Danone (Netherlands)

Emulsion-filled protein gels are interesting composite materials widely applied in food, cosmetic and pharmaceutical industries. Incorporating emulsion droplets in a protein gel matrix can sometimes undesirably weaken the gel. This study introduces a strategy to counteract this defect using protein pre-aggregation induced by heating. Faba bean globulin (Fg) gels were studied as an example, by comparing a lab-extracted Fg that was mostly native, Fg heated for 10 or 60 min at 95°C (causing a controlled degree of pre-aggregation), and a commercial Fg (partially denatured). The 60 min-heated Fg was aggregated more significantly than the 10 min-heated one, and the commercial Fg mainly consisted of large and dense protein aggregates. Emulsion droplets were stabilized by the same type of Fg that formed the gel matrix and proved to act as inactive fillers. They decreased the gel strength by 15-30% at a 6.5% (w/w) oil concentration and shifted the maximum linear strain to lower amplitudes by decreasing the homogeneity of the gel matrix. However, they barely influenced the gelation kinetics and the behavior of all Fg gels in large shear deformations, which were dominated by the gel matrix. Pre-aggregated Fg endowed the emulsion-filled gels with 1.4- to 4.1-fold higher storage modulus and 1.3- to 1.9-fold higher maximum linear strain than untreated Fg, presumably due to stronger hydrophobic protein-protein interactions resulting from enhanced exposed hydrophobicity; moreover, the pre-aggregates may have acted as active fillers. Therefore, pre-aggregation is an effective strategy to improve the mechanical properties of emulsion-filled faba bean globulin gels when the emulsion droplets are inactive fillers. These findings are expected to improve the texture and tailor the mechanical properties of the emulsion-filled-gel-based products in food, cosmetics and pharmaceutical products. • Faba bean globulin-stabilized emulsion droplets acted as inactive fillers. • Protein pre-aggregation can reinforce emulsion-filled faba bean globulin (Fg) gels. • Fg pre-aggregates seemed to act as active fillers. • The gelation kinetics and gels’ nonlinear rheology were dominated by the gel matrices.

It is essential to determine glycan structures in complex biological samples to understand their biology and exploit their diagnostics, therapeutics and nutraceuticals potential. An unresolved analytical challenge is the identification of isomeric glycan structures in complex biological samples. Ion mobility (IM) combined with MS enables separation of isomeric glycans and identification by comparing their intrinsic collision cross section (CCS) values with similar data of synthetic standards. To identify glycans without the need to synthesize all biologically occurring glycans, we describe here an IM-MS de novo sequencing method based on fragment identification and sequence assembly. CCS values of additional fragments from glycans in biological samples result in a self-expanding reference database, gradually facilitating the sequencing of glycans of increasing complexity and expanding the database from an initial 19 standards to 332 unique entries. The methodology is employed to determine structures of human milk oligosaccharides and N-glycans of biotherapeutics.

Consumption of an amino acid-based formula (AAF) with added synbiotics [short-chain oligofructose and long-chain inulin (scFOS/lcFOS, 9:1 ratio) and Bifidobacterium breve M-16V] (AAF-S) beneficially impacts the gut microbiome of infants with cow's milk allergy (CMA). We assessed the effect of consuming AAF with or without synbiotics by children with CMA for 12 months on their fecal (branched) short-chain fatty acids (SCFA/BCFA) concentrations, and on gut barrier and inflammation markers (Netherlands Trial Register NTR3725). Feces and saliva were collected from 161 children (≤13 months) with IgE-mediated CMA at baseline, 6 and 12 months after enrollment, and at 24 and 36 months follow-up. Fecal SCFA and BCFA were analyzed by gas chromatography, and gut barrier and inflammation markers were measured in saliva/feces by ELISA or ImmunoCAP. At 6 months, children receiving AAF-S had significantly lower fecal propionate, valerate and BCFA concentrations compared to children consuming AAF. The percentage of propionate from the total 6 SCFA/BCFA (acetate + butyrate + propionate + valerate + isobutyrate + isovalerate) was significantly lower, while the percentage of acetate from the total 6 SCFA/BCFA was significantly higher in the AAF-S group. There were no significant differences between groups in fecal concentrations of butyrate at 6 months, nor in SCFA or BCFA at baseline and after 12, 24 or 36 months. Intestinal inflammation and barrier markers did not differ between groups. Addition of synbiotics to AAF brings concentrations of key fecal microbial metabolites more in line with patterns observed in healthy breastfed infants. The effects on SCFA and BCFA concentrations were transient and only seen at 6 months.

