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Stefano RAIMONDI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Chimica
Ricercatore Legge 240/10 - t.det.
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103

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Pubblicazioni

2024 - Fermentative processes for the upcycling of xylose to xylitol by immobilized cells of Pichia fermentans WC1507 [Articolo su rivista]
Ranieri, R.; Candeliere, F.; Moreno-García, J.; Mauricio, J. C.; Rossi, M.; Raimondi, S.; Amaretti, A.
abstract

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)(2)SO4 and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, Y-P/S 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While Y-P/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, Y-P/S of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions.

2024 - Genomic and functional analysis of the mucinolytic species Clostridium celatum, Clostridium tertium, and Paraclostridium bifermentans [Articolo su rivista]
Candeliere, Francesco; Musmeci, Eliana; Sola, Laura; Amaretti, Alberto; Raimondi, Stefano; Rossi, Maddalena
abstract

: Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and release monosaccharides that can be assimilated. In this study, we present a characterization on the strains Clostridium celatum WC0700, Clostridium tertium WC0709, and Paraclostridium bifermentans WC0705. These three strains were previously isolated from enrichment cultures on mucin of fecal samples from healthy subjects and can use mucin as sole carbon and nitrogen source. Genome analysis and in vitro functional analysis of these strains elucidated their physiological and biochemical features. C. celatum WC0700 harbored the highest number of glycosyl hydrolases specific for mucin degradation, while P. bifermentans WC0705 had the least. These predicted differences were confirmed growing the strains on 5 mucin-decorating monosaccharides (L-fucose, N-Acetylneuraminic acid, galactose, N-acetylgalactosamine, and N-acetylglucosamine) as only source of carbon. Fermenting mucin, they all produced formic, acetic, propionic, butyric, isovaleric, and lactic acids, and ethanol; acetic acid was the main primary metabolite. Further catabolic capabilities were investigated, as well as antibiotic susceptibility, biofilm formation, tolerance to oxygen and temperature. The potential pathogenicity of the strains was evaluated through in silico research of virulence factors. The merge between comparative and functional genomics and biochemical/physiological characterization provided a comprehensive view of these mucin degraders, reassuring on the safety of these species and leaving ample scope for deeper investigations on the relationship with the host and for assessing if some relevant health-promoting effect could be ascribed to these SCFA producing species.

2024 - Good and bad dispositions between archaea and bacteria in the human gut: New insights from metagenomic survey and co-occurrence analysis [Articolo su rivista]
Candeliere, F.; Sola, L.; Raimondi, S.; Rossi, M.; Amaretti, A.
abstract

Archaea are an understudied component of the human microbiome. In this study, the gut archaeome and bacteriome of 60 healthy adults from different region were analyzed by whole-genome shotgun sequencing. Archaea were ubiquitously found in a wide range of abundances, reaching up to 7.2 %. The dominant archaeal phylum was Methanobacteriota, specifically the family Methanobacteriaceae, encompassing more than 50 % of Archaea in 50 samples. The previously underestimated Thermoplasmatota, mostly composed of Methanomassiliicoccaceae, dominated in 10 subjects (>50 %) and was present in all others except one. Halobacteriota, the sole other archaeal phylum, occurred in negligible concentration, except for two samples (4.6–4.8 %). This finding confirmed that the human gut archaeome is primarily composed of methanogenic organisms and among the known methanogenic pathway: i) hydrogenotrophic reduction of CO2 is the predominant, being the genus Methanobrevibacter and the species Methanobrevibacter smithii the most abundant in the majority of the samples; ii) the second pathway, that involved Methanomassiliicoccales, was the hydrogenotrophic reduction of methyl-compounds; iii) dismutation of acetate or methyl-compounds seemed to be absent. Co-occurrence analysis allowed to unravel correlations between Archaea and Bacteria that shapes the overall structure of the microbial community, allowing to depict a clearer picture of the human gut archaeome.

2023 - Ce-MBGs Loaded with Gentamicin: Characterization and In Vitro Evaluation [Articolo su rivista]
Fraulini, Francesca; Raimondi, Stefano; Candeliere, Francesco; Ranieri, Raffaella; Zambon, Alfonso; Lusvardi, Gigliola
abstract

Mesoporous Bioactive Glasses (MBGs) are biomaterials widely used in tissue engineering, particularly for hard tissue regeneration. One of the most frequent postoperative complications following a biomaterial surgical implant is a bacterial infection, which usually requires treatment by the systemic administration of drugs (e.g., antibiotics). In order to develop biomaterials with antibiotic properties, we investigated cerium-doped MBGs (Ce-MBGs) as in situ-controlled drug delivery systems (DDSs) of gentamicin (Gen), a wide spectrum antibiotic commonly employed against bacteria responsible of postoperative infections. Here we report the optimization of Gen loading on MBGs and the evaluation of the antibacterial properties and of retention of bioactivity and antioxidant properties of the resulting materials. The Gen loading (up to 7%) was found to be independent from cerium content, and the optimized Gen-loaded Ce-MBGs retain significant bioactivity and antioxidant properties. The antibacterial efficacy was verified up to 10 days of controlled release. These properties make Gen-loaded Ce-MBGs interesting candidates for simultaneous hard tissue regeneration and in situ antibiotic release.

2023 - Dual loaded Ce-MBGs with bioactivity, antioxidant and antibacterial properties [Articolo su rivista]
Zambon, Alfonso; Fraulini, Francesca; Raimondi, Stefano; Lusvardi, Gigliola
abstract

2023 - Hemp Biomass Pretreatment and Fermentation with non- Saccharomyces Yeasts: Xylose Valorization to Xylitol [Articolo su rivista]
Raimondi, S.; Ranieri, R.; Leonardi, A.; Ottolina, G.; Rossi, M.; Amaretti, A.
abstract

Hemp hurds are the main byproduct from hemp fibers supply chain and they could represent valuable feedstock of lignocellulosic biomass for biorefineries. The industrial hemp variety "Carmagnola", is characterized by low amounts of ash and high amount of carbohydrates. Alpha-cellulose (44% w/w), hemicellulose (25%), and lignin (23%) were fractionated using an organosolv pretreatment. The enzymatic hydrolysis of the cellulose fraction yielded up to 60% of glucose, that can easily find application as substrate for industrial fermentations. On the other hand, the black liquor originating from hemicellulose contains mainly xylose and minor amounts of other sugars. In the perspective of finding an application of black liquor, 50 yeasts belonging to 24 ascomycetous species were screened both in aerobiosis and anaerobiosis for the production of ethanol and the sugar alcohols xylitol and arabitol from xylose. Pichia fermentans WC 1507, Wickerhamomyces anomalus WC 1501, and Kluyveromyces bacillosporus WC 1404 were found to consume xylose, yielding xylitol in aerobic conditions. In particular, aerobic flask cultures of P. fermentans WC 1507 containing 120 g/L xylose showed the highest xylitol production values, yielding 63.5 g/L xylitol with a YP/S of 71.5%. Black liquor, exploited as a carbon source, has been successfully utilized by the three selected yeast strains at a concentration of 20 g/L in the culture medium, resulting in comparable or higher yields of biomass and xylitol compared to the medium containing pure xylose. A higher concentration of black liquor in the culture medium (to reach 120 g/l of xylose) has determined an inhibition of the growth of yeasts indicating the need for treatments for the removal of inhibitors. These preliminary results can be considered promising for the microbial valorization of lignocellulosic hemp feedstock toward the production of xylitol.

2023 - Profiling of the intestinal community of Clostridia: taxonomy and evolutionary analysis [Articolo su rivista]
Candeliere, Francesco; Musmeci, Eliana; Amaretti, Alberto; Sola, Laura; Raimondi, Stefano; Rossi, Maddalena
abstract

: Aim: Clostridia are relevant commensals of the human gut due to their major presence and correlations to the host. In this study, we investigated intestinal Clostridia of 51 healthy subjects and reconstructed their taxonomy and phylogeny. The relatively small number of intestinal Clostridia allowed a systematic whole genome approach based on average amino acid identity (AAI) and core genome with the aim of revising the current classification into genera and determining evolutionary relationships. Methods: 51 healthy subjects' metagenomes were retrieved from public databases. After the dataset's validation through comparison with Human Microbiome Project (HMP) samples, the metagenomes were profiled using MetaPhlAn3 to identify the population ascribed to the class Clostridia. Intestinal Clostridia genomes were retrieved and subjected to AAI analysis and core genome identification. Phylogeny investigation was conducted with RAxML and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithms, and SplitsTree for split decomposition. Results: 225 out of 406 bacterial taxonomic units were ascribed to Bacillota [Firmicutes], among which 124 were assigned to the class Clostridia. 77 out of the 124 taxonomic units were referred to a species, altogether covering 87.7% of Clostridia abundance. According to the lowest AAI genus boundary set at 55%, 15 putative genera encompassing more than one species (G1 to G15) were identified, while 19 species did not cluster with any other one and each appeared to belong to a diverse genus. Phylogenetic investigations highlighted that most of the species clustered into three main evolutive clades. Conclusion: This study shed light on the species of Clostridia colonizing the gut of healthy adults and pinpointed several gaps in knowledge regarding the taxonomy and the phylogeny of Clostridia.

2023 - Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures [Articolo su rivista]
Nasuti, C.; Ruffini, J.; Sola, L.; Di Bacco, M.; Raimondi, S.; Candeliere, F.; Solieri, L.
abstract

: The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.

2022 - Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of D-arabitol from pure glycerol [Articolo su rivista]
Raimondi, Stefano; Foca, Giorgia; Ulrici, Alessandro; Destro, Lorenza; Leonardi, Alan; Buzzi, Raissa; Candeliere, Francesco; Rossi, Maddalena; Amaretti, Alberto
abstract

D-Arabitol, a five-carbon sugar alcohol, represents a main target of microbial biorefineries aiming to valorize cheap substrates. The yeast Wickerhamomyces anomalus WC 1501 is known to produce arabitol in a glycerol-based nitrogen-limited medium and preliminary fed-batch processes with this yeast were reported to yield 18.0 g/L arabitol.

2022 - Indole and p-cresol in feces of healthy subjects: Concentration, kinetics, and correlation with microbiome [Articolo su rivista]
Candeliere, Francesco; Simone, Marta; Leonardi, Alan; Rossi, Maddalena; Amaretti, Alberto; Raimondi, Stefano
abstract

Indole and p-cresol are precursors of the most important uremic toxins, generated from the fermentation of amino acids tryptophan and tyrosine by the proteolytic community of intestinal bacteria. The present study focused on the relationship between the microbiome composition, the fecal levels of indole and p-cresol, and their kinetics of generation/degradation in fecal cultures. The concentration of indole and p-cresol, the volatilome, the dry weight, and the amount of ammonium and carbohydrates were analyzed in the feces of 10 healthy adults. Indole and p-cresol widely differed among samples, laying in the range of 1.0–19.5 μg/g and 1.2–173.4 μg/g, respectively. Higher fecal levels of indole and p-cresol were associated with lower carbohydrates and higher ammonium levels, that are markers of a more pronounced intestinal proteolytic metabolism. Positive relationship was observed also with the dry/wet weight ratio, indicator of prolonged intestinal retention of feces. p-cresol and indole presented a statistically significant negative correlation with OTUs of uncultured Bacteroidetes and Firmicutes, the former belonging to Bacteroides and the latter to the families Butyricicoccaceae (genus Butyricicoccus), Monoglobaceae (genus Monoglobus), Lachnospiraceae (genera Faecalibacterium, Roseburia, and Eubacterium ventriosum group). The kinetics of formation and/or degradation of indole and p-cresol was investigated in fecal slurries, supplemented with the precursor amino acids tryptophan and tyrosine in strict anaerobiosis. The presence of the precursors bursted indole production but had a lower effect on the rate of p-cresol formation. On the other hand, supplementation with indole reduced the net rate of formation. The taxa that positively correlated with fecal levels of uremic toxins presented a positive correlation also with p-cresol generation rate in biotransformation experiments. Moreover other bacterial groups were positively correlated with generation rate of p-cresol and indole, further expanding the range of taxa associated to production of p-cresol (Bacteroides, Alistipes, Eubacterium xylanophylum, and Barnesiella) and indole (e.g., Bacteroides, Ruminococcus torques, Balutia, Dialister, Butyricicoccus). The information herein presented contributes to disclose the relationships between microbiota composition and the production of uremic toxins, that could provide the basis for probiotic intervention on the gut microbiota, aimed to prevent the onset, hamper the progression, and alleviate the impact of nephropaties.

2022 - Investigation on the antimicrobial properties of cerium-doped bioactive glasses [Articolo su rivista]
Raimondi, S.; Zambon, A.; Ranieri, R.; Fraulini, F.; Amaretti, A.; Rossi, M.; Lusvardi, G.
abstract

Cerium-doped bioactive glasses (Ce-BGs) are implant materials that present high biocompatibility, modulate the levels of reactive oxygen species, and exert antimicrobial activity. The potential of BGs, 45S5, and K50S derived glasses doped with CeO2 (1.2, 3.6, and 5.3 mol%) to inhibit the growth of pathogen microbes was thoroughly investigated according to the ISO 22196:2011 method properly adapted. A significant reduction of the E. coli charge was detected in all glasses, including the BGs without cerium. The evolution of pH of the medium not inoculated following the immersion of the Ce-BGs was monitored. The presence of cerium did not affect markedly the pH trend, which increased rapidly for both compositions. The change of pH was strongly mitigated by the presence of 200 mM phosphate buffer pH 7.0 (PB) in the medium. In media buffered by PB, the growth of E. coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and C. albicans was not affected by the presence of BGs doped or not with cerium, suggesting that the antibacterial activity of Ce-BGs is linked to the increase of environmental pH rather than to specific ion effects. However, Ce-BGs resulted promising biomaterials that associate low toxicity to normal cells to a considerable antimicrobial effect, albeit the latter is not directly associated with the presence of cerium.

2022 - Microbiota Survey of Sliced Cooked Ham During the Secondary Shelf Life [Articolo su rivista]
Spampinato, Gloria; Candeliere, Francesco; Amaretti, Alberto; Licciardello, Fabio; Rossi, Maddalena; Raimondi, Stefano
abstract

2022 - Phylogenomic analysis of the genus Leuconostoc [Articolo su rivista]
Raimondi, Stefano; Candeliere, Francesco; Amaretti, Alberto; Costa, Stefania; Vertuani, Silvia; Spampinato, Gloria; Rossi, Maddalena
abstract

Leuconostoc is a genus of saccharolytic heterofermentative lactic acid bacteria that inhabit plant-derived matrices and a variety of fermented foods (dairy products, dough, milk, vegetables, and meats), contributing to desired fermentation processes or playing a role in food spoilage. At present, the genus encompasses 17 recognized species. In total, 216 deposited genome sequences of Leuconostoc were analyzed, to check the delineation of species and to infer their evolutive genealogy utilizing a minimum evolution tree of Average Nucleotide Identity (ANI) and the core genome alignment. Phylogenomic relationships were compared to those obtained from the analysis of 16S rRNA, pheS, and rpoA genes. All the phylograms were subjected to split decomposition analysis and their topologies were compared to check the ambiguities in the inferred phylogenesis. The minimum evolution ANI tree exhibited the most similar topology with the core genome tree, while single gene trees were less adherent and provided a weaker phylogenetic signal. In particular, the 16S rRNA gene failed to resolve several bifurcations and Leuconostoc species. Based on an ANI threshold of 95%, the organization of the genus Leuconostoc could be amended, redefining the boundaries of the species L. inhae, L. falkenbergense, L. gelidum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Two strains currently recognized as L. mesenteroides were split into a separate lineage representing a putative species (G16), phylogenetically related to both L. mesenteroides (G18) and L. suionicum (G17). Differences among the four subspecies of L. mesenteroides were not pinpointed by ANI or by the conserved genes. The strains of L. pseudomesenteroides were ascribed to two putative species, G13 and G14, the former including also all the strains presently belonging to L. falkenbergense. L. lactis was split into two phylogenetically related lineages, G9 and G10, putatively corresponding to separate species and both including subgroups that may correspond to subspecies. The species L. gelidum and L. gasicomitatum were closely related but separated into different species, the latter including also L. inhae strains. These results, integrating information of ANI, core genome, and housekeeping genes, complemented the taxonomic delineation with solid information on the phylogenetic lineages evolved within the genus Leuconostoc.

