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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.

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.

Background 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. Results Fed-batch fermentations with W. anomalus WC 1501 were optimized using central composite design (CCD). Dissolved oxygen had not a significant effect, while optimum values were found for glycerol concentration (114.5 g/L), pH (5.9), and temperature (32.5 °C), yielding 29 g/L d-arabitol in 160 h, a conversion yield of 0.25 g of arabitol per g of consumed glycerol, and a volumetric productivity of 0.18 g/L/h. CCD optimal conditions were the basis for further improvement, consisting in increasing the cellular density (3✕), applying a constant feeding of glycerol, and increasing temperature during production. The best performing fed-batch fermentations achieved 265 g/L d-arabitol after 325 h, a conversion yield of 0.74 g/g, and a volumetric productivity of 0.82 g/L/h. Conclusion W. anomalus WC 1501 confirmed as an excellent producer of d-arabitol, exhibiting a remarkable capability of transforming pure glycerol. The study reports among the highest values ever reported for microbial transformation of glycerol into d-arabitol, in terms of arabitol titer, conversion yield, and productivity. Graphical Abstract

Sliced cooked ham packaged in a modified atmosphere is a popular ready-to-eat product, subjected to abundant microbial contamination throughout its shelf life that can lead to deterioration of both sensorial properties and safety. In this study, the microbial load and the chemical–physical features of cooked ham of five producers were monitored for a period of 12 days after the opening of the packages (i.e., the secondary shelf life), during which the products were stored in a domestic refrigerator at 5.2 ± 0.6°C. The sensorial properties presented a perceivable decay after 8 days and became unacceptable after 12 days. High-performance liquid chromatography analysis and solid-phase microextraction coupled with gas chromatography profiling of volatile metabolites indicated that lactic acid, ethanol, acetic acid, acetoin, 3-methyl-1-butanol, and 2-3 butanediol were the main metabolites that characterized the evolution of the analyzed cooked ham. The microbiota was monitored by 16S ribosomal RNA gene profiling and culture-dependent techniques. Already at the opening of packages, all the products presented high microbial load, generally dominated by lactic acid bacteria, with evident differences among the products. The increase of lactic acid bacteria somehow protected samples from abundant contamination by other bacteria, concurring with the evolution of more safe products. This role was exerted by numerous Latilactobacillus, Leuconostoc, and Carnobacterium species, among which the most frequently detected were Latilactobacillus sakei, Latilactobacillus sakei carnosum, Leuconostoc mesenteroides, and Carnobacterium divergens. Some products presented more complex communities that encompassed Proteobacteria such as Moellerella wisconsensis, Proteus hauseri, Brochothrix thermosphacta, and less frequently Pseudomonas, Erwinia, and Massilia. Opportunistic pathogenic bacteria such as Escherichia coli and Vibrio sp. were found in small quantities. The yeasts Kazachstania servazzii and Debaryomyces hansenii occurred already at 0 days, whereas various species of Candida (Candida zeylanoides, Candida sake, Candida norvegica, and Candida glaebosa) were abundant only after 12 days. These results indicated that the microbiological contaminants overgrowing during the secondary shelf life did not derive from environmental cross-contamination at the opening of the tray but were already present when the packages were opened, highlighting the phases of production up to the packaging as those crucial in managing the safety risk associated to this product.

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 (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.

Additional file 1: Figure S1. 1H NMR (400 MHz, DMSO-d6) of the lyophilized supernatant of W.anomalus WC 1501 fed batch culture. δ 3.27 – 3.49 (m, 5H, 1-5), 3.59 (d, J = 10.9 Hz, 1H, 6 or 7),3.66 (d, J = 6.9 Hz, 1H 6 or 7), 4.12 (d, J = 7.2 Hz, 1H 8), 4.19 (bs, J = 5.8 Hz, 1H, 9 or 10), 4.30 (bs,J = 6.6 Hz, 1H, 9 or 10), 4.43 (bs, J = 6.1, 6.7 Hz, 2H, 11, 12).

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.

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.

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 ano-rectal 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.

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 in the hydrolysis of di-, oligo-, or polysaccharides are exploited to study and compare substrate preference of bifidobacteria for candidate prebiotics.

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.

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.

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.

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.

Dextrans are homopolysaccharides of D-glucose units produced by lactic acid bacteria. They have several technological applications and potential utilisation in positively modulating gut microbiota is attracting increasing attention. Whereas the prebiotic activity of low polymerisation degree (DP) dextrans has been established, high DP dextrans still deserve deeper investigation. In the present study, a long linear chain dextran produced by

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.

