Tatiana Bernardi

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

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

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates.

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

Green electrochemical approach for delignification of wheat straw in second-generation bioethanol production

Ethanol is considered as one of the most promising next-generation automotive fuels, as it is carbon-neutral and can be produced from renewable resources, like lignocellulosic materials. However, owing to biomass characteristics, some technological barriers, such as delignification pretreatment, need to be addressed for an efficient conversion of wood to bioethanol. This paper describes a mild and harmless delignification of wheat straw, carried out at room temperature and requiring only 10 min, using an environmentally-friendly HOCl-containing electrochemically activated water solution, synthesized from diluted brines. Holocellulose (i.e. the sum of cellulose and hemicellulose) rich materials can be obtained with approximately 64% reduction of the initial lignin content. Both the absence of lignin degradation products, which could act as inhibitors during fermentation, and a promising, non-optimized ethanol yield (68% w/w on theoretical value) confirm the potential of this alternative approach towards resolving the challenges to develop a sustainable and low-cost process.

Detection of oligosaccharides in sugar products using planar chromatography

Abstract Oligosaccharides, and in particular raffinose and kestoses, are of great importance not only in the field of cane and beet processing but also in respect of the analyses of a number of agricultural raw materials and trade products. The authors judge it to be useful to have at ones disposal a simple and reliable analytical technique to be used for the detection of oligosaccharides in different materials. Modern planar chromatography can be utilized, bearing in mind that HPTLC (high performance thin layer chromatography) plates, Automated Multiple Development, completely automated elution systems, sample positioning and spots detection apparatuses, are available. Details on the analytical methodology adopted for molasses and other sugar products are presented and discussed also giving statistical data about its accuracy and precision. Some examples of practical application of the proposed methodology are described.

One-Pot, Four-Step Organocatalytic Asymmetric Synthesis of Functionalized Nitrocyclopropanes

The asymmetric synthesis of functionalized nitrocyclopropanes has been achieved by a one-pot, four-step method catalyzed by (S)-diphenylprolinol TMS ether, which joins two sequential domino reactions, namely a domino sulfa-Michael/aldol condensation of α,β-unsaturated aldehydes with 1,4-dithiane-2,5-diol, and a domino Michael/α-alkylation reaction of the derived chiral dihydrothiophenes with bromonitromethane. The title compounds were obtained in 27-45% yields, with high levels of diastereoselectivity (93:7 to 100:0 dr) and generally good enantioselectivities (up to 95:5 er).

Relative acidity scale of glycine- and taurine-conjugated bile acids through ESI-MS measurements

The most important bile acids, in the form of glycine and taurine conjugates, have been ordered in terms of relative acidity scale. The measurements have been carried out using mass spectrometric techniques. The group of taurine conjugates confirm the superior acidity over the glycine derivatives. Rationale of the differences found in gas-phase and comparison with the data reported in solution-phase are discussed with the support of theoretical calculations. The study has been completed with the acidity sequence of mixed oxo-hydroxy bile acids.

Separation and quantitative determination of aldoses and alditols by over-pressured layer chromatography (OPLC)

Anew OPLC method has been established for separation and quantitative determination of three alditols (d-xylitol, L-arabitol, and Dglucitol) and four aldoses (d-xylose, L-arabinose, d-glucose, and Lrhamnose). The aldoses are present in hemicellulose hydrolyzates used as substrates in the production of d-xylitol from d-xylose by yeast, and all seven sugars could be found in the final fermented broth. The separation was performed in approximately sixteen minutes, on aluminum foil-backed silica gel OPLC-HPTLC plates with overrunning elution. Acetonitrile-acetic acid-water, 63 + 33 + 5 (v/v), was used as mobile phase. The upper limits of linearity were in the range 1402-600 ng and detection limits were 152-50 ng per spot. The method has been used successfully to screen fermentation samples for aldoses and alditols.

Research Progress in the Modification of Quercetin Leading to Anticancer Agents

The flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) is widely distributed in plants, foods, and beverages. This polyphenol compound exhibits varied biological actions such as antioxidant, radical-scavenging, anti-inflammatory, antibacterial, antiviral, gastroprotective, immune-modulator, and finds also application in the treatment of obesity, cardiovascular diseases and diabetes. Besides, quercetin can prevent neurological disorders and exerts protection against mitochondrial damages. Various in vitro studies have assessed the anticancer effects of quercetin, although there are no conclusive data regarding its mode of action. However, low bioavailability, poor aqueous solubility as well as rapid body clearance, fast metabolism and enzymatic degradation hamper the use of quercetin as therapeutic agent, so intense research efforts have been focused on the modification of the quercetin scaffold to obtain analogs with potentially improved properties for clinical applications. This review gives an overview of the developments in the synthesis and anticancer-related activities of quercetin derivatives reported from 2012 to 2016.

Separation and quantitative determination of carbohydrates in microbial submerged cultures using different planar chromatography techniques (HPTLC, AMD, OPLC).

Carbohydrates are the principal sources of nutrient and energy in large-scale submerged fermentation processes. A method for detection and quantification of sugar levels are very advantageous because they can be considered as key indicators in determining the yields and the productivity of the process. For this reason, the objective of this study is to develop and validate a simple and relatively economical analytical method for detecting complex sugar mixtures in fermentation broth based on High-Performance Thin-Layer Chromatography (HPTLC). HPTLC is a widely used, fast and accurate method of separating complex mixtures. The proposed method involved the chromatographic separations of xilo-, galacto-, fructo-oligosaccharides mixtures at different molecular weights, tri- and disaccharides (raffinose, sucrose, lactose), and the corresponding monosaccharides (xylose, fructose, galactose) on HPTLC plates, using different eluent mixtures and elution conditions. The documentation of plates was performed using TLC visualization device and the images of plates were processed using a digital processor. HPTLC methods development using instrumental techniques as OPLC (Over Pressure Liquid Chromatography) and AMD (Automated Multiple Development) has been also described, as to simultaneously monitor several samples in the same elution, with significant time and solvent savings. Four different carbohydrates complex mixtures were analyzed using HPTLC techniques, as to optimize the quality of the separation among components. The methods set up were then applied for quantitative determination of sugars. As a model of submerged fermentations, a strain of Bifidobacterium spp. was used, a saccharolytic bacterium with probiotic activities in the human gut, able to anaerobically ferment complex sugar mixtures. Results could be easily extended to other fermentation processes.

An HPTLC-AMD method for understanding the metabolic behavior of microorganisms in the presence of mixed carbon sources. The case of Bifidobacterium adolescentis MB 239

The intestinal microflora Bifidobacteria has been adapted to utilize, as a source of energy, complex carbohydrates that escape hydrolysis by human digestive enzymes, for example fructo-oligosaccharides (FOS). The ability of intestinal microflora to utilize carbohydrates in complex mixtures is of particular interest, even though few data have been reported. This paper presents the results obtained using an innovative analytical approach based on instrumental HPTLC-AMD (automated multiple development) to investigate the metabolic behavior of Bifidobacterium adolescentis MB 239 as a case study. Raffinose, FOS (sucrose, 1-kestose, nystose, fructosyl-nystose), lactose, and their monomeric moieties glucose, galactose, and fructose were simultaneously present as carbon sources in the solution to be fermented by the bacterium. The method proposed has enabled quantitative monitoring of sugar concentrations during the entire time-course of the processes. The complex sugar mixtures were separated by use of acetonitrile-acetone-water mobile phases on diol layers derivatized with 4-aminobenzoic acid.