In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.
Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.
Oenococcus oeni; proteome; two-dimensional electrophoresis
Currently, the majority of prebiotics in the market are derived from non-digestible oligosaccharides. Very few studies have focused on non-digestible long chain complex polysaccharides in relation to their potential as novel prebiotics. Cereals β-glucans have been investigated for immune-modulating properties and beneficial effects on obesity, cardiovascular diseases, diabetes, and cholesterol levels. Moreover, β-glucans have been reported to be highly fermentable by the intestinal microbiota in the caecum and colon, and can enhance both growth rate and lactic acid production of microbes isolated from the human intestine. In this work, we report the effects of food matrices containing barley β-glucans on growth and probiotic features of four Lactobacillus strains. Such matrices were able to improve the growth rate of the tested bacteria both in unstressed conditions and, importantly, after exposure to in vitro simulation of the digestive tract. Moreover, the effect of β-glucans-containing food on bacterial adhesion to enterocyte-like cells was analyzed and a positive influence on probiotic-enterocyte interaction was observed.
barley β-glucans; probiotics; prebiotics; pasta; Lactobacillus
Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions.
An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, was used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells.
L. brevis IOEB 9809 can produce tyramine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.
Biogenic amines; Lactic acid bacteria; Putrescine; Tyramine; Food safety; Food toxicity
Biogenic amines (BA) in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analyzed for their ability to degrade BA commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the BA and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analyzed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade BA in wine.
lactic acid bacteria; amine degradation; biogenic amines; malolactic fermentation; wine; Lactobacillus plantarum; putrescine; tyramine
Cereals-derived foods represent a key constituent in the diet of many populations. In particular, pasta is consumed in large quantities throughout the world in reason of its nutritive importance, containing significant amounts of complex carbohydrates, proteins, B-vitamins, and iron. Lactic acid bacteria (LAB) are a heterogeneous group of bacteria that play a key role in the production of fermented foods and beverages with high relevance for human and animal health. A wide literature testifies the multifaceted importance of LAB biotechnological applications in cereal-based products. Several studies focused on LAB isolation and characterization in durum wheat environment, in some cases with preliminary experimental applications of LAB in pasta-making. In this paper, using sourdough as a model, we focus on the relevant state-of-art to introduce a LAB-based biotechnological step in industrial pasta-making, a potential world driver of innovation that might represent a cutting-edge advancement in pasta production.
lactic acid bacteria; pasta; sourdough; quality; microbial cell factories
Probiotics, prebiotics and synbiotics are frequently-used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-β-d-glucan of non-dairy bacterial origin has a prebiotic effect on three probiotic strains. Moreover, the presence of this β-d-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells.
β-glucans; probiotics; prebiotics; Lactobacillus plantarum
Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.
ClpE; CtsR; heat; Lactobacillus plantarum; stress
Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5′ noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.
RT-qPCR; Lactobacillus acidophilus; probiotic; small heat shock gene; stress
Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the ΔctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.
FtsH proteins have dual chaperone-protease activities and are involved in protein quality control under stress conditions. Although the functional role of FtsH proteins has been clearly established, the regulatory mechanisms controlling ftsH expression in gram-positive bacteria remain largely unknown. Here we show that ftsH of Lactobacillus plantarum WCFS1 is transiently induced at the transcriptional level upon a temperature upshift. In addition, disruption of ftsH negatively affected the growth of L. plantarum at high temperatures. Sequence analysis and mapping of the ftsH transcriptional start site revealed a potential operator sequence for the CtsR repressor, partially overlapping the −35 sequence of the ftsH promoter. In order to verify whether CtsR is able to recognize and bind the ftsH promoter, CtsR proteins of Bacillus subtilis and L. plantarum were overproduced, purified, and used in DNA binding assays. CtsR from both species bound specifically to the ftsH promoter, generating a single protein-DNA complex, suggesting that CtsR may control the expression of L. plantarum ftsH. In order to confirm this hypothesis, a ΔctsR mutant strain of L. plantarum was generated. Expression of ftsH in the ΔctsR mutant strain was strongly upregulated, indicating that ftsH of L. plantarum is negatively controlled by CtsR. This is the first example of an ftsH gene controlled by the CtsR repressor, and the first of the low-G+C gram-positive bacteria where the regulatory mechanism has been identified.
Stress responses are of particular importance to microorganisms, because their habitats are subjected to continual changes in temperature, osmotic pressure, and nutrients availability. Stressors (and stress factors), may be of chemical, physical, or biological nature. While stress to microorganisms is frequently caused by the surrounding environment, the growth of microbial cells on its own may also result in induction of some kinds of stress such as starvation and acidity. During production of fresh-cut produce, cumulative mild processing steps are employed, to control the growth of microorganisms. Pathogens on plant surfaces are already stressed and stress may be increased during the multiple mild processing steps, potentially leading to very hardy bacteria geared towards enhanced survival. Cross-protection can occur because the overlapping stress responses enable bacteria exposed to one stress to become resistant to another stress. A number of stresses have been shown to induce cross protection, including heat, cold, acid and osmotic stress. Among other factors, adaptation to heat stress appears to provide bacterial cells with more pronounced cross protection against several other stresses. Understanding how pathogens sense and respond to mild stresses is essential in order to design safe and effective minimal processing regimes.
stress; stressors; fresh cut; pathogens