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.

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. (C) 2015 Elsevier Ltd. All rights reserved.

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 degrees 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 log(10)(cfu/g), increased to 7.7 log(10)(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.

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.

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., This study was partially supported by Christian Hansen, Denmark, by grants to AM from the Spanish Ministry of Economy and Competitiveness (SAF2015-65878-R) and Generalitat Valenciana (PrometeoII/2014/065), and by the European Regional Development Fund (ERDF).

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.

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.

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.

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., Andrea Quartieri was supported with a research grant from the SPINNER group, Regione Emilia Romagna and European Social Found.

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.

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.

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.

Analisi critica della pronuncia con cui la Corte costituzionale ha riconosciuto la conformità alla Carta Repubblicana della legge che, in vista della crisi di Alitalia, sospendeva temporaneamente la vigenza della disciplina in materia di concentrazioni.

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.

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.

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.

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed alpha-galactosidase, beta-galactosidase, and beta-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.

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.

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.

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg(-1)) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h(-1)), aeration pressure (0.3 vs 1.2 bar) and H(2)O(2) (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H(2)O(2) resulted in an increase in CuZn-SOD up to 475 U mg(-1). When a regulation of oxygen saturation was applied during batch cultures, CuZn-SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg(-1), respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg(-1), respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.

Fructooligosaccharides (FOS) and inulin are food grade non-digestible carbohydrates that exert beneficial nutritional effect. This paper describes the suitability of high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and capillary zone electrophoresis (CZE) to evaluate fermentation properties of FOS and inulin in pure Bifidobacterium cultures; and to study their effects on faecal cultures (microbial population and short-chain fatty acids). Prebiotic effectiveness of FOS and inulin of different degrees of polymerization was evaluated monitoring the changes in their molecular weight distribution during the in vitro growth of selected Bifidobacterium strains. The qualitative analysis of the residual soluble oligosaccharides or polysaccharides from Raftilose Synergy, Raftiline HP and Raftilose P95 was carried out by HPAEC-PAD, using a CarboPac PA 100 column and an appositely optimized gradient elution program. Under the optimized gradient elution conditions, glucose, fructose, sucrose were resolved from each other and from fructans with a DP ranging from 3 (1-kestose) to 60. The chromatographic profiles of the spent broths pointed out that almost every strain presented a different capability to ferment fructan chains of variable DP, indicating wide strain to strain differences. To explore the prebiotic effect of FOS and inulin, related to of short chain fatty acids (SCFAs) accumulation in faecal cultures due to fermentative metabolism of intestinal microflora, analysis of SCFAs, acetic and lactic acid was achieved by co-electroosmotic capillary electrophoresis, where the electrophoretic mobility of the anionic analytes and electroosmotic flow (EOF) were similarly directed. Moreover, the use of UV detection for the analyses of our organic anions required a running electrolyte which allowed indirect detection. The optimization of the capillary electrophoretic conditions was carried out by applying a chemometric study based on the use of the experimental design, the effects of three parameters, i.e. temperature, voltage and percentage of methanol added to the background electrolyte were investigated.

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. A strict 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.

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.

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.

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.

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.

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 biobased 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.

Aims: To determine the antioxidative activity, glutathione production, acid and bile tolerance and carbohydrate preferences of Lactobacillus plantarum LP 1, Streptococcus thermophilus Z 57 and Bifidobacterium lactis B 933. Methods and Results: The intact bacteria exhibited antioxidative capacity against linolenic acid and ascorbate oxidation. The antioxidative activity of cell-free extracts was determined by chemiluminescent assay and agreed with total glutathione content. Superoxide dismutase was negligible in all the strains. Bile and gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. Bifidobacterium lactis B 933 and L. plantarum LP 1 were more acid tolerant than S. thermophilus Z 57. All the strains were resistant to bile. Among 13 indigestible carbohydrates, galacto-oligosaccharides and fructo-oligosaccharides were utilized by all the strains and did not affect survival in human gastric juice. Conclusions: These potential probiotic strains exhibited antioxidative properties and good viability in gastric juice and bile may indicate tolerance to the transit through the upper gastrointestinal tract. Galacto-oligosaccharides and fructo-oligosaccharides are the most appropriate prebiotics to be used in effective synbiotic formulations. Significance and Impact of the Study: These results outline promising strains with antioxidative properties. Carbohydrate preferences can be exploited in order to develop synbiotic products.

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.