Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Reconstruction of Simplified Microbial Consortia to Modulate Sensory Quality of Kombucha Tea.

Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation-acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)-is mainly found embedded in an extracellular cellulosic matrix located at the liquid-air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains' metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance.

Investigating the biotechnological potential of lactic acid bacteria strains isolated from different Algerian dairy and farm sources.

Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

New insights into the variability of lactic acid production in Lachancea thermotolerans at the phenotypic and genomic level.

Non-conventional yeasts are increasingly applied in fermented beverage industry to obtain distinctive products with improved quality. Among these yeasts, Lachancea thermotolerans has multiple features of industrial relevance, especially the production of l(+)-lactic acid (LA), useful for the biological acidification of wine and beer. Since few information is available on this peculiar activity, the current study aimed to explore the physiological and genetic variability among L. thermotolerans strains. From a strain collection, mostly isolated from wine, a huge phenotypic diversity was acknowledged and allowed the selection of a high (SOL13) and a low (COLC27) LA producer. Comparative whole-genome sequencing of these two selected strains and the type strain CBS 6340T showed a high similarity in terms of gene content and functional annotation. Notwithstanding, target gene-based analysis revealed variations between high and low producers in the key gene sequences related to LA accumulation. More in-depth investigation of the core promoters and expression analysis of the genes ldh, encoding lactate dehydrogenase, indicated the transcriptional regulation may be the principal cause behind phenotypic differences. These findings highlighted the usefulness of whole-genome sequencing coupled with expression analysis. They provided crucial genetic insights for a deeper investigation of the intraspecific variability in LA production pathway.

Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking.

The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.

Antimicrobial spectrum activity of bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk.

Bacteriocins have attracted great attention as potential alternatives to antibiotics and chemical food additives. In the present study, 243 Staphylococcus isolates from milk samples (n = 110) of goat and sheep herds located in Fars province, Iran, were screened for antimicrobial substance production. Twenty-eight isolates showed an antagonistic activity against the indicator strain Micrococcus luteus ATCC 4698. The susceptibility of all antimicrobial substances to proteolytic enzymes allowed us to consider them as bacteriocin-like inhibitory substances (BLIS). The term BLIS is applied to uncharacterized proteinaceous antimicrobials produced by gram-positive bacteria. Based on molecular identification methods, the isolates belonged to the species Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus pseudintermedius, Staphylococcus aureus, and Staphylococcus agnetis. Pulsed-field gel electrophoresis revealed a high level of genotype diversity among the Staph. chromogenes isolates. All of the isolates harbored nukA or bsaA2 genes, suggesting that their BLIS were related to nukacin or Bsa. The antimicrobial compounds from test strains were not effective against gram-negative pathogens, including Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Klebsiella pneumonia as well as the indicator mold Aspergillus fumigatus. All the gram-positive targets, including Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis Ef37 (a tyramine-producer strain), Lactobacillus saerimneri 30a (a histamine-producer strain), and methicillin-resistant Staph. epidermidis, were inhibited by the Staph. chromogenes isolates. Staphylococcus haemolyticus 4S12 was able to inhibit the majority of gram-positive bacteria. Listeria monocytogenes strains were the only indicators sensitive to the antimicrobial agents produced by Staph. agnetis 4S97B. The other Staphylococcus strains were ineffective on all the organisms tested. Based on their inhibitory capacities, the BLIS produced by the Staph. chromogenes isolates seem to be interesting candidates for developing novel antimicrobial agents.

Safety hazards in bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk.

In this study, 28 bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk were subjected to the PCR detection of enterotoxin genes (sea-see), enterotoxin-like toxin Q gene (selq), toxic shock syndrome toxin gene (tst1), and antibiotic resistance genes. They were also evaluated for phenotypic resistance against 10 antibiotics and hemolytic activity. The tyramine and histamine production was investigated using the agar plate assay and capillary zone electrophoretic analysis (CZE). Twenty-five isolates harbored at least one enterotoxin gene. The gene sec was the most frequent (89%). The gene tst1 was found in 84% of sec-positive isolates. The occurrence of antibiotic resistance genes was in the order of blaZ/tetK (100%), mecA/ermB (86%), ermC (50%), and tetM (18%). The genes ermA, aac(6')Ie-aph(2″)Ia, vanA, and vanB were absent in all the isolates. Nineteen isolates were phenotypically susceptible to all the antibiotics. The only isolate with phenotypic resistance to penicillin G and oxacillin was S. epidermidis 4S93 which had a different SmaI-PFGE profile from those of the other S. epidermidis strains. All the S. haemolyticus and S. pseudintermedius isolates were not susceptible to trimethoprim. Twenty-five isolates showed complete or partial hemolytic activity. None of the isolates was able to decarboxylate tyrosine, while CZE analysis revealed histamine formation activity in S. haemolyticus 4S12. The occurrence of safety risks in the isolates reinforces the need for regular monitoring of food-producing animals to mitigate the risks of multidrug resistant and zoonotic pathogens. Moreover, none of the isolates fulfilled the safety criteria to be used as starter cultures or biopreservatives.

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.

Draft Genome Sequence of the Probiotic Yeast Kluyveromyces marxianus fragilis B0399.

Here, we report the draft genome sequence of Kluyveromyces marxianus fragilis B0399, the first yeast approved as a probiotic for human consumption not belonging to the genus Saccharomyces The genome is composed of 8 chromosomes, with a total size of 11.44 Mb, including mitochondrial DNA.

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.

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.

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.

Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

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.

Modeling the aminogenic potential of Enterococcus faecalis EF37 in dry fermented sausages through chemical and molecular approaches.

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.

A genetic insight into peptide and amino-acid utilization by Propionibacterium freudenreichii LMG 16415.

In this note the genetic characterization of the peptide degrading system of Propionibacterium freudenreichii was addressed. Genomic fragments of P. freudenreichii subsp. freudenreichii LMG 16415 were cloned in Escherichia coli XL1 Blue, and those leading to an increase in peptidase-like activity using chromogenic substrates aminoacyl-beta-naphtylamides (aminoacyl-betaNA) were isolated and sequenced. This strategy allowed the identification of partial gene regions of P. freudenreichii LMG 16415 with significant similarity to proteins directly or indirectly involved in peptide and amino acid metabolism, i.e., an oligopeptide transporter, a D-amino acid oxidase, a muropeptidase, and an ABC transporter involved in osmoregulation similar to glycine betaine transporters.