BACKGROUND: The purpose of this research was to assess the growth, tolerance, and compliance outcomes associated with the consumption of a hydrolyzed rice infant formula (HRF) enriched with 2\'-Fucosyllactose (2\'-FL) a Human Milk Oligosaccharide (HMO), and nucleotides in an intended population of infants.
METHODS: This was a non-randomized single-group, multicenter study. The study formula was a hypoallergenic HRF with 2\'-FL, Docosahexaenoic acid (DHA), Arachidonic acid (ARA), and nucleotides. Infants 0-90 days of age who were formula fed and experiencing persistent feeding intolerance symptoms, symptoms of suspected food protein (milk and/or soy) allergy, or other conditions where an extensively hydrolyzed infant formula was deemed an appropriate feeding option were recruited by pediatricians from their local populations. The primary outcome was maintenance of weight-for-age z-score. Weight, length, head circumference, formula intake, tolerance measures, clinical symptoms and questionnaires were collected. Thirty-three infants were enrolled, and 27 completed the study, on study product.
RESULTS: Weight-for-age z-scores of infants showed a statistically significant improvement from Visit 1 to Visit 4 (p = 0.0331). There was an adequate daily volume intake of 762 ± 28 mL/day, average daily number of stools of 2.1 ± 0.3, and mean rank stool consistency of 2.38 ± 0.18. After 28 days of switching to a HRF, 86.8 ± 5.9% of the symptoms resolved or got better by Visit 4 as reported by parents.
CONCLUSIONS: HRF with 2\'-FL HMO was safe, well tolerated, and supported weight gain in infants with suspected cow\'s milk allergy or persistent feeding intolerance.

Growing evidence indicates that human milk oligosaccharides (HMOs) are important bioactive compounds that enhance health and developmental outcomes in breastfed babies. Maternal dietary intake likely contributes to variation in HMO composition, but studies identifying diet-HMO relationships are few and inconsistent. This study aimed to investigate how the maternal intake of macronutrients and micronutrients-specifically proteins, fats, vitamins, and minerals-associated with HMOs at 1 month (n = 210), 6 months (n = 131), and 12 months postpartum (n = 84). Several associations between maternal dietary factors and HMO profiles were identified utilizing partial correlation analysis. For example, maternal free sugar (rho = -0.02, p < 0.01), added sugar (rho = -0.22, p < 0.01), and sugary sweetened beverage (rho = -0.22, p < 0.01) intake were negatively correlated with the most abundant HMO, 2\'-fucosyllactose (2\'-FL), at 1 month, suggesting that higher sugar consumption was associated with reduced levels of 2\'-FL. Further, vitamins D, C, K, and the minerals zinc and potassium were positively correlated with 2\'-FL at 1 month (pAll < 0.05). For the longitudinal analysis, a mixed-effects linear regression model revealed significant associations between maternal vitamin intake and HMO profiles over time. For example, for each unit increase in niacin intake, there was a 31.355 nmol/mL increase in 2\'-FL concentration (p = 0.03). Overall, the results provide additional evidence supporting a role for maternal nutrition in shaping HMO profiles, which may inform future intervention strategies with the potential of improving infant growth and development through optimal HMO levels in mothers\' milk.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5\'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N\',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Bacterial infections lack reliable, specific, and quick detection methods, which incur substantial costs to patients and caretakers. Our team conjugated the FDA-approved fluorescent dye indocyanine green (ICG) with a maltotriose sugar, resulting in two highly specific imaging agents (ICG-DBCO-1-Maltotriose and ICG-Amide-1-Maltotriose) for detecting bacterial infections. We then evaluated the two derivatives using fluorescence imaging (FLI), bioluminescence imaging (BLI), and photoacoustic imaging (PAI) in bacterial infection murine models. Our findings indicate that both imaging agents can correlate with and reliably detect the infection site using FLI and PAI for both Gram-negative and Gram-positive strains, with various bacterial loads. Furthermore, the differences in pharmacokinetic (PK) properties between the two agents allow for one to be used for immediate imaging (2-4 h postinjection), while the other is more effective for longitudinal studies (18-40 h postinjection).

Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first time, much-applied spectrophotometric methods relying on the quantification of certain functional groups (i.e., DNS, GOPOD) were compared to chromatographic quantification of starch metabolites (HPLC-ELSD). The estimated rate constant and linked initial rates of amylolysis were highly correlated for DNS, GOPOD, and HPLC-ELSD. However, absolute amylolysis levels depended on the applied method and sample-specific metabolite formation patterns. Multiresponse modelling was employed to further investigate HPLC-ELSD metabolite formation patterns. This delivered insight into the relative importance of different amylolysis reactions during in vitro digestion of pulse ICC, proving that maltotriose and maltose formation determined the overall amylolysis rate in this case. Multiresponse reaction rate constants of maltotriose and maltose formation were highly correlated to single response amylolysis rate constants (and initial rates) obtained for all three quantification methods.

As the evidence supporting the beneficial effects of human milk oligosaccharides (HMOs) grows, so does the commercial interest in their inclusion in infant formula products. This also requires analytical methods capable of their quantification from finished infant formula products as well as from premixed ingredients in some cases. The objective of the present study was the development and single-laboratory validation of a method that can be used for this purpose for seven HMOs: 2\'-fucosyllactose (2\'FL), 3-fucosyllactose (3FL), difucosyllactose (DFL), 3\'-sialyllactose (3\'SL), 6\'-sialyllactose (6\'SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). The present method uses labeling by reductive amination, with 4-aminobenzoic acid ethyl ester (benzocaine) as the labeling reagent and picoline borane as the reducing agent, then applies HPLC separation with UV detection. The seven HMOs could be analyzed from infant formula and premix samples with recoveries between 91 and 108 %, relative standard deviations of 4.3 % or lower across all replicates, and limits of quantitation between 0.001 % and 0.004 % of powder sample by weight. The method was found to be rapid and reliable, with a runtime of only 14 min per injection, in contrast to other methods found in literature which typically use nearly or more than an hour. In addition, it uses instrumentation that\'s readily available in most analytical laboratories.

Oligosaccharides, namely, chitosan oligosaccharides (COS), fructooligosaccharides (FOS), and 2\'-fucosyllactose (2-FL) were used to prevent the dextran sulfate sodium (DSS)-induced colitis in vivo based on antioxidant properties and anti-inflammatory activities, further comparing their alleviating effects to investigate the optimal anti-inflammatory agent. The results showed COS demonstrated the highest antioxidant properties, with a DPPH scavenging rate of 37.4% and an ABTS scavenging rate of 46.4% in these oligosaccharides. Consequently, COS exhibited the best anti-inflammatory activities on inflamed RAW 264.7 cells. Furthermore, the COS intervention demonstrated the best attenuated effects on decrease in the body weight and increase in DAI score, as well as on the overexpressed inflammatory factors and underexpressed short-chain fatty acids (SCFAs) compare to FOS and 2-FL. Therefore, these beneficial changes help prevent the damage to the inflammatory lesions in colonic histopathology. Additionally, COS significantly increased the diversity of gut microbiota and the ratio of Firmicutes/Bacteroidetes at phylum level. It also up-regulated the abundance of Lactobacillaceae and down-regulated Helicobacteraceae and Desulfovibrionaceae more effectively at family level to maintain oral tolerance against DSS. In short, COS intervention could be a promising nutritional strategy for alleviating colitis.

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 μg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.