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Lignocellulosic biomasses have the potential to generate by-products with biological activity (i. e., polyphenols) as well as biopolymers (i. e., cellulose, hemicellulose, pectins, lignin). The wine industry is one of the pillars of Italian agri-food sector. Nevertheless, large quantities of by-products such as grape stems are produced, which are usually disposed of at a cost, and therefore represent an attractive negative-cost feedstock for biorefinery. In this work, a sequential protocol for biomass valorization is proposed, characterized by a multidisciplinary strategy using enabling technologies and subcritical water as a green solvent, where physical/chemical treatments synergistically interact with biological treatments. The first phase involved the sequential fractionation of grape stalks, obtaining several product streams rich in polyphenols, hemicellulose, pectin (13.15 % of cumulative yield on biomass), lignin and cellulose. A membrane treatment was employed to recycle materials within the process. Finally, the cellulose-rich residue was exploited as a fermentation substrate for the last step, producing up to 5.8 g/L of lactic acid by harnessing suitably engineered Clostridium thermocellum strains. The polyphenolic fraction successfully inhibited the growth of Brettanomyces bruxellensis and Acetobacter pasteurianus, microorganisms responsible for major wine off-flavors. Globally, this study represents a proof-of-concept of a second-generation biorefining process based on locally available waste biomass., (© 2025 The Author(s). ChemSusChem published by Wiley-VCH GmbH.)

Scope: Plant phenolics, known to exert beneficial effects on human health, were supplemented to cultures of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) to assess their effect on its adhesive capacity and the abundancy of individual proteins., Methods and Results: The presence of resveratrol and ferulic acid during bacterial growth stimulated adhesion of NCFM to mucin and human intestinal HT-29 cells, while tannic acid improved adhesion only to HT-29 cells and caffeic acid had very modest effect overall. Some dosage dependence was found for the four phenolics supplemented at 100, 250, and 500 μg mL -1 to the cultures. Notably, 500 μg mL -1 ferulic acid only stimulated adhesion to mucin. Analyses of differential whole-cell as well as surface proteomes revealed relative abundancy changes for a total of 27 and 22 NCFM proteins, respectively. These changes include enzymes acting in metabolic pathways, such as glycolysis, nucleotide metabolism, and stress response, as well as known moonlighting or surface-associated proteins., Conclusion: The five plant phenolics found in various foods stimulate the adhesive capacity of NCFM in diverse ways and elicit relative abundancy changes of specific proteins, providing molecular level insight into the mechanism of the putative beneficial effects of the polyphenols., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H 2 O 2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H 2 O 2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide-reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein-refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Lb26 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 Lb26 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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence.

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline pI's, because of the limits inherent in 2-DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pI's; the most crowded areas lying between pI 4-7 and 9-11. The aim of the present research was to set up a general, simple, and standardizable 2-DE protocol suitable for studying the alkaline proteome of Lactobacillus hilgardii, a Gram-positive bacillus isolated from wine. The method has also been tested on a Gram-negative bacterium able to degrade aromatic pollutants, Acinetobacter radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup loading), and different reducing agents (DTT and bis(2-hydroxyethyl) disulfide (HED)) were tested and compared. The proposed protocol was found to resolve efficiently alkaline proteins from both of our Lactobacillus and Acinetobacter strains, in spite of their different external layers, thus, enabling a more comprehensive study of their proteomes.

All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers' health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermenting microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide transport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different M(r), pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.

Study of the bacterial membrane proteome, though in its early stages, is a field of growing interest in the search for information about nutrient transport and processing. We tested different strategies and chemical compounds to extract proteins from the membranes (inner and outer) of Acinetobacter radioresistens S13, a Gram-negative bacterium selected for its ability to degrade aromatics. A. radioresistens S13 was monitored under different growth substrate conditions, using acetate, benzoate or phenol as sole carbon source. Two-dimensional gel electrophoresis map analysis of membrane extracts from benzoate- and phenol-grown cells reveals differences versus controls (acetate-grown cultures). Primarily, a different pattern of spots was observed and, in particular, some proteins were only expressed in the presence of aromatic substrate. Among these, we detected a Na(+)/H(+) antiporter, whose function is likely to be regulation of intracellular pH, and an ABC type sugar transport system, probably involved in capsular polysaccharide translocation. We also identified other proteins, detectable in acetate-grown but over-expressed in aromatic-grown cells. These include: (1) an outer membrane protein ascribable to an OmpA-like protein, recently described in the literature as "alasan", a bioemulsifying agent involved in solubilizing and enhancing bioavailability of hydrocarbons; (2) a trimeric porin of the PhoE family also belonging to the outer membrane and involved in facilitating the transport of anions (especially phosphate); and (3) two glycosyl transferases probably involved in capsules and/or lipopolysaccharide biosynthesis. Study of the bacterial membrane proteome helps to elucidate the role of the membrane as modulable site enabling communication between internal and external environments.

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the beta-ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol-1,2-dioxygenase isoenzymes (C-1,2-O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis-muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (D) and C-1,2-O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C-1,2-O and D, while phenol is the best growth substrate to obtain PH.