2022 - β-Glucuronidase Pattern Predicted From Gut Metagenomes Indicates Potentially Diversified Pharmacomicrobiomics [Articolo su rivista]
Candeliere, Francesco; Raimondi, Stefano; Ranieri, Raffaella; Musmeci, Eliana; Zambon, Alfonso; Amaretti, Alberto; Rossi, Maddalena
abstract

: β-glucuronidases (GUS) of intestinal bacteria remove glucuronic acid from glucoronides, reversing phase II metabolism of the liver and affecting the level of active deconjugated metabolites deriving from drugs or xenobiotics. Two hundred seventy-nine non-redundant GUS sequences are known in the gut microbiota, classified in seven structural categories (NL, L1, L2, mL1, mL2, mL1,2, and NC) with different biocatalytic properties. In the present study, the intestinal metagenome of 60 healthy subjects from five geographically different cohorts was assembled, binned, and mined to determine qualitative and quantitative differences in GUS profile, potentially affecting response to drugs and xenobiotics. Each metagenome harbored 4-70 different GUS, altogether accounting for 218. The amount of intestinal bacteria with at least one GUS gene was highly variable, from 0.7 to 82.2%, 25.7% on average. No significant difference among cohorts could be identified, except for the Ethiopia (ETH) cohort where GUS-encoding bacteria were significantly less abundant. The structural categories were differently distributed among the metagenomes, but without any statistical significance related to the cohorts. GUS profiles were generally dominated by the category NL, followed by mL1, L2, and L1. The GUS categories most involved in the hydrolysis of small molecules, including drugs, are L1 and mL1. Bacteria contributing to these categories belonged to Bacteroides ovatus, Bacteroides dorei, Bacteroides fragilis, Escherichia coli, Eubacterium eligens, Faecalibacterium prausnitzii, Parabacteroides merdae, and Ruminococcus gnavus. Bacteria harboring L1 GUS were generally scarcely abundant (<1.3%), except in three metagenomes, where they reached up to 24.3% for the contribution of E. coli and F. prausnitzii. Bacteria harboring mL1 GUS were significantly more abundant (mean = 4.6%), with Bacteroides representing a major contributor. Albeit mL1 enzymes are less active than L1 ones, Bacteroides likely plays a pivotal role in the deglucuronidation, due to its remarkable abundance in the microbiomes. The observed broad interindividual heterogeneity of GUS profiles, particularly of the L1 and mL1 categories, likely represent a major driver of pharmacomicrobiomics variability, affecting drug response and toxicity. Different geographical origins, genetic, nutritional, and lifestyle features of the hosts seemed not to be relevant in the definition of glucuronidase activity, albeit they influenced the richness of the GUS profile.

2021 - Comparative Genomics of Leuconostoc carnosum [Articolo su rivista]
Candeliere, F.; Raimondi, S.; Spampinato, G.; Tay, M. Y. F.; Amaretti, A.; Schlundt, J.; Rossi, M.
abstract

Leuconostoc carnosum is a known colonizer of meat-related food matrices. It reaches remarkably high loads during the shelf life in packaged meat products and plays a role in spoilage, although preservative effects have been proposed for some strains. In this study, the draft genomes of 17 strains of L. carnosum (i.e., all the strains that have been sequenced so far) were compared to decipher their metabolic and functional potential and to determine their role in food transformations. Genome comparison and pathway reconstruction indicated that L. carnosum is a compact group of closely related heterofermentative bacteria sharing most of the metabolic features. Adaptation to a nitrogen-rich environment, such as meat, is evidenced by 23 peptidase genes identified in the core genome and by the autotrophy for nitrogen compounds including several amino acids, vitamins, and cofactors. Genes encoding the decarboxylases yielding biogenic amines were not present. All the strains harbored 1–4 of 32 different plasmids, bearing functions associated to proteins hydrolysis, transport of amino acids and oligopeptides, exopolysaccharides, and various resistances (e.g., to environmental stresses, bacteriophages, and heavy metals). Functions associated to bacteriocin synthesis, secretion, and immunity were also found in plasmids. While genes for lactococcin were found in most plasmids, only three harbored the genes for leucocin B, a class IIa antilisterial bacteriocin. Determinants of antibiotic resistances were absent in both plasmids and chromosomes.

2021 - Draft genome sequence of the mucin degrader clostridium tertium wc0709 [Articolo su rivista]
Musmeci, E.; Candeliere, F.; Amaretti, A.; Rossi, M.; Raimondi, S.
abstract

The draft genome sequence of Clostridium tertium WC0709, a gut bacterium able to use mucin in pure culture as the sole carbon and nitrogen source, is presented here. The genome sequence of C. tertium will provide valuable references for comparative genome analysis and for studying the relationship with the host.

2021 - Identification of mucin degraders of the human gut microbiota [Articolo su rivista]
Raimondi, S.; Musmeci, E.; Candeliere, F.; Amaretti, A.; Rossi, M.
abstract

Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.

2021 - In Vitro Assessment of Prebiotic Activity [Capitolo/Saggio]
Amaretti, Alberto; Raimondi, Stefano; Volpi, Nicola; Rossi, Maddalena
abstract

Bifidogenic effect is a main target for the assessment of prebiotic activity. pH-controlled batch processes of bifidobacteria and fecal microbiota are herein presented. Growth of bifidobacteria, carbohydrate breakdown and consumption, organic acid production, and activity of specific glycosyl hydrolases involved inthe hydrolysis of di-, oligo-, or polysaccharides are exploited to study and compare substrate preference of bifidobacteria for candidate prebiotics.

2021 - Multivariate Analysis in Microbiome Description: Correlation of Human Gut Protein Degraders, Metabolites, and Predicted Metabolic Functions [Articolo su rivista]
Raimondi, S.; Calvini, R.; Candeliere, F.; Leonardi, A.; Ulrici, A.; Rossi, M.; Amaretti, A.
abstract

Protein catabolism by intestinal bacteria is infamous for releasing many harmful compounds, negatively affecting the health status, both locally and systemically. In a previous study, we enriched in protein degraders the fecal microbiota of five subjects, utilizing a medium containing protein and peptides as sole fermentable substrates and we monitored their evolution by 16S rRNA gene profiling. In the present study, we fused the microbiome data and the data obtained by the analysis of the volatile organic compounds (VOCs) in the headspace of the cultures. Then, we utilized ANOVA simultaneous component analysis (ASCA) to establish a relationship between metabolites and bacteria. In particular, ASCA allowed to separately assess the effect of subject, time, inoculum concentration, and their binary interactions on both microbiome and volatilome data. All the ASCA submodels pointed out a consistent association between indole and Escherichia–Shigella, and the relationship of butyric, 3-methyl butanoic, and benzenepropanoic acids with some bacterial taxa that were major determinants of cultures at 6 h, such as Lachnoclostridiaceae (Lachnoclostridium), Clostridiaceae (Clostridium sensu stricto), and Sutterellaceae (Sutterella and Parasutterella). The metagenome reconstruction with PICRUSt2 and its functional annotation indicated that enrichment in a protein-based medium affected the richness and diversity of functional profiles, in the face of a decrease of richness and evenness of the microbial community. Linear discriminant analysis (LDA) effect size indicated a positive differential abundance (p < 0.05) for the modules of amino acid catabolism that may be at the basis of the changes of VOC profile. In particular, predicted genes encoding functions belonging to the superpathways of ornithine, arginine, and putrescine transformation to GABA and eventually to succinyl-CoA, of methionine degradation, and various routes of breakdown of aromatic compounds yielding succinyl-CoA or acetyl-CoA became significantly more abundant in the metagenome of the bacterial community.

2021 - Phenotypic Traits and Immunomodulatory Properties of Leuconostoc carnosum Isolated From Meat Products [Articolo su rivista]
Raimondi, S.; Spampinato, G.; Candeliere, F.; Amaretti, A.; Brun, P.; Castagliuolo, I.; Rossi, M.
abstract

Twelve strains of Leuconostoc carnosum from meat products were investigated in terms of biochemical, physiological, and functional properties. The spectrum of sugars fermented by L. carnosum strains was limited to few mono- and disaccharides, consistently with the natural habitats of the species, including meat and fermented vegetables. The strains were able to grow from 4 to 37C with an optimum of approximately 32.5C. The ability to grow at temperatures compatible with refrigeration and in presence of up to 60 g/L NaCl explains the high loads of L. carnosum frequently described in many meat-based products. Six strains produced exopolysaccharides, causing a ropy phenotype of colonies, according to the potential involvement on L. carnosum in the appearance of slime in packed meat products. On the other side, the study provides evidence of a potential protective role of L. carnosum WC0321 and L. carnosum WC0323 against Listeria monocytogenes, consistently with the presence in these strains of the genes encoding leucocin B. Some meat-based products intended to be consumed without cooking may harbor up to 108 CFU/g of L. carnosum; therefore, we investigated the potential impact of this load on health. No strains survived the treatment with simulated gastric juice. Three selected strains were challenged for the capability to colonize a mouse model and their immunomodulatory properties were investigated. The strains did not colonize the intestine of mice during 10 days of daily dietary administration. Intriguingly, despite the loss of viability during the gastrointestinal transit, the strains exhibited different immunomodulatory effect on the maturation of dendritic cells in vivo, the extent of which correlated to the production of exopolysaccharides. The ability to stimulate the mucosal associated immune system in such probiotic-like manner, the general absence of antibiotic resistance genes, and the lack of the biosynthetic pathways for biogenic amines should reassure on the safety of this species, with potential for exploitation of selected starters.

2021 - Vaginal and anal microbiome during chlamydia trachomatis infections [Articolo su rivista]
Raimondi, S.; Candeliere, F.; Amaretti, A.; Foschi, C.; Morselli, S.; Gaspari, V.; Rossi, M.; Marangoni, A.
abstract

Background. Chlamydia trachomatis (CT) is the agent of the most common bacterial sexually transmitted infection worldwide, with a significant impact on women’s health. Despite the increasing number of studies about the vaginal microbiome in women with CT infections, information about the composition of the anal microbiome is still lacking. Here, we assessed the bacterial community profiles of vaginal and anal ecosystems associated or not with CT infection in a cohort of Caucasian young women. Methods. A total of 26 women, including 10 with a contemporary vaginal and anorectal CT infection, were enrolled. Composition of vaginal and anal microbiome was studied by 16S rRNA gene profiling. Co-occurrence networks of bacterial communities and metagenome metabolic functions were determined. Results. In case of CT infection, both vaginal and anal environments were characterized by a degree of dysbiosis. Indeed, the vaginal microbiome of CT-positive women were depleted in lactobacilli, with a significant increase in dysbiosis-associated bacteria (e.g., Sneathia, Parvimonas, Megasphaera), whereas the anal microbiota of CT-infected women was characterized by higher levels of Parvimonas and Pseudomonas and lower levels of Escherichia. Interestingly, the microbiome of anus and vagina had numerous bacterial taxa in common, reflecting a significant microbial ‘sharing’ between the two sites. In the vaginal environment, CT positively correlated with Ezakiella spp. while Gardnerella vaginalis co-occurred with several dysbiosis-related microbes, regardless of CT vaginal infection. The vaginal microbiome of CT-positive females exhibited a higher involvement of chorismate and aromatic amino acid biosynthesis, as well as an increase in mixed acid fermentation. Conclusions. These data could be useful to set up new diagnostic/prognostic tools, offering new perspectives for the control of chlamydial infections.

2020 - Antibiotic resistance, virulence factors, phenotyping, and genotyping of non–escherichia coli enterobacterales from the gut microbiota of healthy subjects [Articolo su rivista]
Amaretti, A.; Righini, L.; Candeliere, F.; Musmeci, E.; Bonvicini, F.; Gentilomi, G. A.; Rossi, M.; Raimondi, S.
abstract

Non-Escherichia coli Enterobacterales (NECE) can colonize the human gut and may present virulence determinants and phenotypes that represent severe heath concerns. Most information is available for virulent NECE strains, isolated from patients with an ongoing infection, while the commensal NECE population of healthy subjects is understudied. In this study, 32 NECE strains were isolated from the feces of 20 healthy adults. 16S rRNA gene sequencing and mass spectrometry attributed the isolates to Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter kobei, Citrobacter freundii, Citrobacter amalonaticus, Cronobacter sp., and Hafnia alvei, Morganella morganii, and Serratia liquefaciens. Multiplex PCR revealed that K. pneumoniae harbored virulence genes for adhesins (mrkD, ycfM, and kpn) and enterobactin (entB) and, in one case, also for yersiniabactin (ybtS, irp1, irp2, and fyuA). Virulence genes were less numerous in the other NECE species. Biofilm formation was spread across all the species, while curli and cellulose were mainly produced by Citrobacter and Enterobacter. Among the most common antibiotics, amoxicillin-clavulanic acid was the sole against which resistance was observed, only Klebsiella strains being susceptible. The NECE inhabiting the intestine of healthy subjects have traits that may pose a health threat, taking into account the possibility of horizontal gene transfer.

2020 - Draft Genome Sequences of 12 Leuconostoc carnosum Strains Isolated from Cooked Ham Packaged in a Modified Atmosphere and from Fresh Sausages [Articolo su rivista]
Candeliere, Francesco; Raimondi, Stefano; Spampinato, Gloria; Tay, Moon Yue Feng; Amaretti, Alberto; Schlundt, Joergen; Rossi, Maddalena
abstract

Leuconostoc carnosum is a lactic acid bacterium that preferentially colonizes meat. In this work, we present the draft genome sequences of 12 Leuconostoc carnosum strains isolated from modified-atmosphere-packaged cooked ham and fresh sausages. Three strains harbor bacteriocin genes.