Microbiome research focusing on late and moderate preterm infants (LMPT; 32 to 36 weeks gestation) is limited, despite rising LMPT births, large healthcare burdens, and increased risks of multiple morbidities, potentially microbially related. In this longitudinal cohort study, 16S rRNA gene sequencing was used to analyze 371 stool and 402 saliva samples from 160 LMPT infants, collected at five time points between birth and 12 months corrected age (CA), to describe spatial and temporal variability in gut and oral microbiomes. Paired stool and saliva samples (n = 337) were analyzed for potential microbial relationships. Early LMPT samples (up to 60 days of life; DOL) were also compared with data from seven extremely preterm infants (EP; <28 weeks gestation; stool n = 14, saliva n = 14). LMPT stool and saliva were composed of distinct microbial communities at each time point, and both sample types showed increasing alpha diversity over time. Stool was initially dominated by Escherichia/Shigella, Klebsiella, and Streptococcus, with Bifidobacterium becoming dominant from term equivalent age (TEA). Contrarily, saliva was dominated by Streptococcus throughout the first year, with early contributions from Staphylococcus and later Veillonella. LMPT infants had higher stool and lower saliva diversity compared with EP infants. Both sample types from EP infants were taxonomically distinct from LMPTs, with Escherichia/Shigella dominating both EP sample types throughout the first 60 DOL. The results highlight the unique trajectories of LMPT microbiomes and emphasize the role of gestational maturity in shaping microbial communities.IMPORTANCEThe oral and gut microbiome develops from birth and plays important roles in health. This has been well studied in extremely preterm infants (EP; born <32 weeks gestation) and term infants (born >38 weeks gestation), but there is a paucity of research describing oral and gut microbiome development in late and moderate preterm infants (LMPT; 32 to 36 weeks gestation). Our study analyzed microbiome development in 160 LMPT infants from birth to 12 months corrected age. The results showed distinct microbial communities in stool and saliva, with increasing alpha diversity and niche specification over time. LMPT infants' gut microbiome became dominated by Bifidobacterium by month 3, while the oral community was consistently dominated by Streptococcus. These results highlight that LMPT infants have gut and oral microbiome development that is more like term infants than EP infants, which has important implications for the care of LMPT infants.

This Review Article summarizes outcomes from the ILSI Europe expert workshop on plant-based proteins in infant formula, held in November 2024. Experts from academia, clinical nutrition, and food science evaluated the current use and future potential of plant-based protein sources in infant formula, considering nutritional adequacy, allergenicity, sustainability, processing technologies, and regulatory constraints. While soy and hydrolyzed rice proteins are already approved and in use, emerging sources such as pea, lentil, and faba beans show promise but require further validation of their amino acid profiles, digestibility, safety, and suitability for infants. Key research priorities identified include the development of improved protein extraction methods, in vitro digestion and allergy modeling, and targeted clinical studies. This review synthesizes current evidence and expert perspectives to support the development of sustainable, nutritionally adequate plant-based infant formulas.

ABSTRACTFood allergies (FAs) are common in society with limited treatment options available. Therefore, there is an urgent need for new targets and treatment options. Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) are mostly inhibitory receptors differentially expressed on all immune cells. There are many different types of Siglecs, and they are involved in the regulation of several signalling pathways. The specific role that Siglecs can have on various diseases, including cancer and immune‐driven disorders, is gaining interest. This review focusses on the current knowledge of the role of Siglecs on immune cells involved in FA sensitisation and elicitation and how targeting these Siglecs could possibly prevent or treat FA. Most research is focussed on targeting Siglecs expressed by mast cells and basophils, and how this can dampen the activation and/or degranulation of these cells. Targeting Siglecs on cells involved in the sensitisation phase of FA could be an interesting option to intervene earlier on in the allergic response, thus preventing the onset of FA rather than treating it. Siglec‐2 on B cells is already of great interest for the treatment of FA, and results seem promising as B cell receptor signalling and antibody production were inhibited. Siglecs on other cell types in the sensitisation phase, such as dendritic cells, seem promising, but functional assays with human cells are lacking so far. Overall, Siglecs are broadly expressed on all immune cells involved in the allergic response, which support the hypothesis that Siglecs are involved in the allergic response itself and may act as a potential target in the treatment of FA. Especially, treatments focussed on targeting Siglecs on multiple immune cell types may have great potential, as this could enhance both efficacy and safety.

