sausages and their size and starter culture

The aroma profiles of 10 traditional Italian fermented sausages were evaluated. The volatile organic compounds (VOCs) obtained by solid-phase microextraction and gas chromatograph-mass spectrometry were analysed using principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed an acceptable separation but some sausage typologies were not well separated. On the other hand, the supervised approach of LDA allowed a clear grouping of the samples in relation to sausage size and starter culture. In spite of the extreme variability of the volatile profiles of the sausage typologies, this work showed the influence of diameter on VOC profile. The differences observed can be related to the effects that some fundamental physicochemical characteristics (such as water loss kinetics and oxygen availability) have on the results of ripening processes. Differences in VOC profiles were also observed due to the lactic acid bacteria used as starter cultures, with differences mainly attributable to compounds deriving from pyruvate metabolism.

Control of tyramine and histamine accumulation by lactic acid bacteria using bacteriocin forming lactococci.

The aim of this study was to evaluate the competitive effects of three bacteriocin producing strains of Lactococcus lactis subsp. lactis against two aminobiogenic lactic acid bacteria, i.e. the tyramine producing strain Enterococcus faecalis EF37 and the histamine producing strain Streptococcus thermophilus PRI60, inoculated at different initial concentrations (from 2 to 6 log cfu/ml). The results showed that the three L. lactis subsp. lactis strains were able to produce bacteriocins: in particular, L. lactis subsp. lactis VR84 and EG46 produced, respectively, nisin Z and lacticin 481, while for the strains CG27 the bacteriocin has not been yet identified, even if its peptidic nature has been demonstrated. The co-culture of E. faecalis EF37 in combination with lactococci significantly reduced the growth potential of this aminobiogenic strain, both in terms of growth rate and maximum cell concentration, depending on the initial inoculum level of E. faecalis. Tyramine accumulation was strongly reduced when E. faecalis EF37 was inoculated at 2 log cfu/ml and, to a lesser extent, at 3 log cfu/ml, as a result of a lower cell load of the aminobiogenic strain. All the lactococci were more efficient in inhibiting streptococci in comparison with E. faecalis EF37; in particular, L. lactis subsp. lactis VR84 induced the death of S. thermophilus PRI60 and allowed the detection of histamine traces only at higher streptococci inoculum levels (5-6 log cfu/ml). The other two lactococcal strains did not show a lethal action against S. thermophilus PRI60, but were able to reduce its growth extent and histamine accumulation, even if L. lactis subsp. lactis EG46 was less effective when the initial streptococci concentration was 5 and 6 log cfu/ml. This preliminary study has clarified some aspects regarding the ratio between bacteriocinogenic strains and aminobiogenic strains with respect to the possibility to accumulate BA and has also showed that different bacteriocins can have different effects on BA production on the same strain. This knowledge is essentially aimed to use bacteriocinogenic lactococci as a predictable strategy against aminobiogenic bacteria present in cheese or other fermented foods.

Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression

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.

The Capability of Tyramine Production and Correlation between Phenotypic and Genetic Characteristics of Enterococcus faecium and Enterococcus faecalis Strains

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.

Physico-chemical and microbiological characterisation of Italian fermented sausages in relation to their size

BACKGROUND The aim of this work was to study the physico-chemical and microbiological parameters of 10 Italian fermented sausages, produced industrially, in order to highlight the differences in relation to their size. The sausages were classified as small, medium and large and the data concerning every feature considered were analysed with some statistical explorative tools: ANOVA, principal component analysis (PCA) and linear discriminant analysis (LDA). RESULTS Significant differences in relation to the sausage size were found regarding microbial populations (in particular, enterobacteria and staphylococci). The pH was higher in the small sausages, and consequently the presence of lactate and acetate was higher in the bigger one. Also the biogenic amine content (particularly tyramine, cadaverine and putrescine) was influenced by the size. CONCLUSIONS In spite of the extreme variability of the sausage types at the end of ripening, this work showed that it was possible to find a strict relationship between sausages diameter and some essential physico-chemical parameters. Thus, the cross-sectional size of the product is an essential parameter, which can drive the biochemical processes during ripening, not only by affecting the kinetics of water losses, but also by influencing the microbiota enzymatic activity.

Growth, biogenic amine production and tyrDC transcription of Enterococcus faecalis in synthetic medium containing defined amino acid concentrations

The tyraminogenic potential of the strains Enterococcus faecalis EF37 and ATCC 29212 was investigated in a synthetic medium containing defined amounts of tyrosine and phenylalanine at different temperatures.

Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects

Few information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant‐derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2‐phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations.