Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of α-lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since α-lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using α-l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

β-glucans come from cereals that have been located within compounds with prebiotic activity. They have presented several bioactivities that have determined their high functional value. The aim of this study was to identify the influence of oat β-glucan on the survival and proteolytic activity of Lactobacillus rhamnosus GG in a milk fermentation through an experimental design to optimize the process. For β-glucan extraction after dry milling of oats, two methods were applied: with and without enzymatic inactivation of the semolina. The highest extraction yield (45.25 g/L) was obtained with enzymatic inactivation. For the optimization of survival and proteolytic activity, a central design composed of axial points with two factors on three levels was used. Control factors were β-glucan and inoculum concentrations. According to response surface, the best survival growth rate of probiotic was observed with 4.38% of inoculum and 22.46 g/L of β-glucan, and the highest production of free amino groups was observed with 4.18% of inoculum and 22.71 g/L of β-glucan. Thus, β-glucan promotes the proteolytic activity of Lb. rhamnosus GG in milk fermentation.

Sweet whey can be valorized through fermentation and subsequent production of Requeson, a fresh whey cheese obtained by heat-induced protein precipitation. This study evaluated the physicochemical (moisture, water activity, protein, ash, chloride, and EPS content), textural (hardness, cohesiveness, adhesiveness, springiness, chewiness, and fracturability), sensory (overall liking and JAR saltiness), and in vitro bioactive characteristics of Requeson produced from sweet whey fermented with exopolysaccharide (EPS)-producing Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 and Streptococcus thermophilus SY-102, individually or in co-culture. Unfermented, citric-acid-acidified whey served as the control. The co-culture (FC) showed the highest EPS content (3.5 mg/g), higher moisture, lower hardness and fracturability, and greater springiness than the control. Consumer evaluation (n = 130) showed favorable acceptance of FC. Among FC formulations containing 0.5–1.5% NaCl, 0.5% NaCl yielded the highest overall liking, although it did not differ significantly from 1.0%. FC showed the highest DPPH radical-scavenging (22.76%) and ACE-I inhibitory (31.00%) activities. Overall, co-culture fermentation represents a potential strategy for producing value-added Requeson with modified technological and sensory properties and measurable in vitro bioactivities.

α-L-Fucosidases are versatile glycoside hydrolases capable of catalyzing both hydrolysis and transfucosylation reactions, making them valuable biocatalysts for the synthesis of fucosylated oligosaccharides (FucOS). However, transfucosylation efficiency is often limited by competing hydrolysis, and the factors governing this balance remain incompletely understood. In this study, we investigated the effect of divalent cations on the catalytic behavior of the α-L-fucosidase from Thermotoga maritima (FUC- Tm ) by combining enzymatic assays with molecular dynamics (MD) simulations and binding energy analyses. Among the tested ions, Ca ²⁺ produced the most pronounced effect, increasing both hydrolytic and transfucosylation activities, with the highest transfucosylation efficiency observed at intermediate CaCl ₂ concentrations. MD simulations indicated that Ca ²⁺ does not induce major global conformational changes but is associated with reduced flexibility in loop regions surrounding the catalytic pocket, particularly Loop-B, and with improved positioning of catalytic residues. These effects are consistent with enhanced substrate interactions, as supported by binding energy analyses. However, the increase in transfucosylation efficiency coincided with a decrease in water activity, suggesting that the observed behavior arises from a combination of structural and physicochemical factors. Therefore, Ca ²⁺ -mediated modulation of FUC- Tm activity is best interpreted as a multifactorial process involving changes in local protein dynamics and in the reaction environment. These findings provide insight into ion-dependent modulation of glycoside hydrolases and highlight the importance of integrating structural dynamics and solvent effects to improve enzymatic synthesis of FucOS. Key points: Calcium enhances both hydrolytic and transfucosylation activities of α-L-fucosidase from Thermotoga maritima under specific conditions. Molecular dynamics simulations indicate that Ca ²⁺ is associated with reduced flexibility in loop regions adjacent to the catalytic site, contributing to the maintenance of catalytic pocket geometry. The modulation of catalytic behavior is influenced by a combination of structural effects and physicochemical factors, including changes in water activity and ionic strength.