Erika Mangiapane

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb2 BM for nutraceutical applications.

Selenium (Se), Se‐cysteines and selenoproteins have received growing interest in the nutritional field as redox‐balance modulating agents. The aim of this study was to establish the Se‐concentrating and Se‐metabolizing capabilities of the probiotic Lactobacillus reuteri Lb2 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4–7 and 6–11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb2 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo‐ergonic reactions balanced by an increase of substrate‐level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Se‐concentrating probiotics.

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (78Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Enantioselective lactic acid production by an Enterococcus faecium strain showing potential in agro-industrial waste bioconversion: Physiological and proteomic studies

The growing demand of biodegradable plastic polymers is increasing the industrial need of enantiospecific l-lactic acid (l-LA), the building block to produce polylactides. The most suitable industrial strategy to obtain high amounts of LA is the microbial fermentation of fruit and vegetable wastes by lactic acid bacteria (LAB). In this paper seven LAB strains from our laboratory collection, were screened for their ability to produce the highest amount of pure l-LA. A strain of Enterococcus faecium (LLAA-1) was selected and retained for further investigations. E. faecium LLAA-1 was grown in different culture media supplemented with the most abundant sugars present in agricultural wastes (i.e., glucose, fructose, cellobiose and xylose) and its ability to metabolize them to l-LA was evaluated. All tested sugars proved to be good carbon sources for the selected strain, except for xylose, which resulted in unsatisfactory biomass and LA production. Growth under aerobic conditions further stimulated l-LA production in fructose supplemented cultures with respect to anoxic-grown cultures. Proteomic profiles of E. faecium LLAA-1 grown in aerobiosis and anoxia were compared by means of two-dimensional electrophoresis followed by MALDI-TOF mass spectrometry. Seventeen proteins belonging to three main functional groups were differentially expressed: the biosynthesis of 6 proteins was up-regulated in aerobic-grown cultures while 11 proteins were biosynthesized in higher amounts in anoxia. The de novo biosynthesis of the f-subunit of alkyl hydroperoxide reductase involved in the re-oxidation of NADH seems the key element of the global re-arrangement of E. faecium LLAA-1 metabolism under aerobic conditions. An improved oxidative catabolism of proteinaceous substrates (i.e., protein hydrolisates) seems the main phenomenon allowing both higher biomass growth and improved LA production under these conditions.

'Ropy' phenotype, exopolysaccharides and metabolism: Study on food isolated potential probiotics LAB

Lactic acid bacteria are fully recognized for their industrial applications among which the production and release of exopolysaccharides. In the present investigation, we screened fifteen Lactobacilli in order to find ropy strains, quantify exopolysaccharides and detect proteins specifically associated with the ropy-exopolysaccharide production. The highest ropy-exopolysaccharide producer (L. helveticus 6E8), was grown in stimulating and basal condition (10% and 2% lactose) and subjected to comparative proteomic analysis. The levels of 4 proteins were found significantly increased in the membrane fraction under stimulating conditions: a specific exopolysaccharide biosynthetic protein, a stress-induced protein, a protein involved in secretion and an ATP-synthase subunit. Conversely, several enzymes involved in anabolism and protein synthesis were decreased. These results suggest a general shift from growth to exopolysaccharide-mediated protection from the hyperosmotic environment. Due to the great interest in exopolysaccharides with novel features, the identification of these proteins could have implications for future improvements of industrial strains.