María Pía Taranto

Upper respiratory tract infections (URTI) are highly prevalent worldwide. Although probiotics have shown potential in preventing URTI, evidence in healthy adults remains limited. Lacticaseibacillus rhamnosus CRL 1505 is a strain with immunomodulatory effects in preclinical studies and benefits in healthy children. Based on this evidence, a randomized, double-blind, placebo-controlled, parallel-group clinical trial was conducted in healthy adults, receiving either L. rhamnosus CRL 1505 (1 bln/day) or placebo for 12 weeks, with a 4-week follow-up. The primary endpoint was the proportion of participants experiencing URTI episodes (at least one, two, or three episode(s)). The secondary endpoints included: number and duration of URTI episodes, URTI-free time, symptom severity, use of symptomatic medication, salivary IgA levels, and safety outcomes. Results show that L. rhamnosus CRL 1505 significantly reduced the number of participants experiencing ≥3 URTI episodes at 16 weeks compared with placebo. The probiotic group experienced fewer URTI episodes per participant, a shorter cumulative duration of URTIs, and a higher URTI-free time rate. Probiotic supplementation significantly reduced the use of symptomatic medications. In conclusion, daily supplementation with L. rhamnosus CRL 1505 reduced the burden of URTI in healthy adults, specifically of those experiencing more episodes, by decreasing infection frequency, duration, and medication use.

The high potential of the probiotic L. reuteri CRL 1098, as hypocholesterolemic and vitamin B₁₂ producer strain, would require addressing freeze-drying studies of the microorganism as well as its stability during storage. L. reuteri CRL 1098 was freeze-dried with glutamate, maltodextrin and trehalose as lyoprotectants and an accelerated inactivation assays under isothermal conditions (50, 60 and 70°C) was used to evaluate the stability of lyophilizates. Results obtained were analyzed with informatic GInaFiT Ap-inn and traditional predictive tools (D/z and Arrhenius models) for describing both, the inactivation process and the bacterial behaviour. The predicted shelf life of the lyophilized probiotic to obtain 1 × 10⁶ UFC/g was experimentally tested at 30°C. Regardless of the lyoprotectant used, the survival of L. reuteri CRL1098 fitted acceptably to the weilbulian model of Marfat in all heat treatments; the data described curves with up dawn concavities (p < 1) and allowed to identify a more sensitive bacterial population through the first decimal reduction time (δ-value) specially at 50°C in presence of maltodextrin. From the second reduction time, survivors of L. reuteri CRL 1098 fitted to first kinetic order reactions (lineal distribution of data), so D-values (decimal reduction time), k-values (inactivation rate constant), and z-values (intrinsic resistance) as well as the activation energy Ea, were calculated through D/z model and Arrhenius model. From these, a resistant subpopulation of L. reuteri CRL 1098 could be described, mainly when trehalose was used as lyoprotectant, which was characterized by high z-values. These results correlated with higher D-values but lower k-values and Ea in trehalose, which did not show the best stabilizing property along the storage tested at 30°C. Maltodextrine proved to be the best stabilizer lyoprotectant, especially when the shelf life is predicted at low temperatures of conservation.

Lacticaseibacillus (L.) rhamnosus CRL75 is a lactic acid bacterium (LAB) isolated from local dairy products, demonstrating significant adaptation in skimmed milk (FM75). In this context, CRL75 exhibited high microbial growth (3.63 ± 0.18 log CFU·mL⁻¹), strong acidification (9.20 ± 0.10 g·L⁻¹ lactic acid, and 2.40 ± 0.10 pH units), and increased viscosity in FM75 after 16 h of fermentation. Additionally, this LAB strain produces both capsular polysaccharides (CPS+) and extracellular polysaccharides (EPS75), contributing to a ropy phenotype (>10 cm). The purified EPS75 (70.70 ± 3.25 mg·L⁻¹) displayed low molecular weight (12.7 kDa), with galactose and glucose as its primary monomers in a 4:1 ratio. In aqueous environments, EPS75 exhibited an extended size (147 nm), a random coil structure, and macromolecular aggregation. Furthermore, vibrational spectroscopy confirmed the presence of a neutral EPS with high thermal stability. Additionally, EPS75 exhibited dose-dependent antioxidant activity, effectively reducing metal ions (Fe³⁺, Mo⁶⁺, and Mn⁷⁺) and stabilizing radicals (ABTS^•+, HO^•, O₂^•−, and HOO^•). The biopolymer also demonstrated immunostimulatory and anti-inflammatory effects in RAW 264.7 cells. In vivo assays using Balb/c mice indicated that both EPS75 and FM75 prevented constipation, suggesting their potential as natural and safe agents for constipation-related disorders. Due to its viscosifying and health-promoting attributes, CRL75 offers promising applications for functional dairy products.

