Production of arabitol from glycerol by immobilized cells of Wickerhamomyces anomalus WC 1501.

Polyalcohols such as arabitol are among the main targets of biorefineries aiming to upcycle wastes and cheap substrates. In previous works Wickerhamomyces anomalus WC 1501 emerged as an excellent arabitol producer utilizing glycerol. Arabitol production by this strain is not growth associated, therefore, in this study, pre-grown cells were entrapped in calcium alginate beads (AB) and utilized for glycerol transformation to arabitol. Flasks experiments aimed to assess the medium composition (i.e., the concentration of inorganic and organic nitrogen sources and phosphates) and to establish the appropriate carrier-to-medium proportion. In flasks, under the best conditions of ammonium limitation and the carrier:medium ratio of 1:3 (w/v), 82.7 g/L glycerol were consumed in 168 h, yielding 31.2 g/L arabitol, with a conversion of 38% and volumetric productivity of 186 mg/mL/h. The process with immobilized cells was transferred to laboratory scale bioreactors with different configurations: stirred tank (STR), packed bed (PBR), fluidized bed (FBR), and airlift (ALR) bioreactors. The STR experienced oxygen limitation due to the need to maintain low stirring to preserve AB integrity and performed worse than flasks. Limitations in diffusion and mass transfer of oxygen and/or nutrients characterized also the PBR and the FBR and were partially relieved only in ALR, where 89.4 g/L glycerol were consumed in 168 h, yielding 38.1 g/L arabitol, with a conversion of 42% and volumetric productivity of 227 mg/mL/h. When the ALR was supplied with successive pulses of concentrated glycerol to replenish the glycerol as it was being consumed, 117 g/L arabitol were generated in 500 h, consuming a total of 285 g/L glycerol, with a 41% and 234 mg/L/h. The study strongly supports the potential of W. anomalus WC 1501 for efficient glycerol-to-arabitol conversion using immobilized cells. While the yeast shows promise by remaining viable and active for extended periods, further optimization is required, especially regarding mixing and oxygenation. Improving the stability of the immobilization process is also crucial for reusing pre-grown cells in multiple cycles, reducing dead times, biomass production costs, and enhancing the economic feasibility of the process.

Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of D-arabitol from pure glycerol.

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.

Indole and p-cresol in feces of healthy subjects: Concentration, kinetics, and correlation with microbiome.

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.

Multivariate Analysis in Microbiome Description: Correlation of Human Gut Protein Degraders, Metabolites, and Predicted Metabolic Functions.

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.

Comparison of gluten peptides and potential prebiotic carbohydrates in old and modern Triticum turgidum ssp. genotypes.

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.

Microbiota of sliced cooked ham packaged in modified atmosphere throughout the shelf life: Microbiota of sliced cooked ham in MAP.

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.

Bacterial community of industrial raw sausage packaged in modified atmosphere throughout the shelf life.

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.

Characterization of the peptide fraction from digested Parmigiano Reggiano cheese and its effect on growth of lactobacilli and bifidobacteria.

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.

Conjugated Linoleic Acid Production by Bifidobacteria: Screening, Kinetic, and Composition.

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.

Detection of novel metabolites of flaxseed lignans in vitro and in vivo.

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.

Hydrolysis of the rutinose-conjugates flavonoids rutin and hesperidin by the gut microbiota and bifidobacteria.

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.

Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii.

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.

In vitro transformation of chlorogenic acid by human gut microbiota.

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.

Potential impact of probiotic consumption on the bioactivity of dietary phytochemicals.

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, ß-glucosidases are the most pertinent, because many phytochemicals are glucoconjugates. ß-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.

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria.

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.

Zinc uptake by lactic Acid bacteria.

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.

Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350: kinetics, metabolism, and ß-xylosidase activities.

Xylo-oligosaccharides (XOS) are sugar oligomers of ß-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, ß-xylosidase activity was present in the cytosol, but it occurred in the supernatant as well. A ß-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-ß-1,4-xylanase, but has not been characterized so far. This ß-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.

Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside.

Lignans are ubiquitous plant polyphenols, which have relevant health 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 ß-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 Bifidobacterium 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 ß-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.

Single cell oils of the cold-adapted oleaginous yeast Rhodotorula glacialis DBVPG 4785.

BACKGROUND: The production of microbial lipids has attracted considerable interest during the past decade since they 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 psychrophilic basidiomycetous 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 not influence the yield coefficients of both biomass and lipids production, but had positive effect on the growth rate and thus on volumetric productivity of lipid. In glucose-based media, cellular multiplication occurred first, while the lipogenic phase followed whenever the culture was limited by a nutrient other than glucose. The extent of the carbon excess had positive effects on triacylglycerols production, that was maximum with 120 g L-1 glucose, in terms of lipid concentration (19 g L-1), lipid/biomass (68%) and lipid/glucose yields (16%). Both glucose concentration and growth temperature influenced the composition of fatty acids, whose unsaturation degree decreased when the temperature or glucose excess increased. CONCLUSIONS: This study is the first proposed biotechnological application for Rhodotorula glacialis species, whose oleaginous biomass accumulates high amounts of lipids within a wide range of temperatures through appropriate cultivation C:N ratio. Although R. glacialis DBVPG 4785 is a cold adapted yeast, lipid production occurs over a broad range of temperatures and it can be considered an interesting microorganism for the production of single cell oils.

Rapid method for screening enoate reductase activity in yeasts.

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.