Human milk oligosaccharides (HMOs) represent the thirdmost abundantfraction of biomolecules in human milk (HM) and play a crucial rolein infant health and development. The unique contributions of HMOsto healthy development of breast-fed infants are assumed to rely onthe extraordinary complexity and diversity of HMO isomeric structures,which in turn still cause a huge analytical challenge. Many contemporaryanalytical methods aiming for more detailed HMO characterization combineion mobility (IM) with LC-MS for enhanced structural resolution butare typically lacking the robustness necessary for application toHM cohorts with hundreds of samples. To overcome these challenges,we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MSmethod integrating four analytical dimensions: high-resolution LCseparation, IM drift time, accurate mass precursor, and fragment ionmeasurements. This four-dimensional (4D) analytical characterizationis sufficient for resolving various HMO structural isomers in an efficientway. Thereby, up to 200 HMO compounds with a maximum degree of polymerizationof 13 could be simultaneously identified and relatively quantified.We devised two methods using this 4D analytical approach. One intendedfor in-depth characterization of multiple known but also novel HMOstructures and the second is designed for robust, increased-throughputanalyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected beforein HM, as well as additional difucosyl-lacto-N-heaoseisomers (DF-LNHs), were revealed and structures fully elucidated byAIF and IM. This exemplifies the potential of our method for in-depthcharacterization of novel complex HMO structures. Furthermore, theincreased-throughput method featuring a shorter LC gradient was appliedto real-world HM samples. Here, we could differentiate the HM typesI–IV based on a broader range of partly new marker HMOs. Wecould also derive valuable new insights into variations of multipleand rare HMOs up to DP 11 across lactational stages. Overall, ourAIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confidentidentification of a broad array of distinct and simple to very complexHMOs. We envision this robust AIF LC-ESI-IM-MS approach to advanceHMO research by facilitating the characterization of a broad rangeof HMOs in high numbers of HM samples. This may help to further extendour understanding about HMOs structure–function relationshipsrelevant for infants’ healthy development

ABSTRACT The intestinal mucosal barrier is a dynamic system that allows nutrient uptake, stimulates healthy microbe–host interactions, and prevents invasion by pathogens. The mucosa consists of epithelial cells connected by cellular junctions that regulate the passage of nutrients covered by a mucus layer that plays an important role in host–microbiome interactions. Mimicking the intestinal mucosa for in vitro assays, particularly the generation of a mucus layer, has proven to be challenging. The intestinal cell-line Caco-2 is widely used in academic and industrial laboratories due to its capacity to polarize, form an apical brush border, and reproducibly grow into confluent cell layers in different culture systems. However, under normal culture conditions, Caco-2 cultures lack a mucus layer. Here, we demonstrate for the first time that Caco-2 cultures can form a robust mucus layer when cultured under air–liquid interface (ALI) conditions on Transwell inserts with addition of vasointestinal peptide (VIP) in the basolateral compartment. We demonstrate that unique gene clusters are regulated in response to ALI and VIP single stimuli, but the ALI-VIP combination treatment resulted in a significant upregulation of multiple mucin genes and proteins, including secreted MUC2 and transmembrane mucins MUC13 and MUC17. Expression of tight junction proteins was significantly altered in the ALI-VIP condition, leading to increased permeability to small molecules. Commensal Lactiplantibacillus plantarum bacteria closely associated with the Caco-2 mucus layer and differentially colonized the surface of the ALI cultures. Pathogenic Salmonella enterica were capable of invading beyond the mucus layer and brush border. In conclusion, Caco-2 ALI-VIP cultures provide an accessible and straightforward way to culture an in vitro intestinal mucosal model with improved biomimetic features. This novel in vitro intestinal model can facilitate studies into mucus and epithelial barrier functions and in-depth molecular characterization of pathogenic and commensal microbe–mucus interactions.

Deviations from normal postnatal growth patterns in infancy and early childhood are indicators to predict future negative health outcomes and to direct early interventions, such as changes in feeding regimens. The objective of our study was to examine potential differences in BMI trajectory during the first year of life between infants of different birth weight categories. A pooled, fully anonymized dataset was constructed based on individual subject data of 4,777 participants from 12 clinical studies in which infant growth outcome measurements were collected at several timepoints during their first year of life. A BMI model was constructed using a joint bivariate growth model for weight and height (and their deterministic relationship). A Bayesian approach was used to explore birthweight category-specific differences in BMI development. Our main finding is that the BMI trajectories of the birthweight categories (particularly the small vs large groups) continue to show differences over time, although these differences become less apparent after the first months of age. Both weight and length continue to show consistent differences between categories across the first year of life. A more detailed analysis including the effect of region and gender, showed differential BMI patterns among the three birthweight categories over time across the different subgroups (combinations of region and gender).