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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.

In this work, autochthonous lactic acid bacteria (LAB) were isolated from chia ( L.) dough and selected on the basis of the kinetics of acidification and proteolytic activity. Strain no. C8, identified as 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 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 ( 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.

The context of food science and biotechnology is undergoing a profound transformation, characterized by an evolutionary shift from conventional large-scale fermentation to precision biomanufacturing, positioning Lactic Acid Bacteria (LAB) as versatile cellular biofactories for next-generation functional foods. This review analyzes the evolutionary role of LAB, their utilization as probiotics, and the technological advances driving this shift. This work also recognizes the fundamental contributions of pioneering women in the field of biotechnology. The primary methodology relies on the seamless integration of synthetic biology (CRISPR-Cas editing), Multi-Omics analysis, and advanced Artificial Intelligence/Machine Learning, enabling the precise, rational design of LAB strains. This approach has yielded significant findings, including successful metabolic flux engineering to optimize the biosynthesis of high-value nutraceuticals such as Nicotinamide Mononucleotide and N-acetylglucosamine, and the development of Live Biotherapeutic Products using native CRISPR systems for the expression of human therapeutic peptides (e.g., Glucagon-like Peptide-1 for diabetes). From an industrial perspective, this convergence enhances strain robustness and supports the digitalized circular bioeconomy through the valorization of agri-food by-products. In conclusion, LAB continue to consolidate their position as central agents for the development of next-generation functional foods.

ObjectivesTo elucidate the molecular mechanisms involved in the substrate interaction of the bile salt hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH) with bile acids (BAs) and to evaluate potential enzyme inhibitors based on computer and in vitro modeling assays.ResultsAsp19, Asn79, and Asn171 participated in the LrBSH interaction with all BAs tested while Leu56 and Glu 222 played an important role in the interaction with glyco- and tauro-conjugated BAs, respectively. A great percentage of hydrophobic and polar interactions were responsible for the binding of LrBSH with glyco- and tauro-conjugated BAs, respectively. Remarkably, the four binding pocket loops participated in the substrate binding site of LrBSH unlike most of the reported BSHs. Inhibition assays showed that ascorbic acid, citric acid, penicillin G, and ciprofloxacin decreased LrBSH activity by 47.1%, 40.14%, 28.8%, and 9%, respectively. Docking analysis revealed that tetracycline and caffeic acid phenethyl ester had the low binding energy (−7.32 and −7.19 kcal/mol, respectively) and resembled the interaction pattern of GDCA (−6.88 kcal/mol) while penicillin (−6.25 kcal/mol) and ascorbic acid (−5.98 kcal/mol) interacted at a longer distance.ConclusionThis study helps to delve into the molecular mechanisms involved in the recognition of substrates and potential inhibitors of LrBSH.

Goat milk can be a vehicle for beneficial microorganisms, such as probiotic lactic acid bacteria (LAB). During lactic fermentation, the hydrolysis of milk proteins can improve their nutritional properties and sensory attributes and even have beneficial health effects. The objective of this study was to evaluate the caseinolytic activity of LAB strains with probiotic potential and to monitor the changes induced by fermentation and during storage in milk components using Fourier transform infrared (FTIR) spectroscopy. First, the proteolytic activity of 36 LAB strains isolated from dairy products was qualitatively assessed. Then, 17 strains with probiotic potential and moderate to high proteolytic activity were selected for further analysis. Casein proteolysis was found to be strain-dependent, with a decrease in total protein concentration ranging from 28% to 87% and an increase in amino acids ranging from 29% to 88%. Furthermore, a notable difference was observed in the amide bands in the FTIR spectra between the beginning and end of incubation, showing a decrease in the intensities of the bands attributed to proteins. In fermented goat milk, LAB growth resulted in a final count between 0.62 and 2.6 log CFU/mL, a 0.29 to 2.0 drop in pH, and lactic acid production between 0.20 and 1 g/L. FTIR spectra revealed time-dependent modifications in amide I and II bands accompanied by a marked reduction in carbohydrate content and an increase in lactic acid signal. After 21 days of storage, the viability of the strains, pH, and lactic acid in the fermented milks were not substantially modified. These results highlight the potential of lactic fermentation with strains selected for their probiotic potential as an approach to producing value-added goat milk products, as well as the usefulness of FTIR spectroscopy for characterizing complex systems such as goat milk.

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 WORK: To 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.

PURPOSE OF WORK: To 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.

In recent years, more and more scientific community, food producers, and food industry show increased interest in functional foods containing probiotics, which is a big challenge. The consumption of probiotics in the context of a balanced diet through the consumption of functional foods or through the intake of pharmaceutical preparations has proven to contribute to the improvement of human health, even contributing to the prevention of diseases. In order for probiotics to be considered suitable for consumption, they must contain a minimum concentration of viable cells, namely, at least 10 7 colony forming units of beneficial microbes per gram. Ensuring the viability of bacterial cells until the moment of consumption is the overriding priority of functional probiotic food manufacturers. Probiotic bacteria are subject to stress conditions not only during food manufacturing but also during gastrointestinal passage, which limit or even compromise their functionality. This paper first examines all the stressful conditions faced by probiotic cells in their production stages and related to the conditions present in the bioreactor fermentation and drying processes as well as factors related to the food matrix and storage. The stress situations faced by probiotic microorganisms during the gastrointestinal transit especially during stomach and intestinal residence are also analyzed. In order to understand the adaptation mechanisms of probiotic bacteria to gastrointestinal stress, intrinsic and adaptive mechanisms identified in probiotic strains in response to acid stress and to bile and bile acid stress are analyzed. In addition, improvement strategies for multiple stress tolerance of lactic acid bacteria through directions dealing with stress, accumulation of metabolites, use of protectants, and regulation of technological parameters are examined. Finally, the definition of postbiotics, inanimate microorganisms and/or their components conferring health benefits, is also introduced. Postbiotics include cell lysates, enzymes, and cell wall fragments derived from probiotic bacteria and may represent an alternative to the use of probiotics, when they do not tolerate stressful conditions.

PURPOSE OF WORK: To 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.

Fil: Taranto, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina