► Three different polysaccharides were produced by 4 different lactic acid bacteria strains. ► Different methods were developed to purify the bacterial polysaccharides. ► After purification they were ...used for structural analysis. ► After structural analysis they were identified as homo and heteropolysaccharides.
Lactic acid bacteria (LAB) produce homopolysaccharides (HoPS) and heteropolysaccharides (HePS) with potential functional properties. In this work, we have performed a comparative analysis of production and purification trials of these biopolymers from bacterial culture supernatants. LAB strains belonging to four different genera, both natural as well as recombinant, were used as model systems for the production of HoPS and HePS. Two well characterized strains carrying the gft gene were used for β-glucan production, Pediococcus parvulus 2.6 (P. parvulus 2.6) isolated from cider, and the recombinant strain Lactococcus lactis NZ9000pGTF (L. lactis NZ9000pGTF). In addition, another cider isolate, Lactobacillus suebicus CUPV225 (L. suebicus CUPV225), and Leuconostoc mesenteroides RTF10 (L. mesenteroides RTF10), isolated from meat products were included in the study. Chemical analysis of the EPS revealed that L. mesenteroides produces a dextran, L. suebicus a complex heteropolysaccharide, and the β-glucan producing-strains the expected 2-substituted (1,3)-β-glucan.
Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus ...STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.
•Fermentative activities of LAB and bifidobacteria adapt to carbohydrates in the media.•L. casei shifts metabolism during growth on fructooligosaccharides.•L. acidophilus competes against enteropathogens for intestinal cell colonization.
•Purified bacterial 2-substituted (1–3)-β-d-glucan activated human macrophages.•This activation was indicative of an anti-inflammatory response.•The activation differed markedly from that caused by ...bacterial lipopolysaccharide.•This glucan from natural and recombinant strains had the same biological activity.
β-glucans produced by eukaryotic cells and by microorganisms are known to modulate immune responses by affecting macrophage activation. Here, we have investigated the effect of purified 2-substituted (1–3)-β-d-glucan, produced by either Pediococcus parvulus 2.6 or Lactococcus lactis NZ9000pNGTF, on the effector functions of human PMA-differentiated THP-1 cells and M1 pro-inflammatory monocyte-derived macrophages. The results reveal that this kind of β-D-glucan activates macrophages and has an anti-inflammatory effect.
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.
Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug ...resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such β-amino alkanol derivatives using a set of 15 N-mono- and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylaminododecan-1-ol, had a 50% effective concentration (EC50) of 0.3 μM and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other β-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted β-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.
Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these β-glucans have immunomodulating properties. We show that the overproduction of the ...Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter⁻¹) of a position 2-substituted (1rightward arrow3)-β-D-glucan that has potential use as a food additive.
•Leuconostoc mesenteroides CM9, CM30 and SM34 synthesise dextran.•These dextrans have very high molecular mass 1–4×108Da.•These dextrans have a low percentage of branching and a flexible ...configuration.•These dextrans could down-regulate inflammatory response to bacterial pathogens.•These dextrans could diminish over-response to pathogens by the innate immunity.
Dextrans synthesised by three Leuconostoc mesenteroides strains, isolated from mammalian milks, were studied and compared with dextrans produced by Lc. mesenteroides and Lactobacillus sakei strains isolated from meat products. Size exclusion chromatography coupled with multiangle laser light scattering detection analysis demonstrated that the dextrans have molecular masses between 1.74×108Da and 4.41×108Da. Rheological analysis of aqueous solutions of the polymer revealed that all had a pseudoplastic behaviour under shear conditions and a random, and flexible, coil structure. The dextrans showed at shear zero a difference in viscosity, which increased as the concentration increased. Also, the purified dextrans were able to immunomodulate in vitro human macrophages, partially counteracting the inflammatory effect of Escherichia coli O111:B4 lipopolysaccharide.
During prolonged incubation on a solid medium containing sucrose, dextran-producing bacteria showed two distinct phenotypes not related to the genus or species to which they belonged. Colonies of Lc. mesenteroides CM9 from milk and Lb. sakei MN1 from meat formed stable and compact mucoid colonies, whereas the colonies of the other three Leuconostoc strains became diffuse after 72h. This differential behaviour was also observed in the ability of the corresponding strains to bind to Caco-2 cells. Strains forming compact mucoid colonies showed a high level of adhesion when grown in the presence of glucose, which decreased in the presence of sucrose (the condition required for dextran synthesis). However no influence of the carbon source was detected for the adhesion ability of the other Lc. mesenteroides strains, which showed variable levels of binding to the enterocytes.
The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on ...transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain
Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from
Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains
L. lactis T1 and
L. lactis IFPL730, showing branched chain amino acid aminotransferase and α-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain
L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by
L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.
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.
This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. ...bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells.
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