2020 - Effect of Rearing Temperature on Growth and Microbiota Composition of Hermetia illucens [Articolo su rivista]
Raimondi, Stefano; Spampinato, Gloria; Macavei, Laura Ioana; Lugli, Linda; Candeliere, Francesco; Rossi, Maddalena; Maistrello, Lara; Amaretti, Alberto
abstract

The potential utilization of black soldier fly (Hermetia illucens) as food or feed is interesting due to the nutritive value and the sustainability of the rearing process. In the present study, larvae and prepupae of H. illucens were reared at 20, 27, and 33 °C, to determine whether temperature affects the whole insect microbiota, described using microbiological risk assessment techniques and 16S rRNA gene survey. The larvae efficiently grew across the tested temperatures. Higher temperatures promoted faster larval development and greater final biomass but also higher mortality. Viable Enterobacteriaceae, Bacillus cereus, Campylobacter, Clostridium perfringens, coagulase-positive staphylococci, Listeriaceae, and Salmonella were detected in prepupae. Campylobacter and Listeriaceae counts got higher with the increasing temperature. Based on 16S rRNA gene analysis, the microbiota of larvae was dominated by Providencia (>60%) and other Proteobateria (mainly Klebsiella) and evolved to a more complex composition in prepupae, with a bloom of Actinobacteria, Bacteroidetes, and Bacilli, while Providencia was still present as the main component. Prepupae largely shared the microbiota with the frass where it was reared, except for few lowly represented taxa. The rearing temperature was negatively associated with the amount of Providencia, and positively associated with a variety of other genera, such as Alcaligenes, Pseudogracilibacillus, Bacillus, Proteus, Enterococcus, Pediococcus, Bordetella, Pseudomonas, and Kerstersia. With respect to the microbiological risk assessment, attention should be paid to abundant genera, such as Bacillus, Myroides, Proteus, Providencia, and Morganella, which encompass species described as opportunistic pathogens, bearing drug resistances or causing severe morbidity.

2020 - Potential of wickerhamomyces anomalus in glycerol valorization [Articolo su rivista]
Amaretti, A.; Russo, B.; Raimondi, S.; Leonardi, A.; Foca, G.; Mucci, A.; Zambon, A.; Rossi, M.
abstract

Five-carbons polyalcohols, such as xylitol and arabitol, and microbial oils are important targets for biotechnological industries. Polyalcohols can find application as low-calories sweeteners and as building block in the synthesis of valuable compounds, while lipids are interesting for both biofuel and food industry. The osmophilic yeast Wickerhamomyces anomalus WC 1501 was preliminary known to produce arabitol from glycerol. Production kinetics were investigated in this study. Production was not growth-associated and occurred during a nitrogen-limited stationary phase, in presence of an excess of carbon source. Typical bioreactor batch cultures, carried out with 160 g/L glycerol, yielded 16.0 g/L arabitol in 160 h. A fed-batch process was developed, in which growth is carried out batchwise in a balanced medium containing 20 g/L glycerol, and arabitol production is induced at the entrance into the stationary phase with a pulse of concentrated glycerol to provide the remaining 140 g/L carbon source. At the end of the process 18.0 g/L arabitol were generated. Under these conditions, the yeast also accumulated intracellular triacylglycerols, with fatty acids of 16-18 carbons bearing 0 to 2 unsaturations, reaching up the 23% of biomass dry weight. Therefore, W. anomalus WC 1501 is a good candidate for the development of a fermentative process yielding arabitol and has potential also as oleaginous yeast for producing lipids, further improving the interest in this strain for glycerol biorefinery. The utilization of a fed-batch process allows to carry out distinct growth and production phases and thus allows the optimization of both phases separately, in order to achieve the highest concentration of catalytic biomass during growth and the maximum efficiency during production. This strain deserves further investigation to better exploit its biotechnological potential in the valorization of glycerol.

2020 - Potential prebiotic effect of a long-chain dextran produced by Weissella cibaria: an in vitro evaluation [Articolo su rivista]
Amaretti, Alberto; Bottari, Benedetta; Morreale, Federico; Savo Sardaro, Maria Luisa; Angelino, Donato; Raimondi, Stefano; Rossi, Maddalena; Pellegrini, Nicoletta
abstract

2020 - Riboflavin Biosynthesis and Overproduction by a Derivative of the Human Gut Commensal Bifidobacterium longum subsp. infantis ATCC 15697 [Articolo su rivista]
Solopova, A.; Bottacini, F.; Venturi degli Esposti, E.; Amaretti, A.; Raimondi, S.; Rossi, M.; van Sinderen, D.
abstract

Riboflavin or vitamin B2 is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Despite increased interest in microbial synthesis of this water-soluble vitamin, the metabolic pathway for riboflavin biosynthesis has been characterized in just a handful of bacteria. Here, comparative genome analysis identified the genes involved in the de novo biosynthetic pathway of riboflavin in certain bifidobacterial species, including the human gut commensal Bifidobacterium longum subsp. infantis (B. infantis) ATCC 15697. Using comparative genomics and phylogenomic analysis, we investigated the evolutionary acquisition route of the riboflavin biosynthesis or rib gene cluster in Bifidobacterium and the distribution of riboflavin biosynthesis-associated genes across the genus. Using B. infantis ATCC 15697 as model organism for this pathway, we isolated spontaneous riboflavin overproducers, which had lost transcriptional regulation of the genes required for riboflavin biosynthesis. Among them, one mutant was shown to allow riboflavin release into the medium to a concentration of 60.8 ng mL–1. This mutant increased vitamin B2 concentration in a fecal fermentation system, thus providing promising data for application of this isolate as a functional food ingredient.

2019 - Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of E. coli Isolated from the Feces of Healthy Subjects [Articolo su rivista]
Raimondi, S; Righini, L; Candeliere, F; Musmeci, Eliana; Bonvicini, F; Gentilomi, G; STARCIC ERJAVEC, Marjanca; Amaretti, A; Rossi, M
abstract

Escherichia coli may innocuously colonize the intestine of healthy subjects or may instigate infections in the gut or in other districts. This study investigated intestinal E. coli isolated from 20 healthy adults. Fifty-one strains were genotyped by molecular fingerprinting and analyzed for genetic and phenotypic traits, encompassing the profile of antibiotic resistance, biofilm production, the presence of surface structures (such as curli and cellulose), and their performance as recipients in conjugation experiments. A phylogroup classification and analysis of 34 virulence determinants, together with genes associated to the pks island (polyketide-peptide genotoxin colibactin) and conjugative elements, was performed. Most of the strains belonged to the phylogroups B1 and B2. The different phylogroups were separated in a principal coordinate space, considering both genetic and functional features, but not considering pulsed-field gel electrophoresis. Within the B2 and F strains, 12 shared the pattern of virulence genes with potential uropathogens. Forty-nine strains were sensitive to all the tested antibiotics. Strains similar to the potential pathogens innocuously inhabited the gut of healthy subjects. However, they may potentially act as etiologic agents of extra-intestinal infections and are susceptible to a wide range of antibiotics. Nevertheless, there is still the possibility to control infections with antibiotic therapy.

2019 - Comparison of gluten peptides and potential prebiotic carbohydrates in old and modern Triticum turgidum ssp. genotypes [Articolo su rivista]
Ficco, Donatella Bianca Maria; Prandi, Barbara; Amaretti, Alberto; Anfelli, Igor; Leonardi, Alan; Raimondi, Stefano; Pecchioni, Nicola; De Vita, Pasquale; Faccini, Andrea; Sforza, Stefano; Rossi, Maddalena
abstract

Old wheat genotypes are perceived by consumers as healthier than modern ones. The release of gluten peptides with in vitro digestion and the content of potentially prebiotic carbohydrates (i.e. resistant fraction of starch and cell-wall associated dietary fiber) were evaluated in tetraploid wheats, namely 9 old and 3 modern Triticum turgidum ssp. genotypes. Simulated digestion of wholemeal flours yielded 152 major peptides, 59 of which were attributed a sequence. Principal component analysis revealed that peptide profiles were variable in old genotypes, unlike in modern ones. Digestion of old genotypes generally yielded peptides in greater concentration. In particular, 5 peptides of γ-gliadin, known to trigger the adaptive immune reaction, and two peptides of α-gliadin, known to be toxic to celiac patients, were particularly abundant in some old varieties. Resistant starch (RS) was negligible in modern genotypes (<0.6%), but it was remarkably abundant in some old varieties, reaching the highest value in Dauno III (8.5%, P < 0.05). Dauno III also presented the highest amount of soluble fiber (4.2%, P < 0.05). Pasta was made with an old and a modern genotype (Dauno III and PR22D89, respectively) with opposite RS content. Pasta making and cooking affected starch digestibility, overtaking differences between genotypes and yielding the same amount of RS for both the varieties (approx. 1.7%). The data herein presented suggest that the wholemeal flours of old tetraploid wheat genotypes could not boast particular claims associated to a lower exposure to gluten peptides and, if cooked, to a prebiotic potential.

2019 - Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization [Articolo su rivista]
Raimondi, Stefano; Amaretti, Alberto; Gozzoli, Caterina; Simone, Marta; Righini, Lucia; Candeliere, Francesco; Brun, Paola; Ardizzoni, Andrea; Colombari, Bruna; Paulone, Simona; Castagliuolo, Ignazio; Cavalieri, Duccio; Blasi, Elisabetta; Rossi, Maddalena; Peppoloni, Samuele
abstract

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.

2019 - Microbiota of sliced cooked ham packaged in modified atmosphere throughout the shelf life: Microbiota of sliced cooked ham in MAP [Articolo su rivista]
Raimondi, Stefano; Luciani, Rosaria; Sirangelo, Tiziana Maria; Amaretti, Alberto; Leonardi, Alan; Ulrici, Alessandro; Foca, Giorgia; D'Auria, Giuseppe; Moya, Andrés; Zuliani, Véronique; Seibert, Tim Martin; Søltoft-Jensen, Jakob; Rossi, Maddalena
abstract

Fourteen lots of cooked ham in modified atmosphere packaging (CH) were analyzed within a few days from packaging (S) and at the end of the shelf-life (E), after storage at 7 °C to simulate thermal abuse. Five more lots, rejected from the market because spoiled (R), were included in the study. Quality of the products was generally compromised during the shelf life, with only 4 lots remaining unaltered. Analysis of 16S rRNA gene amplicons resulted in 801 OTUs. S samples presented a higher diversity than E and R ones. At the beginning of the shelf life, Proteobacteria and Firmicutes dominated the microbiota, with Acinetobacter, Brochothrix, Carnobacterium, Lactobacillus, Prevotella, Pseudomonas, Psychrobacter, Weissella, Vibrio rumoiensis occurring frequently and/or abundantly. E and R samples were dominated by Firmicutes mostly ascribed to Lactobacillales. It is noteworthy the appearance of abundant Leuconostoc, negligible in S samples, in some E and R samples, while in other LAB were outnumbered by V. rumoiensis or Brochothrix thermosphacta. The microbiota of spoiled and R samples could not be clustered on the basis of specific defects (discoloration, presence of slime, sourness, and swollen packages) or supplemented additives. LAB population of S samples, averaging 2.9 log10(cfu/g), increased to 7.7 log10(cfu/g) in the E and R samples. Dominant cultivable LAB belonged to the species Lactobacillus sakei and Leuconostoc carnosum. The same biotypes ascribed to different species where often found in the corresponding S and R samples, and sometime in different batches provided from the same producer, suggesting a recurrent contamination from the plant of production. Consistently with growth of LAB, initial pH (6.26) dropped to 5.74 in E samples. Volatiles organic compound (VOCs) analysis revealed that ethanol was the major metabolite produced during the shelf life. The profile of volatile compounds got enriched with other molecules (e.g. 2-butanone, ethyl acetate, acetic acid, acetoin, butanoic acid, ethyl ester, butanoic acid, and 2,3-butanediol) mainly ascribed to microbial metabolism.

2019 - Profiling of Protein Degraders in Cultures of Human Gut Microbiota [Articolo su rivista]
Amaretti, Alberto; Gozzoli, Caterina; Simone, Marta; Raimondi, Stefano; Righini, Lucia; Pérez-Brocal, Vicente; García-López, Rodrigo; Moya, Andrés; Rossi, Maddalena
abstract

Unabsorbed proteins reach the colon and are fermented by the microbiota, yielding a variety of harmful metabolites. In the present study, a 16S rRNA gene survey identified the bacterial taxa flourishing in 11 batch fermentations with proteins and peptones as the sole fermentable substrates, inoculated with the feces of six healthy adults. Organic acids, ammonia, and indole resulting from protein breakdown and fermentation accumulated in all of the cultures. Analysis of differential abundances among time-points identified Enterobacteriaceae, Burkholderiaceae, and Desulfovibrionaceae (including Esherichia-Shigella, Sutterella, Parasutterella, and Bilophila) among the bacteria that especially in the cultures with low inoculation load. Lachnospiraceae and Ruminococcaceae also encompassed many taxa that significantly expanded, mainly in cultures inoculated with high inoculation load, and showed the strongest correlation with the production of ammonium, indole, and p-cresol. Anaerotruncus, Dorea, Oscillibacter, Eubacterium oxidoreducens, Lachnoclostridium, Paeniclostridium, and Rombutsia were among them. Other Firmicutes (e.g., Roseburia, Ruminococcus, Lachnospira, Dialister, Erysipelotrichaceae, and Streptococcaceae) and many Bacteroidetes (e.g., Barnesiellaceae, Prevotellaceae, and Rickenelliaceae) decreased. Sequences attributed to Bacteroides, unresolved at the level of species, presented opposite contributions, resulting in no significant changes in the genus. This study sheds light on the multitude of bacterial taxa putatively participating in protein catabolism in the colon. Protein fermentation was confirmed as unfavorable to health, due to both the production of toxic metabolites and the blooming of opportunistic pathogens and pro-inflammatory bacteria.

2018 - Bacterial community of Industrial raw Sausage Packaged in Modified Atmosphere throughout the Shelf Life [Articolo su rivista]
Raimondi, Stefano; Nappi, MARIA ROSARIA; Sirangelo, Tiziana M.; Leonardi, Alan; Amaretti, Alberto; Ulrici, Alessandro; Magnani, Rudy; Montanari, Chiara; Tabanelli, Giulia; Gardini, Fausto; Rossi, Maddalena
abstract

Ten lots of industrial raw sausages in modified atmosphere (CO2 30%, O2 70%), produced in the same plant over 7 months, were analyzed at the day after production (S samples) and at the end of shelf life (E samples), after 12 days storage at 7 °C to simulate thermal abuse. Quality of the products was generally compromised by storage at 7 °C, with only 3 E samples without alterations. During the shelf life, the pH decreased for the accumulation of acetic and lactic acids. A few biogenic amines accumulated, remaining below acceptable limits. The profile of volatile compounds got enriched with alcohols, ketones, and acids (e.g. ethanol, 2,3-butanediol, 2,3-butandione, butanoic acid) originated by bacterial metabolism. Throughout the shelf life, aerobic bacteria increased from 4.7 log to 6.6 log cfu/g, and lactic acid bacteria (LAB) from 3.7 to 8.1 log cfu/g. Staphylococci, enterobacteria, and pseudomonads passed from 3.7, 3.0, and 1.7 to 5.5, 4.8, and 3.0 log cfu/g, respectively. Dominant cultivable LAB, genotyped by RAPD-PCR, belonged to the species Lactobacillus curvatus/graminis and Lactobacillus sakei, with lower amounts of Leuconostoc carnosum and Leuconostoc mesenteroides. Brochothrix thermosphacta was the prevailing species among aerobic bacteria. The same biotypes ascribed to several different species where often found in E samples of diverse batches, suggesting a recurrent contamination from the plant of production. Profiling of 16S rRNA gene evidenced that microbiota of S samples clustered in two main groups where either Firmicutes or Bacteroidetes prevailed, albeit with taxa generally associated to the gastro-intestinal tract of mammals. The microbial diversity was lower in E samples than in S ones. Even though a common profile could not be identified, most E samples clustered together and were dominated by Firmicutes, with Lactobacillaceae and Listeriaceae as the most abundant families (mostly ascribed to Lactobacillus and Brochothrix, respectively). In a sole E sample Proteobacteria (especially Serratia) was the major phylum.

