Ana Yanina Bustos

Relationship between bile salt hydrolase activity, changes in the internal pH and tolerance to bile acids in lactic acid bacteria

The effect of the conjugated bile acid (BA) on the microbial internal pH (pHin) values in lactic acid bacteria with and without ability to hydrolyze bile salts (BSH[+] and BSH[−] strains, respectively) was evaluated. BSH(+) strains showed a gradual increase in the pHin following the addition of conjugated BA; this behavior was more pronounced with GDCA than with TDCA may be due to the higher affinity of BSH for the glyco-conjugates acids. Conversely, the BSH(−) strains showed a decrease in internal pH probably as a consequence of weak acid accumulation. As expected, a decrease in the cytoplasmatic pH affected the cell survival in this last group of strains, while the BSH(+) strains were more resistant to the toxic effect of BA.Purpose of workTo evaluate bile salt hydrolase activities, changes in the internal pH and cell survival to bile acids in lactic acid bacteria to establish the relationship between these parameters.

Efflux of bile acids in Lactobacillus reuteri is mediated by ATP

Purpose of workTo study whether an active bile acid (BA) efflux occurs in Lactobacillus reuteri CRL 1098 as well as the nature (ATP or proton motive force [PMF] mediated primary transport) of the BA extrusion mechanism.BAs are powerful detergents which disorganize the lipid bilayer structure of cellular membranes. Specific bile resistance mechanisms (bile efflux, bile salt hydrolysis, and intrinsic architecture and composition of cell membrane the most prevalent) have been described in intestinal bacteria. L. reuteri, showed a significant degree of resistance to the toxic action of BA and the presence of an active efflux ATP-dependent of conjugated (taurocholic [TCA]) and free (cholic [CA]) BA in the CRL 1098 strain is now reported. However, due the high pKa (5.5) of cholic acid (CA) compared with the conjugated species, a significant fraction (between 35 and 50% at pH 6.5 and 5.2, respectively) of free BA also diffused passively, even in the absence of ATP. To our knowledge, our results represent the first evidence of ATP as the energy source involved in the BA extrusion in L. reuteri.

Application of fluorescent techniques to evaluate the survival of probiotic lactobacilli to bile acid

Purpose of workTo apply a fluorescent dye as an alternative technique to evaluate the survival of potentially probiotic lactobacilli to bile acids (BA) as first step in the design of probiotic functional foods.The use of lactic acid bacteria (LAB) in the functional food design depends on their ability to survive in the gastrointestinal tract where bile is an important natural barrier. Bile is mainly constituted by conjugated BA, which can be hydrolyzed to free BA and taurine or glycine. Changes in the transmembrane electrical potential (ΔΨ) of probiotic LAB strains due to the effect of conjugated and free BA were measured and showed that the majority of the tested LAB strains had greater sensibility to free BA than to their respective conjugated acids. Variations in the ΔΨ of the microorganism correlated well with bacterial viability determined by standard plate count method. We therefore propose the DiSC3-based fluorescent technique as a rapid and effective method to evaluate the resistance of probiotic lactobacilli to bile as first step for strain selection to be included in functional foods.

Lactic Acid Fermentation Improved Textural Behaviour, Phenolic Compounds and Antioxidant Activity of Chia (Salvia hispanica L.) Dough

In this work, autochthonous lactic acid bacteria (LAB) were isolated from chia (Salvia hispanica L.) dough and selected on the basis of the kinetics of acidification and proteolytic activity. Strain no. C8, identified as Lactobacillus plantarum C8, was selected and used as starter to obtain chia sourdough. Lactic acid fermentation increased the organic acid mass fractions (lactic, acetic and phenyl lactic acids to 12.3 g, 1.0 g and 23.8 µg per kg of dough respectively), and antioxidant activities, which increased by approx. 33-40% compared to unfermented chia flour dough. In addition, total phenolic content increased 25% and its composition was strongly modified after 24 h of fermentation by L. plantarum C8. Chlorogenic acid was only found in the fermented dough (2.5 mg/g), while ferulic acid was detected from the beginning of fermentation, being 32% higher in chia sourdough (5.6 mg/g). The use of fermented chia sourdough improved the overall characteristics of white bread, including physical (e.g. reduced hardness and chewiness of the crumb) and antioxidant properties (25% on average), compared to the white bread. These results indicate that the use of chia sourdough could be a promising alternative to improve the technological and antioxidant properties of wheat bread. In addition, this work has shown, for the first time, that lactic acid bacterium is able to ferment chia dough, improving its overall characteristics.

Effects of Lysozyme on the Activity of Ionic of Fluoroquinolone Species

Fluoroquinolones (FQs) constitute an important class of biologically active broad-spectrum antibacterial drugs that are which are in contact with many biological fluids under different acidity conditions. We studied the reactivity of ciprofloxacin (Cpx) and levofloxacin (Lev) and their interaction with lysozyme (Lyz) at different pH values, using UV-visible absorption, fluorescence, infrared spectroscopies supported by DFT calculation and docking. In addition, by antimicrobial assays, the biological consequences of the interaction were evaluated. DFT calculation predicted that the FQ cationic species present at acid pH have lower stabilization energies, with an electric charge rearrangement because of their interactions with solvent molecules. NBO and frontier orbital calculations evidenced the role of two charged centers, NH2+ and COO−, for interactions by electronic delocalization effects. Both FQs bind to Lyz via a static quenching with a higher interaction in neutral medium. The interaction induces a structural rearrangement in β-sheet content while in basic pH a protective effect against the denaturation of Lyz was inferred. The analysis of thermodynamic parameters and docking showed that hydrophobic, electrostatic forces and hydrogen bond are the responsible of Cpx-Lyz and Lev-Lyz associations. Antimicrobial assays evidenced an antagonist effect of Lyz in acid medium while in neutral medium the FQs’ activities were not modified by Lyz.

Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (aw) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parameters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on aw. Greatest growth rate inhibition (46.9%) was obtained at aw=0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllactic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at aw ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at aw=0.971, while inhibition of fungi growth was more evident at aw=0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with aw values between 0.971 and 0.995.

New insights into bacterial bile resistance mechanisms: the role of bile salt hydrolase and its impact on human health

Bile acids (BA), the major components of bile, are biological detergents that facilitate the emulsification and solubilization of dietary lipids and also display potent antimicrobial activity, the bacterial membranes being their main targets. Considering the complicated nature of the stress produced by bile and BA, the microorganism tolerance requires different defence mechanisms including the presence of efflux pumps, bile salt hydrolase (BSH) enzyme, the intrinsic capacity of cells to maintain intracellular homeostasis and modifications in the architecture and composition of the cell membrane. Besides, the expression of proteins involved in carbohydrate and fatty acid metabolism, amino acid and nitrogenous base biosynthesis, and general stress response are commonly affected by the presence of bile. Among the microbial transformations, deconjugation of BA by BSH is the most important. Several studies indicate that BSH activity affects both the host physiology and the microbiota. In fact, it was strongly suggested that BSH could play an important role in the colonization and survival of bacteria in the gut. Also, recent work has shown that BSH and free BA participate in a variety of metabolic processes that include regulation of dietary lipid absorption, cholesterol metabolism, and energy and inflammation homeostasis. In this review we summarize recent advances in the understanding of the mechanisms involved in the tolerance of bacteria to bile, with special emphasis on the contributions of studies applying an omic approach. Besides, the physiological and ecological role of BSH enzyme and its relevance to human health as well as the function of bile acid as metabolic regulator are also discussed.