Background/Objectives: HMOs are the third most abundant solid component after lactose and fats in human milk. This study aimed to examine the relationships between maternal diet and HMO composition and concentration in human milk among lactating women in Latvia. Methods: Pooled 24 h human milk samples, 72 h food diaries, and questionnaires on anthropometric and sociodemographic characteristics were collected from 68 exclusively breastfeeding women residing in Latvia. HMOs were analyzed by UHPLC/FLD, and dietary data were analyzed using the Estonian NutriData program. Results: The eight most abundant HMO structures were determined with total concentration ranging between 178.66 and 32,910.09 mg L-1. 2'-FL was the most prevalent HMO in human milk (median concentration-3647 mg L-1), followed by 3'-FL (1436.74 mg L-1). Participants had an insufficient intake of vegetables, fruits, berries, milk and dairy products, and fish, leading to vitamin A, vitamin C, folate, and iodine intakes lower than recommended for lactating women. Limitation or exclusion of milk and dairy products from the diet was associated with a higher 2'-FL concentration in human milk (p = 0.037). Preference for "zero sugar" products was associated with a higher 3'-FL, 6'-GL, LNnT, 6'-SL, LNDFH II concentration in human milk (p < 0.050). Dietary supplement use (e.g., vitamin D, calcium) was also associated with differences in HMO composition and concentration in milk (p < 0.050). Conclusions: The findings highlight the importance of dietary habits and supplement use in shaping HMO profiles, though more human milk samples and dietary data need to be evaluated to draw further conclusions.

$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.
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α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.