2018 - Changes in Microbiota of Fresh Sausage Throughout Shelf Life [Abstract in Atti di Convegno]
Raimondi, Stefano; Sirangelo, TIZIANA MARIA; Gardini, Fausto; Luciani, Rosaria; Amaretti, Alberto; Magnani, Rudy; Rossi, Maddalena
abstract

Introduction: Fresh sausage is a perishable product easily colonized by spoilage bacteria. Microbial diversity of fresh sausage has been generally investigated using traditional cultural methods. Purpose: The present study aimed to obtain an exhaustive description of the microbiota composition and diversity throughout the shelf life of fresh sausage. Methods: Cultivable bacteria from ten batches of sausage provided by the same factory were enumerated on PCA, MRS, Mac Conkey, ALOA, and PDA plates, at delivery and after 12 days at 7°C. The dominant strains were genotyped by RAPD-PCR fingerprinting, and taxonomic diversity was estimated by partial sequencing of 16S rRNA genes. A comprehensive picture of bacteria was obtained by metataxonomic analysis of Illumina 16S sequences. Results: The mean charge (log of CFU/g) of cultivable aerobic and aerotolerant bacteria started at 5.0 and reached 7.2 after 12 days at 7°C, with a concomitant drop of pH from 5.9 to 5.5. Lactic acid bacteria showed the highest increase starting from 4.1 and reaching 8.6 log, followed by Staphylococci (4.0 to 6.5 log) and by Enterobactericeae (3.2 to 6.2 log). The majority of MRS biotypes belonged to Lactobacillus curvatus, followed by Lactobacillus sakei, Leuconostoc carnosum, and Leuconostoc mesenteroides. The most represented species in PCA was Brochotrix thermosphacta. Metataxonomic analysis revealed that at packaging, fresh sausage was characterized by bacteria generally ascribable to two diverse microbiota associated with gut microbes. At the end of the shelf life, Firmicutes dominated the microbiota, in some cases with prevalence of Lactobacillales. In other samples, Listeriaceae took over, with Brochothrix being the most represented genus. Enterobacteriaceae, generally ascribed to Serratia, were abundant in a few samples at the end of the shelf life. Significance: This study provided a wide overview of microbiota evolution in fresh sausage, shedding light on meat hygiene and safety issues.

2018 - Microbiota of fresh pork sausage in modified atmosphere [Abstract in Rivista]
Sirangelo, TIZIANA MARIA; Luciani, Rosaria; Raimondi, Stefano; Tabanelli, Giulia; Montanari, Chiara; Amaretti, Alberto; Leonardi, Alan; Gardini, Fausto; Rossi, Maddalena
abstract

A comprehensive analysis of microbial population was performed on 9 lots of fresh pork sausage, sampled in a lapse of time of 6 months from a sole production plant.

2018 - Screening of environmental yeasts for the fermentative production of arabitol from lactose and glycerol [Articolo su rivista]
Amaretti, Alberto; Anfelli, Igor; Foca, Giorgia; Ulrici, Alessandro; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena
abstract

Arabitol is a sugar alcohol, stereoisomer to xylitol, which is enlisted among the main target for biorefineries. It can serve as low calorie sweetener and as building block in the enantiopure synthesis of immunosuppressive glycolipids, herbicides, and drugs. Several studies described the fermentative production of arabitol by osmophilic yeasts, cultured with high concentrations of D-glucose. The utilization of cheaper carbon sources, such as glycerol or lactose, is of great interest for biorefinery implementation, but information on exploitation to arabitol production is still scarce. In the present study 50 yeasts belonging to 24 ascomycetous species were screened for the ability to grow and produce arabitol in presence of 80 g/L lactose or glycerol. Production from lactose was generally unsuccessful, the best producer being Kluyveromyces lactis WC 1401 with 0.94 g/L in 160 h. Production from glycerol was promising, with Zygosaccharomyces rouxii WC 1206, Pichia guilliermondii CBS 566, Hansenula anomala WC 1501, and Candida freyschussii ATCC 18737 yielding 3 to 4.5 g/L arabitol, with conversion yield (YP/S) ranging from 11 to 21.7%. Batch growth with high initial glycerol amount (160 g/L) resulted in higher production, with H. anomala WC 1501 yielding 10.0 g/L arabitol (YP/S = 12%) in 160 h. Preliminary bioreactor fermentations with H. anomala WC 1501 indicated that production is not growth associated and revealed some major parameters affecting production, such as the pH and the C:N ratio, that will be the target of following studies aiming at process optimization. Cultivation under controlled oxygenation (DOT = 20%) and pH (= 3.0) resulted in improvement in the performance of H. anomala WC 1501, yielding 16.1 g/L arabitol. Cultivation in a medium with high C:N ratio, lacking inorganic nitrogen yielded 17.1 g/L arabitol. Therefore, this strain was selected for the development of a fed-batch process, aiming to improve the efficiency of the biomass, generated in the growth phase, and increasing the production in the stationary phase.

2017 - Characterization of the peptide fraction from digested Parmigiano Reggiano cheese and its effect on growth of lactobacilli and bifidobacteria [Articolo su rivista]
Bottari, Benedetta; Quartieri, Andrea; Prandi, Barbara; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena; Ulrici, Alessandro; Gatti, Monica; Sforza, Stefano; Nocetti, Marco; Amaretti, Alberto
abstract

Parmigiano Reggiano (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value. Long ripening times allow for extensive proteolysis of milk proteins to yield a number of peptides, some of which have potential healthy bioactive properties. This study aimed to: i) determine the peptide profile of PR cheese subjected to simulated gastrointestinal transit; ii) evaluate in vitro whether the peptides could support growth of beneficial microbial groups of the gut microbiota. PR samples were subjected to in vitro digestion, simulating oral, gastric, and duodenal transit. Liquid chromatography coupled with tandem mass spectrometry revealed that digestion caused the disappearance of the serum proteins and most of the original peptides, while 71 new peptides were found, all ranging from 2 to 24 residues. The digests were given as sole nitrogen source to pure cultures of Bifidobacterium (27 strains) and Lactobacillus (30 strains), and to bioreactor batch cultures of human gut microbiota. Most of bifidobacteria and lactobacilli grew more abundantly on PR digests than on the control peptone, and exhibited strain- or species-specific peptide preferences, as evidenced by principal component analysis. Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptide consumption and the number of peptides consumed. For bifidobacteria, peptide preferences were very diverse, but a core of 10 peptides with 4 or 5 residues were consumed by all the strains. Lactobacilli behaved more homogenously and consumed nearly only the same 6 peptides, mostly dipeptides. The peptide preferences of the different groups of bifidobacteria and lactobacilli could not be ascribed to features such as the length of the peptide or the abundance of residues with peculiar properties (hydrophobicity, polarity, charge) and likely depend on specific proteases and/or peptide transporters preferentially recognizing specific sequence motifs. The cultures of human colonic microbiota confirmed that PR digest promoted the growth of commensal bifidobacteria. This study demonstrated that peptides derived from simulated gastrointestinal digestion of PR supported the growth of most lactobacilli and bifidobacteria.

2017 - Comparison of formula-fed infants with and without colic revealed significant differences in total bacteria, Enterobacteriaceae and faecal ammonia [Articolo su rivista]
Savino, Francesco; Quartieri, Andrea; De Marco, Angela; Garro, Maria; Amaretti, Alberto; Raimondi, Stefano; Simone, Marta; Rossi, Maddalena
abstract

Aim: This study compared the faecal microbial composition of formula-fed infants who did and did not have colic. Methods: Faecal samples from formula-fed infants under 16 weeks of age with (n = 38) and without (n = 39) colic were collected at Department of Pediatrics in Turin, Italy, between February 2014 and October 2015. The pH and faecal ammonia were determined and total bacteria, bifidobacteria, lactic acid bacteria and coliforms were quantified by fluorescent in situ hybridisation (FISH). Results: Faecal ammonia was significantly higher in the colicky infants than in the controls (483 vs. 216 μg/g, p < 0.05). The FISH counts of total bacteria were lower in colicky infants (1.8E10 ± 1.5E10) than in the controls (3.4E10 ± 3.0E10) (p < 0.05). The relative abundance of coliform bacteria was significantly higher in colicky infants (p < 0.05). No differences were observed for the bifidobacteria and lactic acid bacteria counts between the two groups. Conclusion: Our comparison of formula-fed infants with and without colic revealed significant differences in total bacteria, Enterobacteriaceae and faecal ammonia. This study provides the stimulus for further studies of the gut microbiome, using new methods of analysis such as 16S metagenomics sequencing in order to lead to more tailored dietary approaches.

2017 - Evolution of microbial community and chemical properties of a sourdough during the production of Colomba, an Italian sweet leavened baked product [Articolo su rivista]
Raimondi, Stefano; Amaretti, Alberto; Rossi, Maddalena; Fall, Papa Abdoulaye; Tabanelli, Giulia; Gardini, Fausto; Montanari, Chiara
abstract

A sourdough for the industrial production of Colomba, a traditional Italian sweet-leavened baked good, was characterized in terms of microbiota composition, pH, aw, carbohydrates, organic acids, and VOCs. pH of the ripened sourdough was low (3.55), and the content of acetic acid limited (0.77 g/kg). A small biodiversity, in terms of both LAB and yeasts, was observed. Culture dependent analyses identified two biotypes of Lactobacillus sanfranciscensis and a dominant yeast species, Torulaspora delbrueckii. Metataxonomic analysis confirmed bacterial composition, whereas Candida humilis was the most represented yeast. The VOCs profile was strongly affected by bakers’ yeast addition, resulting in higher amounts of acetaldehyde, acetoin, ethanol, and phenethyl alcohol. As a whole, a stable and abundant microbial community loosing competitiveness only after addition of S. cerevisiae was described. This robustness and simplicity can represent an advantage in terms of stable and easier propagation, allowing the production of more reproducible lots of Colomba.

2017 - Evolution of sliced cooked ham microbiota packaged in modified atmosphere [Abstract in Atti di Convegno]
Sirangelo, Tiziana M.; Raimondi, Stefano; Amaretti, Alberto; Leonardi, Alan; Luciani, Rosaria; Zuliani, Veronique; Soeltoft-Jensen, Jakob; Martin Seibert, Tim; Rossi, Maddalena
abstract

Microorganisms colonize cooked ham (CH) packaged in modified atmosphere with a preferential growth of psychrotrophic lactic acid bacteria (LAB). Depending on the strains, the overwhelming colonization can result in a premature spoilage, characterized by pH decrease, gas and slime production, discoloration, and off-flavors formation. The LABs of 15 CH samples coming from different manufacturers were analyzed both at the beginning (T0) and at the end (Tf) of the shelf-life. Randomly selected colonies were subjected to RAPD-PCR clustering, and each biotype was taxonomically characterized through 16S rRNA gene partial sequencing. The evolution of sensorial properties was monitored by evaluation of the samples at T0 and Tf. Seven other CH samples were similarly processed when identified as flawed and withdrawn from the market before the sell-by date. The samples at the end of the shelf life and the spoiled ones shared a similar cultivable microbiota, dominated by Lactobacillus sakei, L. curvatus, and Leuconostoc carnosum. Less recurrent species were Weissella viridescens, Leuconostoc mesenteroides, Enterococcus faecalis, and Enterococcus pseudoavium. The microbiota of the flawed and unflawed (T0 and Tf) samples was further characterized by meta-taxonomic analysis. Initial population was generally represented by consortia of bacteria belonging to the order of Lactobacillales (Lactobacillus, Carnobacterium, Leuconostoc, Enterococcus, Vagococcus, Weissella, Streptococcus), Acinetobacter spp. and other Moraxellaceae, Pseudomonas spp., Bacillus, Prevotella, Weissella, Brochotrix, Vibrio rumoiensis, and Vellionella dispar. Samples that evolved into a spoiled product and samples that were delivered as flawed were often characterized by a dominant population of Lactobacillales. Among the flawed CH samples, overwhelming colonization of Brochothrix or Vibrio rumoiensis was detected.

2017 - MICROBIOTA OF FRESH CURED PORK SAUSAGE OVER THE SHELF-LIFE [Poster]
Luciani, Rosaria; Raimondi, Stefano; Tabanelli, Giulia; Montanari, Chiara; SIRANGELO Tiziana, M.; Amaretti, Alberto; Leonardi, Alan; Ulrici, Alessandro; Foca, Giorgia; Gardini, Fausto; Rossi, Maddalena
abstract

Italian style fresh sausage is a traditional pork food, commonly consumed after cooking. It is a perishable product that over the time can be colonized by spoilage bacteria that render the product inacceptable because of undesirable modifications of sensorial properties, such as appearance, texture, odor, and flavor. Indeed, being fresh meat a matrix with high water activity, slightly acidic pH, and high level of nutrients including glucose, lactic acid, nitrogenous compounds, and amino acids, it allows growth and proliferation of several bacteria. Temperatures and MAP (Modified Atmosphere Packaging) are the most important extrinsic factors affecting growth of microorganism. Generally fresh sausages are conserved refrigerated in MAP to maintain the red colour of the meat. The refrigeration of raw meat slows down growth of bacteria, allowing selection and blooming of psychrotrophic aerobic and aerotolerant species.

2016 - Comparison of culture-dependent and independent approaches to characterize fecal bifidobacteria and lactobacilli [Articolo su rivista]
Quartieri, Andrea; Simone, Marta; Gozzoli, Caterina; Popovic, Mina; D'Auria, Giuseppe; Amaretti, Alberto; Raimondi, Stefano; Rossi, Maddalena
abstract

Different culture-dependent and independent methods were applied to investigate the population of bifidobacteria and lactobacilli in the feces of five healthy subjects. Bacteria were isolated on MRS, a complex medium supporting growth of lactobacilli and bifidobacteria, and on three selective media for bifidobacteria and two for lactobacilli. Taxonomic characterization of the isolates was carried out by RAPD-PCR and partial 16S sequencing. The selectivity of genus-specific media was also investigated by challenging colonies from MRS plates to grow onto each medium. In parallel, a quantitative and qualitative description of bifidobacteria and lactic acid bacteria was obtained by FISH, qPCR, TRFLP, and 16S rRNA gene sequencing. Bifidobacteria did not fail to grow on their specific media and were easily isolated and enumerated, showing comparable quantitative data among culture-dependent and -independent techniques. The Bifidobacterium species identified on plates and those extracted from TRFLP and 16S rRNA gene sequencing were mostly overlapping. Selective media for lactobacilli gave unsuitable results, being too stringent or too permissive. The quantification of lactobacilli through selective plates, qPCR, FISH, and 16S rRNA gene sequencing gave unreliable results. Therefore, unlike bifidobacteria, intestinal lactobacilli are still problematic in terms of quantification and accurate profiling at level of species and possibly of strains by both culture-dependent and culture-independent techniques.

