Non-digestible oligosaccharides for application in infant formula: studying fermentation and immune effects

Mother milk is the best nutrition for newborn infants. It contains a wide range of nutrients and is an energy source for the gut microbiota. Mother milk plays an important role in the maturation of the gut-barrier and also supports the developing immune system in infants directly. The human milk oligosaccharides (HMOs) which are present in mother milk greatly contribute to these processes. For infants where mother-milk is not a feasible option, cow-milk derived formulas supplemented with non-digestible carbohydrates (NDCs) are used. The NDCs are added to mimic the HMO functions. There are already various NDCs available for the application in infant formulas, however, there are many other new NDCs available which might be interesting to apply in these formulas. In addition, there is not much known about how the fermentation of the NDCs by the gut-microbiota affect the immune regulating properties of different NDCs. Therefor, in our study, we selected various NDCs from different sources and fermented them using infant fecal inoculum to examine the fermentation kinetics, the microbiota changes, the production of short-chain fatty acids (SCFAs) and the effect of the digesta on immature immune responses. In addition, we also studied the effect of the infant’s age on these processes and if these processes are similar for infants with different immune backgrounds

Benefits of bacteria-derived exopolysaccharides on gastrointestinal microbiota, immunity and health

The gastrointestinal tract (GIT) is colonized by a broad spectrum of microorganism, of which bacteria are especially recognized for the health effects they exert. However insight in the effector molecules on the bacteria responsible are still largely lacking. Recently, bacterial exopolysaccharides (EPS), which are glycan structures on the bacterial cell wall, have gained considerable attention for their effects on human health. EPS are found to act as polymers that directly influence bacterial interactions with their host, such as bacterial adhesion to the GIT epithelium, their influence on immune responses, and the intestinal microbiota. These effects are highly dependent on the type of EPS structures and might be related to specific monosaccharides or linkages in EPS. Here, current knowledge on structure-specific EPS functions is reviewed in view of possible use of EPS to stimulate intestinal health and the formulation of dietary EPS mixtures or the possible creation of new EPS structures

More than sugar in the milk:human milk oligosaccharides as essential bioactive molecules in breast milk and current insight in beneficial effects

Human milk is the gold standard for newborn infants. Breast milk not only provides nutrients, it also contains bioactive components that guide the development of the infant's intestinal immune system, which can have a lifelong effect. The bioactive molecules in breast milk regulate microbiota development, immune maturation and gut barrier function. Human milk oligosaccharides (hMOs) are the most abundant bioactive molecules in human milk and have multiple beneficial functions such as support of growth of beneficial bacteria, anti-pathogenic effects, immune modulating effects, and stimulation of intestine barrier functions. Here we critically review the current insight into the benefits of bioactive molecules in mother milk that contribute to neonatal development and focus on current knowledge of hMO-functions on microbiota and the gastrointestinal immune barrier. hMOs produced via genetically engineered microorganisms are now applied in infant formulas to mimic the nutritional composition of breast milk as closely as possible, and their prospects and scientific challenges are discussed in depth

2 '-Fucosyllactose impacts the expression of mucus-related genes in goblet cells and maintains barrier function of gut epithelial cells

Scope: Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Methods/results: Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Conclusion: Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells

Distinct fermentation of human milk oligosaccharides 3-FL and LNT2 and GOS/inulin by infant gut microbiota and impact on adhesion of Lactobacillus plantarum WCFS1 to gut epithelial cells

Human milk oligosaccharides (hMOs) are unique bioactive components in human milk. 3-Fucosyllactose (3-FL) is an abundantly present hMO that can be produced in sufficient amounts to allow application in infant formula. Lacto-N-triaose II (LNT2) can be obtained by acid hydrolysis of lacto-N-neotetraose (LNnT). Both 3-FL and LNT2 have been shown to have health benefits, but their impact on infant microbiota composition and microbial metabolic products such as short-chain fatty acids (SCFAs) is unknown. To gain more insight in fermentability, we performed in vitro fermentation studies of 3-FL and LNT2 using pooled fecal microbiota from 12-week-old infants. The commonly investigated galacto-oligosaccharides (GOS)/inulin (9 : 1) served as control. Compared to GOS/inulin, we observed a delayed utilization of 3-FL, which was utilized at 60.3% after 36 h of fermentation, and induced the gradual production of acetic acid and lactic acid. 3-FL specifically enriched bacteria of Bacteroides and Enterococcus genus. LNT2 was fermented much faster. After 14 h of fermentation, 90.1% was already utilized, and production of acetic acid, succinic acid, lactic acid and butyric acid was observed. LNT2 specifically increased the abundance of Collinsella, as well as Bifidobacterium. The GOS present in the GOS/inulin mixture was completely fermented after 14 h, while for inulin, only low DP was rapidly utilized after 14 h. To determine whether the fermentation might lead to enhanced colonization of commensal bacteria to gut epithelial cells, we investigated adhesion of the commensal Lactobacillus plantarum WCFS1 to Caco-2 cells. The fermentation digesta of LNT2 collected after 14 h, 24 h, and 36 h, and GOS/inulin after 24 h of fermentation significantly increased the adhesion of L. plantarum WCFS1 to Caco-2 cells, while 3-FL had no such effect. Our findings illustrate that fermentation of hMOs is very structure-dependent and different from the commonly applied GOS/inulin, which might lead to differential potencies to stimulate adhesion of commensal cells to gut epithelium and consequent microbial colonization. This knowledge might contribute to the design of tailored infant formulas containing specific hMO molecules to meet the need of infants during the transition from breastfeeding to formula

Inhibitory Effects of Dietary N-Glycans From Bovine Lactoferrin on Toll-Like Receptor 8; Comparing Efficacy With Chloroquine

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset, progression, and treatment of a broad spectrum of inflammatory conditions. In this study, we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form, as well as in its partially demannosylated and partially desialylated form, on HEK293 cells expressing TLR-8, and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells, N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent, i.e., chloroquine (CQN), that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8, it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold, p <0.0001), partially demannosylated (0.3-fold, p <0.0001) and partially desialylated (0.4-fold, p <0.0001) N-glycans. Furthermore, the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN, the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune, infections or other inflammatory disorders

Pectins that Structurally Differ in the Distribution of Methyl-Esters Attenuate Citrobacter rodentium-Induced Colitis

Introduction: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity, but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, this study tests pectins that structurally differ in DB in a mouse model with Citrobacter rodentium induced colitis and studies the impact on the intestinal microbiota composition and associated attenuation of inflammation. Methods and Results: Both low and high DB pectins induce a more rich and diverse microbiota composition. These pectins also lower the bacterial load of C. rodentium in cecal digesta. Through these effects, both low and high DB pectins attenuate C. rodentium induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells, which are increased in case of C. rodentium induced colitis, and reduced levels of GATA3+ Tregs, which are related to tissue inflammation. Conclusion: Pectins prevent C. rodentium induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB

Performance Testing of a Homemade Aerosol Generator for Pulmonary Administration of Dry Powder Formulations to Mice

A challenge in the development of dry powder formulations for inhalation is the poor reproducibility of their administration to small laboratory animals. The currently used devices for the pulmonary administration of dry powder formulations to small rodents often function sub-optimally as they use the same puff of air for both powder dispersion and aerosol delivery. As a result, either the air volume and flow rate are too low for complete powder deagglomeration or they are too high for effective aerosol delivery to the lungs of the animal. Therefore, novel and better devices are desired. We here present an aerosol generator designed to administer a pre-generated aerosol to the lungs of mice. By mapping the complex relationship between the airflow rate, delivery time and emitted dose, we were able to control the amount of powder being delivered from the aerosol generator. The emitted aerosol had a size range favorable for lung deposition and could be measured reproducibly. Nevertheless, in vivo fluorescent imaging still revealed considerable differences between the mice in terms of the dose deposited and the distribution of powder over the lungs, suggesting that a certain biological variation in lung deposition is inevitable.</p

Chicory inulin enhances fermentation of 2'-fucosyllactose by infant fecal microbiota and differentially influences immature dendritic cell and T-cell cytokine responses under normal and Th2-polarizing conditions

Scope: Non-digestible carbohydrates (NDCs) such as native chicory inulin and 2′-fucosyllactose (2′-FL) are added to infant formula to mimic some of the human milk oligosaccharide (HMO) functions. It is unknown whether combining inulin and 2′-FL influences their fermentation kinetics and whether the immune-modulatory effects of these NDCs are different under normal and inflammatory-prone Th2-polarizing conditions. Methods and results: We investigated the in vitro fermentation of 2′-FL and native chicory inulin, fermented individually and combined, using fecal inocula of 8-week-old infants. Native inulin was fermented in a size-dependent fashion and expedited the fermentation of 2′-FL. Fermentation of both native inulin and 2′FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. The fermentation digesta of all fermentations differentially influenced both dendritic cell and T-cell cytokine responses under normal culture conditions or in presence of the Th2-polarizing cytokines IL-33 and TSLP, with the most pronounced effect for IL-1β in the presence of TSLP. Conclusions: Our findings show that native inulin can expedite the fermentation of 2′-FL by infant fecal microbiota and that these NDC fermentation digesta have different effects under normal and Th2-polarizing conditions, indicating that infants with different immune backgrounds might benefit from tailored NDC formulations

Endo-1,3(4)-β-Glucanase-Treatment of Oat β-Glucan Enhances Fermentability by Infant Fecal Microbiota, Stimulates Dectin-1 Activation and Attenuates Inflammatory Responses in Immature Dendritic Cells

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, β-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-β-glucanase-treated oat β-glucan using pooled fecal inocula of 2-and 8-week-old infants. While native oat β-glucan was not utilized, both inocula specifically utilized oat β-glucan oligomers containing β(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat β-glucans increased the relative abundance of Enterococcus and attenuated proinflammatory cytokine production (IL-1β, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat β-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-β-glucanase treatment enhances the fermentability of oat β-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat β-glucans have a high potential for supplementation of infant formula.</p