Nuova ricerca

Alan LEONARDI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Chimica

Home |


Pubblicazioni

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2005 - Electrostatic effects on the thermodynamics of protonation of reduced plastocyanin [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Ranieri, Antonio; Leonardi, Alan; Sola, Marco
abstract

The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects.

2005 - Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Gw, Canters; DI ROCCO, Giulia; E., DE WAAL; Y., Arendsen; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic memory of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.

2005 - Reduction thermodynamics of the T1 Cu site in plant and fungal laccases [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Leonardi, Alan; R., Pierattelli; Aj, Vila; A., DE CANDIA; Sola, Marco
abstract

The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV-vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the E-o' of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same E-o', as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.

2004 - Characterization of the solution reactivity of a basic heme peroxidase from Cucumis sativus [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Leonardi, Alan; Sola, Marco
abstract

A basic heme peroxidase has been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and H NMR spectra from pH 3 to 11. The protein, as isolated, contains a high-spin ferriheme which in the low pH region is sensitive to two acid-base equilibria with apparent pK(a) values of approximately 5 and 3.6, assigned to the distal histidine and to a heme propionate, respectively. At high pH, a new low-spin species develops with an apparent pK(a) of 11, likely due to the binding of an hydroxide ion to the sixth (axial) coordination position of the Fe(III). A number of acid-base equilibria involving heme propionates and residues in the distal cavity also affect the binding of inorganic anions such as cyanide, azide, and fluoride to the ferriheme, as well as the catalytic activity. The reduction potentials of the native protein and of its cyanide derivative, determined through UV-Vis spectroelectrochemistry, result to be -0.320 +/- 0.015 and -0.412 +/- 0.010V, respectively. Overall, the reactivity of this protein parallels those of other plant peroxidases, especially horseradish peroxidase. However, some differences exist in the acid-base equilibria affecting its reactivity and in the reduction potential, likely as a result of small structural differences in the heme distal and proximal cavities.

2003 - 1H NMR of native and azide-inhibited laccase from Rhus vernicifera [Articolo su rivista]
Battistuzzi, Gianantonio; DI ROCCO, Giulia; Leonardi, Alan; Sola, Marco
abstract

The H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time.These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in thehigh frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differencesbetween the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structuralproperties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over1 the T1 copper ligands. Overall, these data demonstrate that H NMR can be fruitfully applied to characterize the electronic properties ofthe metal sites of blue oxidases at room temperature.

2002 - Conservation of the free energy change of pH-dependent isomerizations in cytochromes c and blue copper proteins [Poster]
Battistuzzi, Gianantonio; Borsari, Marco; Canters, Gerard W.; DI ROCCO, Giulia; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the conformational transitions occurring at low pH in blue copper proteins (acid transition), and at high pH in cytochromes c (alkaline transition) have been determined through direct electrochemistry experiments carried out at variable pH and temperature. The former transition involves protonation and detachment from the Cu(I) ion of one histidine ligand (1), whereas the latter leads to a conformer in which the axial methione ligand of the ferriheme is substituted by a surface lysine, the transition being triggered by an as yet unidentified deprotonating residue (2). The blue copper proteins investigated were plastocyanins, R. vernicifera stellacyanin, CBP and T. versutus amicyanin. For all species but CBP the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. It is apparent that the thermodynamic “driving force” for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the G of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, but amicyanin, for which the greater exotermicity of the transition can be ascribed to peculiar features of the active site. For cytochromes c, we have found that both transition enthalpy and entropy are remarkably species-dependent, following the order: R.pal cytc2 >> beef (horse) heart cytc > yeast iso-1 cytc. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol-1), and consequently in the apparent pKa value. Therefore, enthalpy/entropy compensation phenomena compensation are common to both transitions, and indicate that solvent reorganization effects play an important role in the thermodynamics of the pH-induced conformational changes.

2002 - Thermodynamics of the acid transition in blue copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; G. W., Canters; E., de Waal; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaHdegrees'(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic driving force for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.