2016 - Conjugated Linoleic Acid Production by Bifidobacteria: Screening, Kinetic, and Composition [Articolo su rivista]
Raimondi, Stefano; Amaretti, Alberto; Leonardi, Alan; Quartieri, Andrea; Gozzoli, Caterina; Rossi, Maddalena
abstract

Conjugated linoleic acids (CLA) are positional and geometric isomers of linoleic acid involved in a number of health aspects. In humans, CLA production is performed by gut microbiota, including some species of potential probiotic bifidobacteria. 128 strains of 31 Bifidobacterium species were screened with a spectrophotometric assay to identify novel CLA producers. Most species were nonproducers, while producers belonged to B. breve and B. pseudocatenulatum. GC-MS revealed that CLA producer strains yielded 9cis,11trans-CLA and 9trans,11trans-CLA, without any production of other isomers. Hydroxylated forms of LA were absent in producer strains, suggesting that the myosin-cross-reactive antigen (MCRA) protein that exerts hydratase activity is not involved in LA isomerization. Moreover, both CLA producer and nonproducer species bear a MCRA homologue. The strain B. breve WC 0421 was the best CLA producer, converting LA into 68.8% 9cis,11trans-CLA and 25.1% 9trans,11trans-CLA. Production occurred mostly during the lag and the exponential phase. For the first time, production and incorporation of CLA in biomass were assessed. B. breve WC 0421 stored CLA in the form of free fatty acids, without changing the composition of the esterified fatty acids, which mainly occurred in the plasmatic membrane.

2016 - Detection of novel metabolites of flaxseed lignans in vitro and in vivo [Articolo su rivista]
Quartieri, Andrea; García Villalba, Rocío; Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena; Tomàs Barberàn, Francisco
abstract

Scope: This study aimed to improve the knowledge of secoisolariciresinol diglucoside (SDG) transformation by human gut microbiota. Methods and results: SDG-supplemented microbiota cultures were inoculated with the feces of five subjects. The same volunteers received a flaxseed supplement for 7 days. SDG metabolites in cultures, feces, and urine were monitored by LC-ESI-QTOF and LC-DAD. In all cultures, SDG was deglycosylated to secoisolariciresinol (SECO) within 12 h. SECO underwent successive dehydroxylations and demethylations yielding enterodiol (4-18% conversion) and enterolactone (0.2-6%) after 24 h. Novel intermediates related to SECO, matairesinol (MATA), and anhydrosecoisolariciresinol (AHS) were identified in fecal cultures. These metabolites were also found after flaxseed consumption in feces and urine (in approximate amounts between 0.01-47.03 μg/g and 0.01-13.49 μg/mL, respectively) in their native form and/or modified by phase II human enzymes (glucuronide, sulfate and sulfoglucuronide conjugates). Conclusions: Derivatives of MATA and AHS are described for the first time as intermediates of SDG biotransformation by intestinal bacteria, providing a more comprehensive knowledge of lignan intestinal metabolism. The transformations observed in vitro seem to occur in vivo as well. The detection in urine of SDG intermediates indicates their gut absorption, opening new perspectives on the study of their systemic biological effects.

2016 - Folate: Relevance of Chemical and Microbial Production [Capitolo/Saggio]
Rossi, Maddalena; Raimondi, Stefano; Costantino, Luca; Amaretti, Alberto
abstract

Folate, water-soluble B9 vitamin, is produced by green plants and some microorganisms. It acts as a coenzyme in the transfer of one-carbon units in many biosynthetic and cellular processes and is essential for cell division, embryonic and foetal development and maintenance of cardiovascular and neurologic functions. Folates occur in a variety of foods, especially in leafy vegetables, mostly as derivatives of the reduced form tetrahydrofolate (THF). Vitamin supplements and food fortification utilise chemically synthetised folic acid (FA), an oxidised form which is more stable and bioavailable. At present, FA is mainly produced via chemical synthesis. In fact, even if numerous studies have shown that certain lactic acid bacteria (LAB) can synthetise the vitamin de novo, the biotechnological production of folate has never been competitive with chemical synthesis. However, appropriate combination of selected LAB, such as Lactococcus lactis and Streptococcus thermophilus, can be used to enrich fermented foods in this vitamin, as successfully demonstrated for dairy products. Furthermore, the use of folate-producing Bifidobacterium strains can be regarded as a specific use of probiotics. Human and animal trials proved that the administration of folate-producing bifidobacteria positively affected folate level in plasma, indicating that the vitamin is produced in vivo by the probiotic strains and absorbed.

2016 - Functional roles of the fatty acid desaturases encoded by KLOLE1, FAD2 and FAD3 in the yeast Kluyveromyces lactis [Articolo su rivista]
de Angelis, Lorenzo; Rinaldi, Teresa; Cirigliano, Angela; Bello, Cristiano; Reverberi, Massimo; Amaretti, Alberto; Montanari, Arianna; Santomartino, Rosa; Raimondi, Stefano; Gonzalez, Alicia; Bianchi, Michele M.
abstract

Functional properties of cell membranes depend on their composition, particularly on the relative amount of saturated, unsaturated and polyunsaturated fatty acids present in the phospholipids. The aim of this study was to investigate the effect of cell membrane composition on cell fitness, adaptation and stress response in Kluyveromyces lactis. To this purpose, we have deleted the genes FAD2 and FAD3 encoding Δ12 and ω3 desaturases in Kluyveromyces lactis, thus generating mutant strains with altered fatty acid composition of membranes. These strains were viable and able to grow in stressing conditions like hypoxia and low temperature. Deletion of the Δ9 desaturase-encoding gene KlOLE1 resulted in lethality, suggesting that this enzyme has an essential role in this yeast. Transcription of the desaturase genes KlOLE1, FAD2 and FAD3 and cellular localization of the corresponding enzymes, have been studied under hypoxia and cold stress. Our findings indicate that expression of these desaturase genes and membrane composition were modulated by hypoxia and temperature stress, although the changes induced by these and other assayed conditions did not dramatically affect the general cellular fitness.

2016 - Lactic acid bacteria as protective cultures in fermented pork meat to prevent Clostridium spp. growth [Articolo su rivista]
Di Gioia, Diana; Mazzola, Giuseppe; Nikodinoska, Ivana; Aloisio, Irene; Langerholc, Tomaz; Rossi, Maddalena; Raimondi, Stefano; Melero, Beatriz; Rovira, Jordi
abstract

In meat fermented foods, Clostridium spp. growth is kept under control by the addition of nitrite. The growing request of consumers for safer products has led to consider alternative bio-based approaches, the use of protective cultures being one of them. This work is aimed at checking the possibility of using two Lactobacillus spp. strains as protective cultures against Clostridium spp. in pork ground meat for fermented salami preparation. Both Lactobacillus strains displayed anti-clostridia activity in vitro using the spot agar test and after co-culturing them in liquid medium with each Clostridium strain. Only one of them, however, namely L. plantarum PCS20, was capable of effectively surviving in ground meat and of performing anti-microbial activity in carnis in a challenge test where meat was inoculated with the Clostridium strain. Therefore, this work pointed out that protective cultures can be a feasible approach for nitrite reduction in fermented meat products.

2016 - Mining metagenomic whole genome sequences revealed subdominant but constant Lactobacillus population in the human gut microbiota [Articolo su rivista]
Rossi, Maddalena; Martínez Martínez, Daniel; Amaretti, Alberto; Ulrici, Alessandro; Raimondi, Stefano; Moya, Andrés
abstract

The genus Lactobacillus includes over 215 species that colonize plants, foods, sewage and the gastrointestinal tract (GIT) of humans and animals. In the GIT, Lactobacillus population can be made by true inhabitants or by bacteria occasionally ingested with fermented or spoiled foods, or with probiotics. This study longitudinally surveyed Lactobacillus species and strains in the feces of a healthy subject through whole genome sequencing (WGS) data-mining, in order to identify members of the permanent or transient populations. In three time-points (0, 670 and 700 d), 58 different species were identified, 16 of them being retrieved for the first time in human feces. L. rhamnosus, L. ruminis, L. delbrueckii, L. plantarum, L. casei and L. acidophilus were the most represented, with estimated amounts ranging between 6 and 8 Log (cells g-1), while the other were detected at 4 or 5 Log (cells g-1). 86 Lactobacillus strains belonging to 52 species were identified. 43 seemingly occupied the GIT as true residents, since were detected in a time span of almost 2 years in all the three samples or in 2 samples separated by 670 or 700 d. As a whole, a stable community of lactobacilli was disclosed, with wide and understudied biodiversity.

2015 - Hydrolysis of the rutinose-conjugates flavonoids rutin and hesperidin by the gut microbiota and bifidobacteria [Articolo su rivista]
Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan; Quartieri, Andrea; Rossi, Maddalena
abstract

Flavonols and flavanones are polyphenols exerting many healthy biological activities. They are often glycosylated by rutinose, which hampers absorption in the small intestine. Therefore they require the gut microbiota to release the aglycone and enable colonic absorption. The role of the gut microbiota and bifidobacteria in the release of the aglycones from two major rutinosides, hesperidin and rutin, was investigated. In bioconversion experiments, the microbiota removed rutinose from both rutin and hesperidin, even though complete hydrolysis was not obtained. To investigate whether bifidobacteria can participate to the hydrolysis of rutinosides, 33 strains were screened. Rutin was resistant to hydrolysis by all the strains. Among six tested species, mostly Bifidobacterium catenulatum and Bifidobacterium pseudocatenultum were able to hydrolyze hesperidin, by means of a cell-associated activity. This result is in agreement with the presence of a putative α-l-rhamnosidase in the genome of B. pseudocatenulatum, while most of the available genome sequences of bifidobacteria aside from this species do not bear this sequence. Even though B. pseudocatenulatum may contribute to the release of the aglycone from certain rutinose-conjugated polyphenols, such as hesperidin, it remains to be clarified whether this species may exert a role in affecting the bioavailability of the rutinoside in vivo.

2015 - Role of bifidobacteria in the hydrolysis of chlorogenic acid [Articolo su rivista]
Raimondi, Stefano; Anighoro, Andrew; Quartieri, Andrea; Amaretti, Alberto; Tomás Barberán, Francisco A.; Rastelli, Giulio; Rossi, Maddalena
abstract

This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds.

2014 - Anti-Listeria Starters: In Vitro Selection and Production Plant Evaluation [Articolo su rivista]
Raimondi, Stefano; Popovic, Mina; Amaretti, Alberto; Diana, Di Gioia; Rossi, Maddalena
abstract

Anti-Listeria bacterial starters are highly demanded by the meat industry. Novel bioprotective anti-Listeria starters were searched among Lactobacillus species strains isolated from artisanal sausages. The screening confirmed that anti-Listeria activity is a strain-specific property and yielded only 1 strain (of 36) exhibiting a satisfactory level of inhibition, L. delbrueckii WC0286. This strain was compared with two commercial bioprotective starters, SafePro B-SF-43 and SafePro B-LC-20, in a model simulating in vitro the first step of the fermentation process. The presence of the bioprotective starters did not modify the pH in such a way that could affect the safety or organoleptic properties of the product. Both SafePro B-SF-43 and SafePro B-LC-20 effected an important reduction of Listeria counts (0.56 and 0.72 log CFU g-1, respectively, in 72 h), while the anti-Listeria effect of L. delbrueckii WC0286 was minor (0.15 log CFU g-1). These results discouraged the utilization of L. delbrueckii WC0286 for a challenge test in a pilot salami production, in favor of the best-performing bioprotective starter, SafePro B-LC-20. The test confirmed that SafePro B-LC-20 did not alter the acidification trend of sausages and was capable of inhibiting Listeria, which decreased by 1.21 log CFU g-1. This information is relevant to address research activity toward the development of new bioprotective starters. The data herein presented demonstrate that the efficacy in Listeria control of potentially bioprotective bacterial starters requires further validation in real meat matrixes, possibly by using in vitro meat fermentation experiments to narrow down the list of candidates before pilot scale challenge tests. Copyright © 2014 International Association for Food Protection.

2014 - Bifidobacteria supplementation: Effects on plasma lipid profiles in dyslipidemic children [Articolo su rivista]
Ornella, Guardamagna; Amaretti, Alberto; Paolo, Emilio Puddu; Raimondi, Stefano; Francesca, Abello; Paola, Cagliero; Rossi, Maddalena
abstract

Objective: Preclinical investigations support the use of probiotics in the treatment of hypercholesterolemia, but clinical evidence is often contrasting. The aim of this study was to evaluate the effects of a probiotic formulation containing three Bifidobacterium strains on lipid profiles in children affected by primary dyslipidemia. Methods: Thirty-eight children with dyslipidemia, ages 10.8 ± 2.1 y, were enrolled in a randomized, double-blind, placebo-controlled cross-over study. After a 4-wk diet run-in period, the children received probiotics (B. animalis subspecies lactis MB 2409, B. bifidum MB 109B, and B. longum subspecies longum BL04) or placebo for 3 mo. After 1 mo, wash-out treatments were switched. Astrict dietary evaluation concerning satured fatty acids and cholesterol content, STEP I diet accordingly, was performed by a dietitian who examined the weekly dietary diary at each visit. Results: Baseline lipid profile was (mean ± SD): total cholesterol (TC) 222.8 ± 23.2 mg/dL, high-density lipoprotein cholesterol (HDL-C) 55.8 ± 12.2 mg/dL, triglycerides (TG) 99.0 ± 61.7 mg/dL, and low-density lipoprotein cholesterol (LDL-C) 147.2 ± 21.9 mg/dL. After 3 mo of probiotic treatment, the lipid profile was: TC 211.9 ± 27.3 mg/dL, HDL-C 60.7 ± 14.2 mg/dL, TG 79.5 ± 34.5 mg/dL, and LDL-C 135.3 ± 24.2 mg/dL. Compared with placebo, probiotics reduced TC by 3.4% (P = 0.02) and LDL-C by 3.8% (P = 0.001). No significant dietary change occurred through the study and no relevant adverse effects were observed. Conclusions: Treatment with a Bifidobacterium probiotic formulation was well tolerated and useful in combination with to diet therapy. Children with dyslipidemia benefited from this approach, although the results need to be confirmed by larger controlled studies.