Bile acids (BAs) are the main endogenous modulators of the composition and metabolic activity of the intestinal microbiota. In the present work, the effect of conjugated (glycodeoxycholic, glycocholic, taurodeoxycholic, taurocholic acids) and free BAs [cholic acid (CA) and deoxycholic acid (DCA)] on the survival, biological molecules, and structural and surface properties of two potential probiotic lactic acid bacteria (LAB) was evaluated. For this, viability assays, Raman spectroscopy, scanning electron microscopy (SEM), and zeta potential (ZP) measurements were employed. Our results evidenced that free BAs were more toxic than conjugates, with CA being significantly more harmful than deoxycholic acid (DCA). RAMAN studies show that BAs modify the bands corresponding to proteins, lipids, carbohydrates, and DNA. SEM showed that BAs cause surface distortions with depressions and fold formation, as well as incomplete cell division. DCA was the one that least altered the ZP of bacteria when compared to CA and taurodeoxycholic acid, with gradual changes towards more positive values. In general, the magnitude of these effects was different according to the BA and its concentration, being more evident in the presence of CA, even at low concentrations, which would explain its greater inhibitory effect. This work provides solid evidence on the effects of BAs on LAB that will allow for the development of strategies by which to modulate the composition of the microbiota positively.

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 10⁷ 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.

Summary: Vegetable and fruit snacks emerge as an innovative non‐dairy functional food of probiotic carriers and a valuable alternative to consumers with dietary restrictions. However, enriching snacks with probiotics is challenging as several factors may affect cell viability making it difficult to maintain adequate microbial doses at consumption time. This article aimed to review the most recent advances in the design of probiotic fruit and vegetable snacks to provide the current state of knowledge on this cutting‐edge topic. In particular, the technologies applied to introduce probiotics into the food matrix, classical and novel drying methods, and strategies to improve microbial survival during processing, storage and gastrointestinal passage, are discussed. Most of the published works report bacterial counts above 7 log cfu g⁻¹ in snacks, which is a proper dose of probiotics to promote health benefits. However, many of these studies did not evaluate the survival rate during storage or considered short periods. The design of fruit‐ or vegetable‐based snacks enriched with probiotics appears to be a worthwhile strategy at laboratory scale but further studies to validate results both at pilot and industrial scale are necessary.

Background: Probiotic bacteria have been emerging as a trustworthy choice for the prevention and treatment of Candida spp. infections. This study aimed to develop and characterize an orodispersible film (ODF) for delivering the potentially probiotic Enterococcus faecium CRL 183 into the oral cavity, evaluating its in vitro antifungal activity against Candida albicans. Methods and Results: The ODF was composed by carboxymethylcellulose, gelatin, and potato starch, and its physical, chemical, and mechanical properties were studied. The probiotic resistance and viability during processing and storage were evaluated as well as its in vitro antifungal activity against C. albicans. The ODFs were thin, resistant, and flexible, with neutral pH and microbiologically safe. The probiotic resisted the ODF obtaining process, demonstrating high viability (>9 log₁₀ CFU·g⁻¹), up to 90 days of storage at room temperature. The Probiotic Film promoted 68.9% of reduction in fungal early biofilm and 91.2% in its mature biofilm compared to the group stimulated with the control film. Those results were confirmed through SEM images. Conclusion: The probiotic ODF developed is a promising strategy to prevent oral candidiasis, since it permits the local probiotic delivery, which in turn was able to reduce C. albicans biofilm formation.

Vitamin B₁₂ or cobalamin is an essential metabolite for humans, which makes it an interesting compound for many research groups that focus in different producer-strains synthesis pathways. In this work, we report the influence of key intermediaries for cobalamin synthesis added to the culture medium in two Lactobacillus (L.) strains, L. reuteri CRL 1098 and L. coryniformis CRL 1001. Here, we report that addition of Co²⁺ and 5,6-dimethylbenzimidazole increased the corrinoid compounds production in both strains while addition of L-threonine increased only the corrinoid compounds production by CRL 1001 strain. Then, we purified and characterized by LC-MS the corrinoid compounds obtained. Physiological studies besides in silico analysis revealed that L. reuteri CRL 1098 and L. coryniformis CRL 1001 follow different pathways for the last steps of the corrinoid compounds synthesis.

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.