Spatiotemporal Distribution of the Environmental Microbiota in Food Processing Plants as Impacted by Cleaning and Sanitizing Procedures: the Case of Slaughterhouses and Gaseous Ozone.

Microbial complexity and contamination levels in food processing plants heavily impact the final product fate and are mainly controlled by proper environmental cleaning and sanitizing. Among the emerging disinfection technologies, ozonation is considered an effective strategy to improve the ordinary cleaning and sanitizing of slaughterhouses. However, its effects on contamination levels and environmental microbiota still need to be understood. For this purpose, we monitored the changes in microbiota composition in different slaughterhouse environments during the phases of cleaning/sanitizing and ozonation at 40, 20, or 4 ppm. Overall, the meat processing plant microbiota differed significantly between secondary processing rooms and deboning rooms, with a greater presence of psychrotrophic taxa in secondary processing rooms because of their lower temperatures. Cleaning/sanitizing procedures significantly reduced the contamination levels and in parallel increased the number of detectable operational taxonomic units (OTUs), by removing the masking effect of the most abundant human/animal-derived OTUs, which belonged to the phylum Firmicutes Subsequently, ozonation at 40 or 20 ppm effectively decreased the remaining viable bacterial populations. However, we could observe selective ozone-mediated inactivation of psychrotrophic bacteria only in the secondary processing rooms. There, the Brochothrix and Pseudomonas abundances and their viable counts were significantly affected by 40 or 20 ppm of ozone, while more ubiquitous genera like Staphylococcus showed a remarkable resistance to the same treatments. This study showed the effectiveness of highly concentrated gaseous ozone as an adjunct sanitizing method that can minimize cross-contamination and so extend the meat shelf life.IMPORTANCE Our in situ survey demonstrates that RNA-based sequencing of 16S rRNA amplicons is a reliable approach to qualitatively probe, at high taxonomic resolution, the changes triggered by new and existing cleaning/sanitizing strategies in the environmental microbiota in human-built environments. This approach could soon represent a fast tool to clearly define which routine sanitizing interventions are more suitable for a specific food processing environment, thus limiting the costs of special cleaning interventions and potential product loss.

Comparative proteomic analyses for elucidating metabolic changes during EPS production under different fermentation temperatures by Lactobacillus plantarum Q823.

Exopolysaccharide (EPS)-producing bacteria are of growing interest in industrial processes, mainly concerning food. Lactic acid bacteria are widely appreciated for their GRAS (generally recognized as safe) status and their ascertained or putative probiotic features. Detailed investigation on what happens at metabolic level during EPS production is scarce in the literature. The facultative heterofermenter Lactobacillus plantarum Q823 was studied in order to compare growth and EPS production at 30°C and 37°C. A higher growth rate was observed at 37°C, whereas, a significantly higher (tenfold increase) EPS amount was produced at 30°C. To understand the molecular mechanisms leading to the different EPS production in the two conditions, a comparative proteomic experiment was performed. The results of the in-gel proteomics revealed that: i) at 37°C a higher abundance of proteins involved in carbon catabolism and nucleic acid biosynthesis together with a significant amount of stress proteins was observed; ii) at 30°C the production of an atypical manganese-containing non-heme catalase (pseudocatalase) was increased, in agreement with previous data reporting that growth-rates of catalase negative Lactobacillus plantarum strains were greater than that of catalase positive strains. Taken together, all these findings provide further insights about the metabolic pathways stimulated during EPS production, and the mechanism that triggers EPS biosynthesis.

Selenium and selenoproteins: an overview on different biological systems.

Selenium (Se) is an essential trace element for humans, plants and microorganisms. Inorganic selenium is present in nature in four oxidation states: selenate, selenite, elemental Se and selenide in decreasing order of redox status. These forms are converted by all biological systems into more bioavailable organic forms, mainly as the two seleno-amino acids selenocysteine and selenomethionine. Humans, plants and microorganisms are able to fix twhese amino acids into proteins originating Se-containing proteins by a simple replacement of methionine with selenomethionine, or "true" selenoproteins if the insertion of selenocysteine is genetically encoded by a specific UGA codon. Selenocysteine is usually present in the active site of enzymes, being essential for their catalytic activity. This review will focus on the strategies adopted by the different biological systems for selenium incorporation into proteins and on the importance of this element for the physiological functions of living organisms. The most known selenoproteins of humans and microorganisms will be listed highlighting the importance of this element and the problems connected with its deficiency.

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.

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis.

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.

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb26 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 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.

Proteomics as a tool for studying energy metabolism in lactic acid bacteria.

Lactic acid bacteria (LAB) are very ancient organisms that can't obtain metabolic energy by respiration without external heme supplementation. Since the gain in ATP from lactic fermentation is inadequate to support efficient growth, they developed alternative strategies for energy production. Three main energy generating routes are present in LAB: amino acid decarboxylation, malate decarboxylation and arginine deimination (ADI pathway). These routes, apart from supplying energy, also play a role in pH control. Lactic fermentation, which leads to lactic acid accumulation, causes a pH decrease that amino acid decarboxylations, originating basic amines, and the ADI pathway, giving rise to ammonia, may partially contrast. In the present mini-review, the reciprocal relationships among these metabolic pathways are considered, on the basis of proteomic results obtained from four different LAB strains, all of which possess the ADI pathway, but express different amino acid decarboxylases. The strains have been isolated and selected from different habitats and the role of some inducing molecules as well as of the growth phases is discussed. The overall results have revealed that LAB are complex biosystems able to set up a sophisticated metabolic regulation through a complex network of proteins that also include stress responses, as well as protease activation or inhibition.

First evidence of a membrane-bound, tyramine and beta-phenylethylamine producing, tyrosine decarboxylase in Enterococcus faecalis: a two-dimensional electrophoresis proteomic study.

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.