2014 - Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii [Articolo su rivista]
Raimondi, Stefano; Rossi, Maddalena; Leonardi, Alan; Michele, Bianchi; Teresa, Rinaldi; Amaretti, Alberto
abstract

BACKGROUND: Microbial lipids represent a valuable alternative feedstock for biodiesel production when oleaginous microbes are cultured with inexpensive substrates in processes exhibiting high yield and productivity. In this perspective, crude glycerol is among the most promising raw materials for lipid production, because it is the costless residual of biodiesel production. Thus, cultivation of oleaginous yeasts in glycerol-based media is attracting great interest and natural biodiversity is increasingly explored to identify novel oleaginous species recycling this carbon source for growth and lipid production. RESULTS: Thirty-three yeasts strains belonging to 19 species were screened for the ability to grow and produce intracellular lipids in a pure glycerol-based medium with high C/N ratio. A minority of them consumed most of the glycerol and generated visible lipid bodies. Among them, Candida freyschussii ATCC 18737 was selected, because it exhibited the highest lipid production and glycerol conversion yield. Lipid production in this strain was positively affected by the increase of C/N ratio, but growth was inhibited by glycerol concentration higher than 40 g/L. In batch cultures, the highest lipid production (4.6 g/L), lipid content of biomass (33% w/w), and lipid volumetric productivity (0.15 g/L/h) were obtained with 40 g/L glycerol, during the course of a 30-h process. Fed-batch cultivation succeeded in preventing substrate inhibition and in achieving a high cell-density culture. The improved lipid production and volumetric productivity reached the remarkable high level of 28 g/L and 0.28 g/L/h, respectively. The lipids accumulated by C. freyschussii ATCC 18737 have similar fatty acid composition of plant oil indicating their potential use as biodiesel feedstock. Calculated physicochemical properties of a biodiesel produced with the lipids from C. freyschussii ATCC 18737 are expected to meet the European and American standards, being equal to those of rapeseed and palm biodiesel. CONCLUSIONS: C. freyschussii ATCC 18737 could be considered an interesting microorganism for utilization in biofuel industry. Cultivation of this yeast in media containing crude glycerol should be investigated deeper in order to evaluate whether it may find application in the valorization of the waste of biodiesel manufacturing.

2014 - In vitro transformation of chlorogenic acid by human gut microbiota [Articolo su rivista]
Francisco, Tomas Barberan; Rocío, García Villalba; Quartieri, Andrea; Raimondi, Stefano; Amaretti, Alberto; Leonardi, Alan; Rossi, Maddalena
abstract

Scope: Chlorogenic acid (3-O-caffeoyl-quinic acid, C-QA), the caffeic ester of quinic acid, is one of the most abundant phenolic acids in Western diet. The majority of C-QA escapes absorption in the small intestine and reaches the colon, where the resident microbiota transforms it into several metabolites. C-QA conversion by the gut microbiota from nine subjects was compared to evaluate the variability of bacterial metabolism. It was investigated whether a potentially probiotic Bifidobacterium strain, capable of C-QA hydrolysis, could affect C-QA fate. Methods and results: Bioconversion experiments exploiting the microbiota from diverse subjects revealed that C-QA was metabolized through a succession of hydrogenation, dexydroxylation and ester hydrolysis, occurring in different order among the subjects. Transformation may proceed also through quinic acid residue breakdown, since caffeoyl-glycerol intermediates were identified (HPLC-MS/MS, Q-TOF). All the pathways converged on 3-(3-hydroxyphenyl)-propanoic acid, which was transformed to hydroxyphenyl-ethanol and/or phenylacetic acid in few subjects. A strain of Bifidobacterium animalis able to hydrolyze C-QA was added to microbiota cultures. It affected microbial composition but not to such an extent that C-QA metabolism was modified. Conclusion: A picture of the variability of microbiota C-QA transformations among subjects is provided. The transformation route through caffeoyl-glycerol intermediates is described for the first time.

2014 - Initial geno-phenotypic characterization of colon mycobiota from healthy donors [Poster]
Rossi, M; Colombari, B; Ardizzoni, A; Raimondi, S; Gozzoli, C; Simone, M; Orsi, Cf; Neglia, Rg; Amaretti, A; Peppoloni, S; Cermelli, C; Blasi, E
abstract

2014 - Isolation of carotenoid-producing yeasts from an alpine glacier [Articolo su rivista]
Amaretti, Alberto; Simone, Marta; Quartieri, Andrea; Masino, Francesca; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena
abstract

Cold-adapted yeasts are increasingly being isolated from glacial environments, including Artic, Antarctic, and mountain glaciers. Psychrophilic yeast isolates mostly belong to Basidiomycota phylum, such as Cryptococcus, Mrakia, and Rhodotorula, and represent an understudied source of biodiversity for potential biotechnological applications. Since some basidiomycetous yeast genera (e.g. Rhodotorula, Phaffia, etc.) were demonstrated to produce commercially important carotenoids (e.g. β-carotene, torulene, torularhodin and astaxanthin), the present study aimed to obtain psychrophilic yeast isolates from the surface ice of two Italian glaciers to identify new pigment-producers. 23 yeast isolates were obtained. Among them, three isolates giving pigmented colonies was subjected to ITS1/ITS2 sequencing and were attributed to the Basidiomycetous yeasts Dioszegia sp., hodotorula mucilaginosa, and Rhodotorula laryngis. The strains were cultured batchwise in a carbon-rich medium at 15°C until the stationary phase was reached, then the pigments were extracted from freeze-dried biomass using DMSO:acetone mixture. Visible spectrum and HPLC-DAD analysis revealed the presence of carotenoid pigments. In batch cultures of Dioszegia sp., carotenoid production was growth-associated and yielded up to 3.4 mg/L of a molecule exhibiting an m/z ratio (568) consistent with the molecular weight of xanthophylls bearing 2 OH groups.

2014 - Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes [Articolo su rivista]
Diego, Romano; Martina, Contente; Francesco, Molinari; Ivano, Eberini; Erica, Ruvutuso; Cristina, Sensi; Amaretti, Alberto; Rossi, Maddalena; Raimondi, Stefano
abstract

Background: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes.Results: Recombinant S. cerevisiae BY4741{increment}Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-β-methyl-β-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity.Conclusions: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.

2014 - The probiotic Bifidobacterium breve B632 inhibited the growth of Enterobacteriaceae within colicky infant microbiota cultures [Articolo su rivista]
Simone, Marta; Gozzoli, Caterina; Quartieri, Andrea; Mazzola, Giuseppe; Di Gioia, Diana; Amaretti, Alberto; Raimondi, Stefano; Rossi, Maddalena
abstract

Infant colic is a common gastrointestinal disorder of newborns, mostly related to imbalances in the composition of gut microbiota and particularly to the presence of gas-producing coliforms and to lower levels of Bifidobacteria and Lactobacilli. Probiotics could help to contain this disturbance, with formulations consisting of Lactobacillus strains being the most utilized. In this work, the probiotic strain Bifidobacterium breve B632 that was specifically selected for its ability to inhibit gas-producing coliforms, was challenged against the Enterobacteriaceae within continuous cultures of microbiota from a 2-month-old colicky infant. As confirmed by RAPD-PCR fingerprinting, B. breve B632 persisted in probiotic-supplemented microbiota cultures, accounting for the 64% of Bifidobacteria at the steady state. The probiotic succeeded in inhibiting coliforms, since FISH and qPCR revealed that the amount of Enterobacteriaceae after 18 h of cultivation was 0.42 and 0.44 magnitude orders lower (P &lt; 0.05) in probiotic-supplemented microbiota cultures than in the control ones. These results support the possibility to move to another level of study, that is, the administration of B. breve B632 to a cohort of colicky newborns, in order to observe the behavior of this strain in vivo and to validate its effect in colic treatment.

2013 - Antioxidant properties of potentially probiotic bacteria: in vitro and in vivo activities [Articolo su rivista]
Amaretti, Alberto; M., di Nunzio; A., Pompei; Raimondi, Stefano; Rossi, Maddalena; A., Bordoni
abstract

Thirty-four strains of lactic acid bacteria (seven Bifidobacterium, 11 Lactobacillus, six Lactococcus, and 10 Streptococcus thermophilus) were assayed in vitro for antioxidant activity against ascorbic and linolenic acid oxidation (TAA(AA) and TAA(LA)), trolox-equivalent antioxidant capacity (TEAC), intracellular glutathione (TGSH), and superoxide dismutase (SOD). Wide dispersion of each of TAA(AA), TAA(LA), TEAC, TGSH, and SOD occurred within bacterial groups, indicating that antioxidative properties are strain specific. The strains Bifidobacterium animalis subsp. lactis DSMZ 23032, Lactobacillus acidophilus DSMZ 23033, and Lactobacillus brevis DSMZ 23034 exhibited among the highest TAA(AA), TAA(LA), TEAC, and TGSH values within the lactobacilli and bifidobacteria. These strains were used to prepare a potentially antioxidative probiotic formulation, which was administered to rats at the dose of 10(7), 10(8), and 10(9) cfu/day for 18 days. The probiotic strains colonized the colon of the rats during the trial and promoted intestinal saccharolytic metabolism. The analysis of plasma antioxidant activity, reactive oxygen molecules level, and glutathione concentration, revealed that, when administered at doses of at least 10(8) cfu/day, the antioxidant mixture effectively reduced doxorubicin-induced oxidative stress. Probiotic strains which are capable to limit excessive amounts of reactive radicals in vivo may contribute to prevent and control several diseases associated with oxidative stress.

2013 - Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria [Articolo su rivista]
Alessandra, Bordoni; Amaretti, Alberto; Leonardi, Alan; Elisa, Boschetti; Francesca, Danesi; Diego, Matteuzzi; Roncaglia, Lucia; Raimondi, Stefano; Rossi, Maddalena
abstract

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSHGCA and BSHTDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.

2013 - Complementary microbial approaches for the preparation of optically pure aromatic molecules [Articolo su rivista]
Contente, Martina; Granato, Tiziana; Remelli, William; Zambelli, Paolo; Raimondi, Stefano; Rossi, Maddalena; Romano, Diego
abstract

Different strategies for stereoselective microbial preparation of various chiral aromatic compounds are described. Optically pure 2-methyl-3-phenyl-1-propanol, ethyl 2-methyl-3-phenylpropanoate, 2-methyl-3-phenylpropanal, 2-methyl-3-phenylpropionic acid and 2-methyl-3-phenylpropyl acetate have been prepared using different microbial biotransformations starting from different prochiral and/or racemic substrates. (S)-2-Methyl-3-phenyl-1-propanol and (S)-2-methyl-3-phenylpropanal were prepared by biotransformation of 2-methyl cinnamaldehyde using the recombinant strain Saccharomyces cerevisiae BY4741ΔOye2Ks carrying a heterologous OYE gene from Kazachstania spencerorum. (R)-2-Methyl-3-phenylpropionic acid was obtained by oxidation of racemic 2-methyl-3-phenyl-1-propanol with acetic acid bacteria. Kinetic resolution of racemic 2-methyl-3-phenylpropionic acid was carried out by direct esterification with ethanol using dry mycelia of Rhizopus oryzaeCBS 112.07 in organic solvent, giving (R)-ethyl 2-methyl-3-phenylpropanoate as major enantiomer. Finally, (R,S)-2-methyl-3-phenylpropyl acetate was enantioselectively hydrolysed employing different bacteria and yeasts having cell-bound carboxylesterases with prevalent formation of (R)- or (S)-2-methyl-3-phenyl-1-propanol, depending on the strain employed.

2013 - Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350: kinetics, metabolism, and β-xylosidase activities. [Articolo su rivista]
Amaretti, Alberto; Bernardi, T; Leonardi, Alan; Raimondi, Stefano; Zanoni, Simona; Rossi, Maddalena
abstract

Xylo-oligosaccharides (XOS) are sugar oligomers of β-1,4-linked xylopyranosyl moieties which exert bifidogenic effect and are increasingly used as prebiotics. The kinetics and the metabolism of Bifidobacterium adolescentis DSMZ 18350 growing on XOS and xylose were investigated. The growth rate was higher on XOS, but greater biomass yield was attained on xylose. Unlike other prebiotics, XOS oligomers were utilized simultaneously, regardless of their chain length. Throughout XOS utilization, xylose concentration slightly increased, being not neatly consumed and remaining unfermented. During growth on XOS, β-xylosidase activity was present in the cytosol, but it occurred in the supernatant as well. A β-1,4-xylolytic enzyme was purified from the supernatant of XOS cultures. The enzyme, a homotetramer of a 39-kDa single protein, was capable of complete XOS hydrolysis and exhibited maximum activity at pH 6.0 and 55 °C. Based on the molecular weight, the protein can be ascribable to the product of the gene BAD_1527, the activity of which has been inferred as an endo-β-1,4-xylanase, but has not been characterized so far. This β-1,4-xylolytic enzyme, found to be active in the cultural supernatant, gives a reason for the never explained accumulation of the monosaccharides in the media of bifidobacterial cultures growing on XOS, without excluding the major role of the intracellular hydrolysis of the imported oligomers.

2013 - Potential Impact of Probiotic Consumption on the Bioactivity of Dietary Phytochemicals [Articolo su rivista]
Rossi, Maddalena; Amaretti, Alberto; Leonardi, Alan; Raimondi, Stefano; Simone, Marta; Quartieri, Andrea
abstract

Many healthy phytochemicals occur in food in the form of esters, glycoconjugates, or polymers, which are not directly bioavailable. Probiotic lactobacilli and bifidobacteria, which have evolved within the colonic ecosystem where indigestible oligo- and polysaccharides are their sole carbon sources, bear several glycosyl-hydrolases and can contribute to release the aglycones from glycoconjugated phytochemicals. Among the glycosyl-hydrolases, β-glucosidases are the most pertinent, because many phytochemicals are glucoconjugates. β-Glucosidase-positive probiotic bacteria were proved to release the aglycones of isoflavones and lignans in vitro, but studies in vivo are scarce. A positive correlation between probiotic consumption and urinary and/or plasma levels of isoflavone or lignan metabolites was not established. However, the strains used in the trials were not validated for the enzymatic properties or for the ability to hydrolyze lignans or isoflavones. Thus, activation of specific phytochemicals by probiotic bacteria still needs substantial efforts to be proved.

2013 - Thermal adaptability of Kluyveromyces marxianus in recombinant protein production [Articolo su rivista]
Raimondi, Stefano; Elena, Zanni; Amaretti, Alberto; Claudio, Palleschi; Daniela, Uccelletti; Rossi, Maddalena
abstract

Background: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.Results: The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively).Conclusions: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.

2013 - Zinc Uptake by Lactic Acid Bacteria [Articolo su rivista]
Leonardi, Alan; Zanoni, Simona; Marzia De, Lucia; Amaretti, Alberto; Raimondi, Stefano; Rossi, Maddalena
abstract

The study aims to investigate zinc biosorption by strains of lactobacilli and bifidobacteria with a view to exploit them as organic matrixes for zinc dietary supplementation. Sixteen human strains of Lactobacillus and Bifidobacterium were assayed for zinc uptake. The minimum inhibitory concentration of zinc salts differed among the strains, but was never below 15 mmol L−1. When cultured in MRS broth containing 10 mmol L−1 ZnSO4, all the strains were capable of accumulating zinc in the range between 11 and 135 μmol g−1. The highest amount of cell-bound zinc was obtained in L. acidophilus WC 0203. pH-controlled batch cultures of this strain revealed that zinc uptake started in the growth phase, but occurred mostly during the stationary phase. Pasteurized and viable cultures accumulated similar amount of zinc, suggesting that a nonmetabolically mediated mechanism is involved in zinc uptake. These results provide new perspectives on the specific use of probiotics, since L. acidophilus WC 0203 could function as an organic matrix for zinc incorporation. The bioavailability of Lactobacillus-bound zinc deserves to be investigated to provide a future basis for optimization of zinc supplementation or fortification.

2012 - CLA production and accumulation by bifidobacteria. [Abstract in Atti di Convegno]
Rossi, Maddalena; Quartieri, Andrea; Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan
abstract

2012 - Candida freyschussii: an oleaginous yeast producing lipids from glycerol [Relazione in Atti di Convegno]
Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena
abstract

A surplus of glycerol is obtained from biodiesel manufacturing and represents a waste product whose applications are lacking. Thus, the use of glycerol as substrate for fermentation processes yielding valuable products is very attractive. In this study, the utilization of glycerol as a growth substrate for the cultivation of oleaginous yeasts was explored with the aim to produce microbial oils. Forty strains of environmental non-conventional yeasts belonging to 19 different species were screened for the ability to grow on glycerol and produce intracellular lipids in a medium containing an excess of this carbon source (C:N = 48:1). Three strains, belonging to the species Candida freyschussii, Pichia farinosa, and Saccharomyces spencerorum, depleted 40 g/L glycerol within 120 h and produced intracellular lipids. C. freyschussii yielded the highest amounts, lipids accounting for the 33 % of biomass on dry basis. 1H-NMR analysis revealed that the lipid extract did not contain detectable free fatty acids and was composed mostly of triacylglycerols. Lipid composition, determined by GC-MS analysis, was similar to plant oils, and may be optimal feedstock for biodiesel production, being dominated by monounsaturated C16 and C18. As in other oleaginous yeasts, lipid production by C. freyschussii sp. increased with the increase of the C:N ratio of the medium, but growth was inhibited at glycerol concentrations higher than 40 g/L. As a result, lipid production was the highest with 40 g/L glycerol, yielding 4.7 g/L lipids, with a mean volumetric productivity of 0.15 g/L/h. In order to prevent growth inhibition over 40 g/L glycerol and extend the lipogenic phase, different fed-batch strategies were tried. The best performing processes took advantage from the feeding with concentrated media exhibiting the same C:N ratio of the basal medium, leading to very productive high cell density cultures. With the continuous feeding of 20X-concentrated medium, 29 g/L lipids (i.e. the 32 % of biomass) were obtained in 100 h of cultivation, with a mean volumetric productivity of 0.30 g/L/h. The values herein reported are among the highest yield and productivity values ever obtained for fermentative processes exploiting oleaginous fungi to produce lipids from glycerol. Therefore, C. freyschussii could be considered as an interesting microorganism to convert glycerol into microbial oils for biofuel industry

2011 - Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae [Articolo su rivista]
Raimondi, Stefano; D., Romano; Amaretti, Alberto; F., Molinari; Rossi, Maddalena
abstract

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselectivetrans-hydrogenation of the double bond, representing a promising alternative to metal-basedcatalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 differentspecies was investigated. Growing cells of most of the strains reduced KIP via OYE and showed highstereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activitywas observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstaniaspencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candidafreyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nineunedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiaeBY4741Oye2, a mutant that showed negligible OYE and CR activities. Compared with the correspondingwild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields ofOYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cellsof recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the resultsherein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably usedas innovative biocatalysts in asymmetric reductions.

2011 - Folate production by probiotic bacteria. [Articolo su rivista]
Rossi, Maddalena; Amaretti, Alberto; Raimondi, Stefano
abstract

Probiotic bacteria, mostly belonging to the genera Lactobacillus and Bifidobacterium, confer a number of health benefits to the host, including vitamin production. With the aim to produce folate-enriched fermented products and/or develop probiotic supplements that accomplish folate biosynthesis in vivo within the colon, bifidobacteria and lactobacilli have been extensively studied for their capability to produce this vitamin. On the basis of physiological studies and genome analysis, wild-type lactobacilli cannot synthesize folate, generally require it for growth, and provide a negative contribution to folate levels in fermented dairy products. Lactobacillus plantarum constitutes an exception among lactobacilli, since it is capable of folate production in presence of para-aminobenzoic acid (pABA) and deserves to be used in animal trials to validate its ability to produce the vitamin in vivo. On the other hand, several folate-producing strains have been selected within the genus Bifidobacterium, with a great variability in the extent of vitamin released in the medium. Most of them belong to the species B. adolescentis and B. pseudocatenulatum, but few folate producing strains are found in the other species as well. Rats fed a probiotic formulation of folate-producing bifidobacteria exhibited increased plasma folate level, confirming that the vitamin is produced in vivo and absorbed. In a human trial, the same supplement raised folate concentration in feces. The use of folate-producing probiotic strains can be regarded as a new perspective in the specific use of probiotics. They could more efficiently confer protection against inflammation and cancer, both exerting the beneficial effects of probiotics and preventing the folate deficiency that is associated with premalignant changes in the colonic epithelia.

2011 - Getting lipids for biodiesel production from oleaginous fungi [Capitolo/Saggio]
Rossi, Maddalena; Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan
abstract

Biomass-based biofuel production represents a pivotal approach to face high energy prices and potential depletion of crude oils reservoirs, to reduce greenhouse gas emissions, and to enhance a sustainable economy (Zinoviev et al., 2010). Microbial lipids can represent a valuable alternative feedstock for biodiesel production, and a potential solution for a bio-based economy.Nowadays, the production of biodiesel is based mostly on plant oils, even though animal fats, and algal oils can also be used. In particular, soybean, rapeseed, and palm oils are adopted as the major feedstock for biodiesel production. They are produced on agricultural land, opening the debate on the impact of the expansion of bioenergy crop cultures, which displace land from food production. Furthermore, their price restricts the large-scale development of biodiesel to some extent. In order to meet the increasing demand of biodiesel production, other oil sources have been explored. Recently, the development of processes to produce single cell oil (SCO) by using heterotrophic oleaginous microorganisms has triggered significant attention (Azocar et al., 2010). These organisms accumulate lipids, mostly consisting of triacylglycerols (TAG), that form the storage fraction of the cell. The occurrence of TAG as reserve compounds is widespread among all eukaryotic organisms such as fungi, plants and animals, whereas it has only rarely been described in bacteria (Meng et al., 2009). In fact, bacteria generally accumulate polyhydroxyalkanoates as storage compound and only few bacterial species, belonging to the actinobacterial genera Mycobacterium, Streptomyces, Rhodococcus and Nocardia produce relevant amounts of lipids (Alvarez & Steinbuchel, 2002).Among heterotrophic microorgansisms, oleaginous fungi, including both molds and yeasts, are increasingly been reported as good TAG producers. This chapter will focus on current knowledge advances in their metabolism, physiology, and in the result achieved in strain improvement, process engineering and raw material exploitation.

2011 - Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside [Articolo su rivista]
L., Roncaglia; Amaretti, Alberto; Raimondi, Stefano; Leonardi, Alan; Rossi, Maddalena
abstract

Lignans are ubiquitous plant polyphenols, which have relevant healthy properties, being the major phytoestrogens occurring in western diets. Secoisolariciresinol (SECO) is the major dietary lignan, mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by B. pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.

2010 - Biocatalytic exploitation of enoate reductase from unconventional yeasts [Abstract in Rivista]
Raimondi, Stefano; Diego, Romano; Raffaella, Gandolfi; Amaretti, Alberto; Francesco, Molinari; Rossi, Maddalena
abstract

Introduction: Old Yellow Enzymes (OYE, EC 1.6.99.1) are flavindependent oxidoreductases that catalyze the stereoselective transhydrogenation of electron-poor alkenes (enoate reductase activity), which can result in important chiral synthons toward synthesis of many pharmaceuticals and chemicals. In this study, environmental yeasts have been screened for OYE activity with the purpose to develop new enoate reductase biocatalyst. Methods: A set of 30 environmental yeasts belonging to 7 genera were screened for OYE activity, based on conversion of ketoisophorone (KIP). Ten new genes encoding OYE enzymes were cloned, sequenced, and overexpressed in S. cerevisiae BY4741(Oye2). The recombinant strains were tested for the reduction of KIP, isophorone, alpha-methyl-transcinnamaldehyde, 3-Methyl-2-cyclohexenone and nitro-phenylpropene. Two OYE genes were overexpressed in Escherichia coli. Results: Based on the amount of KIP reduction products, the strains belonging to the species Kazachstania spp., Candida spp., and Kluyveromyces marxianus exhibited the highest OYE activity, and their genes were cloned. The OYE proteins revealed an identity ranging from 76% to 99% with the protein Oye2 of Saccharomyces cerevisiae. Resting-cell of S. cerevisiae BY4741(Oye2) expressing recombinant OYEs converted up to 1 g l−1 of substrate, in less than 24 hours and with e.e. of 98%. OYE overexpression in E. coli was successful and allowed protein purification. Discussion: These study identified new enoate reductase biocatalysts within unconventional yeasts. Recombinant strains with enhanced OYE activity have been obtained and could be exploited for production of purified enzyme. Furthermore, the data herein reported attract interest due to promising applications in asymmetric synthesis.

2010 - Dietary Isoflavones and Intestinal Microbiota: Metabolism and Transformation into Bioactive Compounds [Capitolo/Saggio]
Rossi, Maddalena; Amaretti, Alberto; Roncaglia, Lucia; Leonardi, Alan; Raimondi, Stefano
abstract

Edible plants provide the human with hundreds of non-nutritional phytochemicals which are recognized as beneficial, such as isoflavones. Likewise other polyphenols, isoflavones may undergo extensive transformations during passage through human digestive tract, especially in the colon, where members of the complex commensal microbiota are capable to carry out synergistically a broad range of metabolic transformations affecting the fate and the biological activity of phytochemicals. Diverse bacterial species occurring in the large intestine hydrolyze the glucose conjugated forms of isoflavones, releasing the corresponding aglycones, which may undergo further microbial conversions (especially reductions) giving rise to a wide spectrum of isoflavone-derived compounds. The end-products of microbial transformations are subjected to substantial person-to-person variation, reflecting the impact of the colonic microbiota, since intestinal bacteria may greatly increase or compromise the biological activity of dietary isoflavones. In fact, certain reduction products (e.g. S-equol) possess superior antioxidant and estrogen-like activities, while others are precursors for C-ring cleavage and lead to isoflavone degradation. Extensive research has been performed to characterize the intestinal bacteria which are responsible for isoflavones transformations. In particular, many efforts are being carried out to identify single bacteria that convert the major soy isoflavone daidzein into S-equol, in the perspective to enrich soy products with such a valuable isoflavone-derived metabolite. Besides, diverse bacteria which are capable of deglycosylation, demethylation, and various reduction reactions are increasingly being identified. Nonetheless, only bifidobacteria and lactobacilli, which produce β-glucosidase that hydrolyze isoflavone glycosides into the corresponding aglycones, are currently exploited to improve the biological activity of soymilk.

2010 - Production of Single Cell Oils by the Cold-Adapted Oleaginous Yeast Rhodotorula glacialis AS 4.7: Effects of the Growth Temperature and the C:N Ratio. [Relazione in Atti di Convegno]
Amaretti, Alberto; Raimondi, Stefano; Sala, Maurizio; Roncaglia, Lucia; DE LUCIA, Marzia; Leonardi, Alan; Rossi, Maddalena
abstract

Rhodotorula glacialis AS 4.7 is an oleaginous psychrophilic yeast which was isolated from glacial environments. Despite its origin, the strain abundantly grew and accumulated lipids up to 20°C. The growth temperature did not influence the yield coefficients of both biomass and lipids production, but had significant effects on the growth rate and thus on volumetric productivity of lipid. 15°C were identified as the optimum temperature for lipid production. As the growth temperature decreased, the abundance of C18 fatty acids (FA) increased at the expenses of C16 FA and the unsaturation degree increased as well. In particular, remarkable amounts of linolenic acid (C18:3 ω-3) were produced at -3°C, accounting for 29% of FA. Lipid production by R. glacialis AS 4.7 especially occurred in carbon rich media, through a two-stages process. The first stage resulted in multiplication of cells and finished with the exhaustion of a nutrient other than the carbon source. During the second stage, the excess glucose was converted into intracellular storage lipids. The extent of the carbon excess had major positive effects on lipid production. The lipid content of biomass, glucose conversion into lipids, lipid concentration, and lipids productivity were all maximum with 120 g L-1 glucose (68%, 16%, 19 g L-1, and 0.054 g L-1 h-1, respectively). The results herein reported suggest that R. glacialis AS 4.7 could be considered as an interesting microorganism for the production of single cell oils and represent the first proposed biotechnological application for this yeast species.

2010 - Production of Single Cell Oils from Glycerol By Oleaginous Yeasts [Abstract in Rivista]
Amaretti, Alberto; Raimondi, Stefano; Rossi, Maddalena
abstract

Introduction: A surplus of crude glycerol is obtained as the by-product of biodiesel manufacturing and has became a waste product whose applications are lacking. Thus, the use of crude glycerol as substrate for fermentation processes yielding valuable products is attractive. In this study, the utilization of glycerol as a growth substrate for the cultivation of oleaginous yeasts has been explored. Methods: 40 environmental yeasts belonging to 19 different species were screened for the ability to grow and produce intracellular lipids at 30 ◦C in a medium containing glycerol excess (C:N = 48:1). The most promising strain was challenged for lipid production under increasing C:N ratios, in batch and fedbatch bioreactor processes. Lipids were extracted from lyophilized biomass and determined gravimetrically. GC-MS analysis of fatty acids methyl-esters was performed. Results: Three out of 40 strains, belonging to the species Candida freyschussii, Pichia farinosa, and Saccharomyces spencerorum, depleted 40 g/L glycerol within 120 h and produced intracellular lipids. C. freyschussii sp. produced the highest amount of lipids, accounting for the 30% of biomass dry weight and the 8% of glycerol conversion. C16 and C18 (C18, C18:1, C18:2, and traces of C18:3) were the most abundant fatty acids, accounting for the 40 and 55%, respectively, with an unsaturation index of 0.9. Lipid production was positively affected by glycerol concentration and was the highest (30% of biomass weight) with 40 g/L glycerol. Since glycerol inhibited the growth of C. freyschussii sp. above 40 g/L, fed-batch processwasdeveloped.When50% glycerolwasfed, lipids increased up to 48% of biomass. Discussion: This study represents the first proposal of a biotechnological application of C. freyschussi, a rare non-albicans Candida. The yeast resulted oleaginous and a good candidate for the production of single cell oils from glycerol.

2010 - Rapid method for screening enoate reductase activity in yeasts [Articolo su rivista]
Raimondi, Stefano; Roncaglia, Lucia; Amaretti, Alberto; Leonardi, Alan; P., Buzzini; Forti, Luca; Rossi, Maddalena
abstract

Old Yellow Enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the asymmetric reduction of electron-poor alkenes (enoate reductase activity). Since OYEs are involved in detoxification of acrolein, a high-throughput method for selecting yeasts expressing high enoate reductase activity, based on their acrolein resistance, was developed. The screening method was based on the measurement of growth in acrolein-supplemented medium, in 96-well microtiter plate cultures. A quantitative descriptor (Acrolein Resistance Factor = ARF) was firstly designed for quantifying the influence of both acrolein concentration and time of exposure on yeast growth. Besides, the efficiency of bioconversion of ketoisophorone (KIP) was exploited to measure OYE activity. In order to validate the method, ARF was correlated with the bioconversion of KIP on thirty yeast strains, belonging to 7 genera. With only a few exceptions, all strains exhibiting the highest ARF also displayed the maximum OYE activity. The presence of OYE genes in the strains showing OYE activity was confirmed by PCR amplification. Based on the results herein reported, this method should be profitably used as a fast screening procedure aimed at selecting outstanding strains for whole-cell bioconversions and could open many possibilities for the isolation and the biocatalytic exploitation of new OYEs from yeasts.

2010 - Secretion of Kluyveromyces lactis Cu/Zn SOD: strategies for enhanced production [Articolo su rivista]
Raimondi, Stefano; D., Uccelletti; Amaretti, Alberto; Leonardi, Alan; C., Palleschi; Rossi, Maddalena
abstract

The Kluyveromyces lactis Cu/Zn SOD gene (SOD1) was fused with the toxin K1 signal sequence to obtain extracellular production of superoxide dismutase. Kluyveromyces marxianus L3 and K. lactis MW98-8C strains were transformed and compared as hosts for the secretion. The effects of the media composition were evaluated: In K. lactis, the highest volumetric activity was obtained in YKK synthetic medium in the presence of Cu(2+)/Zn(2+) cofactors (9.6 kU l(-1)). In K. marxianus, active SOD was produced only in YPD medium supplemented with Cu(2+) and Zn(2+) (8.8 kU l(-1)). In order to improve the production of secreted active SOD in K. lactis, the SOD1 copper carrier (CCS1) was overexpressed and targeted to the secretory apparatus. A positive effect was observed only when K. lactis was grown in a medium without Cu(2+)/Zn(2+) supplement. The best performing culture conditions for K. lactis and K. marxianus recombinant strains were successfully applied to two laboratory-scale fed-batch processes, and volumetric SOD activities increased up to 19.4 and 24.1 kU l(-1), respectively.

2010 - Single cell oils of the cold-adapted oleaginous yeast Rhodotorula glacialis DBVPG 4785 [Articolo su rivista]
Amaretti, Alberto; Raimondi, Stefano; Sala, Maurizio; Roncaglia, Lucia; DE LUCIA, Marzia; Leonardi, Alan; Rossi, Maddalena
abstract

Background: The production of microbial lipids has attracted considerable interest during the past decade sincethey can be successfully used to produce biodiesel by catalyzed transesterification with short chain alcohols.Certain yeast species, including several psychrophilic isolates, are oleaginous and accumulate lipids from 20 to 70%of biomass under appropriate cultivation conditions. Among them, Rhodotorula glacialis is a psychrophilicbasidiomycetous species capable to accumulate intracellular lipids.Results: Rhodotorula glacialis DBVPG 4785 is an oleaginous psychrophilic yeast isolated from a glacial environment.Despite its origin, the strain abundantly grew and accumulated lipids between -3 to 20°C. The temperature did notinfluence the yield coefficients of both biomass and lipids production, but had positive effect on the growth rateand thus on volumetric productivity of lipid. In glucose-based media, cellular multiplication occurred first, while thelipogenic phase followed whenever the culture was limited by a nutrient other than glucose. The extent of thecarbon excess had positive effects on triacylglycerols production, that was maximum with 120 g L-1 glucose, interms of lipid concentration (19 g L-1), lipid/biomass (68%) and lipid/glucose yields (16%). Both glucoseconcentration and growth temperature influenced the composition of fatty acids, whose unsaturation degreedecreased when the temperature or glucose excess increased.Conclusions: This study is the first proposed biotechnological application for Rhodotorula glacialis species, whoseoleaginous biomass accumulates high amounts of lipids within a wide range of temperatures through appropriatecultivation C:N ratio. Although R. glacialis DBVPG 4785 is a cold adapted yeast, lipid production occurs over a broadrange of temperatures and it can be considered an interesting microorganism for the production of single cell oils.

2009 - Bioconversion of soy isoflavones daidzin and daidzein by Bifidobacterium strains. [Articolo su rivista]
Raimondi, Stefano; Roncaglia, Lucia; M., De Lucia; Amaretti, Alberto; Leonardi, Alan; Pagnoni, Ugo Maria; Rossi, Maddalena
abstract

Twenty-two strains of Bifidobacterium, representative of eight major species of human origin, were screened for their ability to transform the isoflavones daidzin and daidzein. Most of the strains released the aglycone from daidzin and 12 gave yields higher than 90%. The kinetics of growth, daidzin consumption, and daidzein production indicated that the hydrolytic activity occurred during the growth. The supernatant of the majority of the strains did not release the aglycone from daidzin, suggesting that cell-associated beta-glucosidases (beta-Glu) are mainly responsible for the metabolism of soybean glyco-conjugates. Cell-associated beta-Glu was mainly intracellular and significantly varied among the species and the strains. The lack of beta-Glu was correlated with the inability to hydrolyze daidzin. Although S-equol production by anaerobic intestinal bacteria has been established, information on S-equol-producing bifidobacteria is contradictory. In this study, 22 bifidobacteria failed to transform daidzein into reduced metabolites under all the experimental conditions, excluding any role in the reductive pathway of daidzein toward the production of S-equol. These results suggest that selected probiotic strains of Bifidobacterium can be used to speed up the release of daidzein, improving its bioavailability for absorption by colonic mucosa and/or biotransformation to S-equol by other intestinal microorganisms.

2009 - Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts. [Articolo su rivista]
Rossi, Maddalena; P., Buzzini; L., Cordisco; Amaretti, Alberto; M., Sala; Raimondi, Stefano; C., Ponzoni; Pagnoni, Ugo Maria; D., Matteuzzi
abstract

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y(X/S)). Obligate and facultative psychrophiles exhibited the highest Y(X/S). Acclimation to lower temperature decreased the lipid yield (Y(L/X)) in mesophilic yeasts, but did not affect Y(L/X) in facultative psychrophilic ones. Similar Y(L/X) were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of alpha-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.

2008 - Adaptation and acclimation to diverse temperatures of obligate and facultative psychrophilic and mesophilic yeasts [Abstract in Atti di Convegno]
Amaretti, Alberto; Cordisco, L; Raimondi, Stefano; Leonardi, A; Buzzini, P; Pagnoni, Um; Rossi, Maddalena
abstract

2008 - Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity [Articolo su rivista]
Raimondi, Stefano; Uccelletti, D; Matteuzzi, D; Pagnoni, Ugo Maria; Rossi, Maddalena; Palleschi, C.
abstract

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 U(SOD) mg(prot) (-1)) and the highest volumetric yield (8.8 x 10(5) U(SOD) l(-1)) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu(2+) and Zn(2+) supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 x 10(6) U(SOD) l(-1).

2008 - Growth parameters of obligate and facultative psychrophilic, and mesophilic yeasts [Abstract in Atti di Convegno]
Amaretti, Alberto; Cordisco, L; Raimondi, Stefano; Turchetti, B; Pagnoni, Um; Matteuzzi, D; Rossi, Maddalena
abstract

2008 - In vitro comparison of the prebiotic effects of two inulin-type fructans [Articolo su rivista]
Pompei, A; Cordisco, L; Raimondi, Stefano; Amaretti, Alberto; Pagnoni, Ugo Maria; Matteuzzi, D; Rossi, Maddalena
abstract

Faecal cultures were used to compare the prebiotic effects of a new fructan containing high solubility inulin (HSI) and of a well-established prebiotic containing oligofructose (OF) with a negative control (CT). Changes in the intestinal microbiota, pH, ammonia, volatile organic acids and lactic acid were monitored during incubation. Molecular techniques for microbial enumeration indicated that both HSI and OF led to a significant increase in bifidobacteria (P≤0.05) and lactobacilli (P≤0.05) compared to the control. Significant changes in the pH and levels of ammonia with both inulin-type fructans were observed, as well as higher levels of acetic, lactic and formic acids (P≤0.05). The fermentative metabolism appeared to be faster on OF than on HSI. Both OF and HSI showed clear prebiotic effects, but had differences in fermentation kinetics because of to the different degree of polymerization (DP). This study provides proof for the prebiotic effectiveness of HSI, and shows that inulin-type fructans with higher DP might have a prolonged bifidogenic effect, thus could extend the saccharolytic metabolism and low pH to the distal parts of the colon.

2008 - SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion [Articolo su rivista]
Raimondi, Stefano; E., Zanni; C., Talora; Rossi, Maddalena; C., Palleschi; D., Uccelletti
abstract

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O(2)(-)) into oxygen and hydrogen peroxide. It is a Cu(2+)/Zn(2+) metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

2007 - Administration of folate-producing bifidobacteria enhances folate status in Wistar rats [Articolo su rivista]
Pompei, A; Cordisco, L; Amaretti, Alberto; Zanoni, S; Raimondi, Stefano; Matteuzzi, D; Rossi, Maddalena
abstract

To develop a probiotic that provides the host with folate, we administered folate-overproducing bifidobacteria (Bifidobacteria adolescentis MB 227, B. adolescentis MB 239, and B. pseudocatenulatum MB 116) to Wistar rats with induced folate deficiency. Four groups of rats were fed a solid, low-folate diet with no supplements, folate-producing bifidobacteria [probiotic (PRO)], oligofructose [prebiotic (PRE)], or PRO plus PRE [symbiotic (SYM)] for 14 d. The SYM group also had a significantly higher (16.4 +/- 3.7 nmol/L) than in the PRO group (9.1 +/- 0.3 nmol/L), which was greater than in the control (4.8 +/- 0.5 nmol/L) and PRE groups (5.3 +/- 1.4 nmol/L). The SYM group also had a significantly higher hepatic folate concentration than in the other groups, whereas the kidney folate concentration did not differ among the groups. In the unsupplemented group, the pH of feces did not change during the trial, whereas diets containing bifidobacteria and/or oligofructose led to significant acidification due to enhanced saccharolytic metabolism. As a consequence of feeding rats PRE, PRO, and SYM diets, lactobacilli and bifidobacteria were significantly greater than in controls, whereas coliforms and enterococci were lower. This experiment showed that B. adolescentis MB 227, B. adolescentis MB 239, and B. pseudocatenulatum MB 116 exert both the beneficial effects of probiotics and produce folate in vivo, positively affecting the folate status of rats. The simultaneous administration of oligofructose and folate-producing bifidobacteria enhance their effectiveness on folate status. This study provides new perspectives on the specific use of probiotics to deliver important vitamins such as folate.

2007 - Laccase catalyzed dimerization of hydroxystilbenes [Articolo su rivista]
Ponzoni, Chiara; E., Beneventi; Cramarossa, Maria Rita; Raimondi, Stefano; G., Trevisi; Pagnoni, Ugo Maria; S., Riva; Forti, Luca
abstract

A series of hydroxystilbenes, analogues of the bioactive phytoalexin resveratrol, were synthesized and submitted to the catalytic action of a laccase from Trametes pubescens in a biphasic system made of ethyl acetate and acetate buffer. Oxidation took place at the 4’-hydroxy (4-hydroxy) position ofthe hydroxystilbenicm oieties, followed by radicalradicalcoupling dimerization reactions. Most of the products were isolated in good yields and fully characterized.Depending on the substrates, three different dimeric products could be identified, the main products usually being 4-O-a-ß-5 (dihydrofuran-like) dimers.

2005 - Enantioselective esterase activity of an industrial glutaryl acylase [Articolo su rivista]
S., Adani; Raimondi, Stefano; Forti, Luca; D., Monti; S., Riva
abstract

The unexpected esterase activity of an industrial glutaryl acylase was investigated. Glutaryl esters of a series of primary and secondary alcohols as well as of phenols were all efficiently hydrolyzed, the only exception being the sterically hindered glutarate of thymol. The enantioselectivities of the acylase, which were evaluated with three of these substrates, were quite low (E values ranging between 1.9 and 7.2), but were significantly improved by substrate and/or solvent engineering. Enantiomerically enriched hydrolyzed alcohols and unreacted glutarates can be easily separated by selective extraction, thus avoiding chromatographic steps.

2004 - Comparison of cyclohexanone monooxygenase as an isolated enzyme and whole cell biocatalyst for the enantioselective oxidation of 1,3-dithiane [Articolo su rivista]
Zambianchi, F.; Raimondi, S.; Pasta, P.; Carrea, G.; Gaggero, N.; Woodley, J. M.
abstract

Both whole cells of recombinant Escherichia coli TOP10, overexpressing cyclohexanone monooxygenase (CHMO) and isolated cyclohexanone monooxygenase, were used to carry out the enantioselective oxidation of 1,3-dithiane (1) to (R)-1,3-dithiane-1-oxide (2). The two biocatalysts were evaluated under various experimental conditions (e.g., shaken flask or bioreactor; non-bound or resin-adsorbed substrate; different substrate concentrations) in terms of volumetric productivity and enantioselectivity. While productivity was similar in the two cases (up to 0.58 g L -1 h -1), the optical purity of the product was much higher with the isolated enzyme (up to 98% e.e.) than with the whole cell biocatalyst (up to 85% e.e.). © 2004 Elsevier B.V. All rights reserved.

2003 - Glutaryl acylases: One-reaction enzymes or versatile enantioselective biocatalysts? [Articolo su rivista]
Raimondi, S.; Monti, D.; Pagnoni, Ugo Maria; Riva, S.
abstract

A significant broad substrate specificity, that crosses over the usual beta-lactam derivatives, has been observed with an industrial glutaryl-7-aminocephalosporanic acid acylase (GA). This enzyme possesses significant enantioselective amidase and even esterase activity, with a stereopreference for the S-enantiomer. The easy separation of products from unreacted reagents, possessing different physical-chemical properties, is achieved by solvent extraction, avoiding chromatography or distillation during reaction work-up.

2003 - Glutaryl-7-ACA acylase: a new tool for the biocatalyzed kinetic resolution of racemic amines and alcohols [Abstract in Rivista]
Raimondi, Stefano; Forti, Luca; D., Monti; S., Riva
abstract

The enzymes D-amino acid oxidase and glutaryl-7-ACAacylase (GA) are currently utilised for the industrial productionof 7-aminocephalosporanic acid (7-ACA, 2), an importantprecursor of semisyntetic cephalosporins. Specifically,GA is devoted to the cleavage of the amide bond betweenglutaric acid and 7-ACA in the intermediate glutaryl-7-ACA(1).The synthetic performances of this enzyme towards 1have been widely investigated, while very little is known onGA substrate specificity.We have found that an industrial GAis very specific for the acyl moiety that has to be released, theglutaryl derivatives being by far the best substrates. On theother hand, this enzyme accepts a wide variety of “leavinggroups”. Not only N-glutarate of b-lactam derivatives, butalso N-glutaryl aminoacids as well as N-glutarylamides (aromaticand aliphatic) could be hydrolysed by GA, which, additionally,showed a significant esterase activity. Morenotably, GA-catalysed hydrolyses were highly enantioselective

2003 - Kinetic resolutions of racemic amines and alcohols catalyzed by an industrial glutaryl-7-aminocephalosporanic acid acylase with unexpected broad substrate specificity [Articolo su rivista]
Raimondi, Stefano; Forti, Luca; D., Monti; S., Riva
abstract

An industrial glutaryl-7-aminocephalosporanic acid acylase (GAR) possesses a significant broad substrate specificity that crosses over the usual cephalosporanic skeleton. Enantioselective amidase and even esterase activities have been observed with all the glutarates of racemic substrates investigated, with a stereopreference for the (S)-enantiomer. The different physical-chemical properties of reagents and products allow their easy separation by solvent extraction, avoiding cumbersome chromatography or distillation processes during reaction work-up. (C) 2003 Elsevier Science Ltd. All rights reserved.