Abstract by Christine Kopka Heerup

In the first six months after birth, exclusive breast-feeding is recommended, but is not achieved in nearly two out of three infants worldwide. In most cases when breast-feeding is not possible or sufficient, infant formula (IF) is used.  Approximately half of the energy from mother’s milk (MM) and IF comes from lipids (predominantly triacylglycerol), why lipid digestion is of particular importance in infants. Despite the continuously improved understanding of infants’ lipid digestion and dietary requirements, IF is still generally less well digested and tolerated by infants than MM. In addition, numerous studies have indicated, that the cognitive development is impaired in infants fed IF compared with breast-fed infants, possibly due to insufficient bioavailability of long chain polyunsaturated fatty acids, which are important building blocks in brain lipids. However, lipid digestion and absorption in infants are complex processes biologically designed for the digestion of MM. Two popular approaches to developing IFs, with regards to lipids, therefore, involve simulating MM in terms of composition and structural organization, and especially the latter has been in focus in the last decade. While the lipid and protein content of modern IFs can be considered an approximation of that of MM, the structural organization of the proteins and lipids is still very different. In MM, the triacylglycerol is packed in the core of the milk fat globules covered by a complex trilayered membrane structure, known as the MFG membrane (MFGM), which is believed to play a critical role during lipid digestion in infants. In contrast with MM, in IFs, the triacylglycerol is packet in the core of lipid droplets covered primarily with protein, which creates a surface that is very different than the MFGM. The use of bovine MFGM-rich ingredients from dairy process streams to make bioinspired lipid droplets, which simulate the milk fat globules in MM, has received increasing attention in the last decade. So far, the enrichment of IF with bovine MFGM has shown great promise to make the outcome of the lipid digestion and absorption in infants fed MM and infants fed IF more similar, with regards to the cognitive development. With the aim of improving the cognitive development in infants fed IF, by developing a new IF optimized for maximal lipid digestibility, the InfantBrain project was established. While the primary hypothesis of the InfantBrain project, in its entirety, was that improved lipid bioavailability has a positive impact on the cognitive development, the current study focused on a subset of hypotheses: I. Improving the lipid digestion also improve the lipid absorption. II. Bioinspired emulsions are more easily digested than emulsions made with soy phospholipid or whey protein. III. In vitro digestion of emulsions containing bioinspired lipid droplets can be used to predict in vivo digestion. The objectives of the current project focused on: I. Establishing an in vitro pediatric gastro-intestinal digestion model simulating lipid digestion in infants. II. Assessment of the lipid digestibility of emulsions containing bioinspired-, or reference lipid droplets in vitro using the established model. III. Assessing the lipid digestibility of pure emulsions and complete formulas containing bioinspired-, or reference lipid droplets in vivo using a preterm piglet model. To investigate the potential of using bioinspired lipid droplets in IFs, simple emulsions containing 3.5% triacylglycerol were made. By using bovine dairy process streams containing MFGM and membrane from extracellular vesicles fragments as emulsifiers, bioinspired emulsions were made. In addition, emulsions were made with reference emulsifiers consisting of soy phospholipid or bovine whey protein isolate since both are common ingredients in IFs. The influence of the emulsifier composition on emulsion characteristics, such as the droplet size and buffer capacity was investigated and primarily showed an association between the emulsifier content of PL and protein on the buffer capacity and an association between the emulsion pH and the average size of the lipid droplets. An in vitro pediatric gastro-intestinal digestion model, suitable for studying lipid digestion of IFs and simple emulsions with recombinant human gastric lipase and porcine pancreatin, was established. The model showed good promise for its in vivo lipid digestion predictability based on a gastric contribution to the observed gastro-intestinal lipid digestion that was considered physiologically relevant. Based on validation with direct quantification of lipid digestion using high performance liquid chromatography, indirect quantification by titration of free fatty acids liberated during digestion, was found to reflect in vitro lipid digestion reliably. In the established model, using titration of liberated free fatty acids as the only quantification method, was, therefore, considered sufficient. The model can thus be used a fast method with low labour intensity to determine in vitro lipid digestion. Using only titrimetric quantification of the lipid digestion, the established in vitro pediatric gastro-intestinal digestion model was used to assess the lipid digestibility of the bioinspired and reference emulsions containing 3.5% triacylglycerol. The emulsions responded differently to the in vitro digestion with recombinant human gastric lipase and pancreatin and the digestibility was found to be correlated with the soy phospholipid and protein content of the emulsifiers used as well as the buffer capacity of the emulsions. Increasing the amount of soy phospholipid in bioinspired emulsions resulted in a decrease in the lipid digestibility, whereas increasing the amount of whey protein generally resulted to an increase. In fact, after 75 minutes of digestion, the emulsion made with a combination of WPC-PL and whey protein isolate had the highest observed mean lipid digestibility, matched only by the emulsion made with WPC-A-EV. The in vitro digestion experiments therefore showed, that depending on the specific emulsifier used, bioinspired lipid droplets can be used to improve the lipid digestibility of emulsions; at least in vitro. To assess the lipid digestion and absorption in vivo, three separate nutrition experiments were conducted in new-born preterm piglets. In contrast with the corresponding in vitro lipid digestions, the in vivo experiments generally showed no or limited immediate effect of the choice of emulsifiers on the lipid digestion and absorption in the piglets. More specifically, in Experiment 1, short-term consumption of complete formulas (with 5.1% triacylglycerol) containing either soy phospholipid or WPC-PL, resulted in equal amounts of fecal lipids and lipid absorption in the piglets, regardless of the diet. In addition, in Experiment 2, following a single bolus feeding of pure emulsion (with 10% triacylglycerol) containing either soy phospholipid or WPC-PL, both test groups had similar plasma triacylglycerol concentrations. However, in Experiment 3, following a single bolus feeding of complete formula (with 10% triacylglycerol), the test group, that had been fed the complete formula containing soy phospholipid, had significant lower plasma triacylglycerol concentration compared the test groups fed complete formulas containing WPC-PL, or WPC-A-EV. The results from Experiment 3, therefore, indicated, that consumption of bioinspired lipid droplets can positively influence the lipid absorption compared with lipid droplets made with soy phospholipids. In conclusion, bioinspired emulsions stabilized with high-protein MFGM-, and EVM-rich emulsifiers from bovine dairy process streams, compared with soy phospholipid, resulted in higher in vitro lipid digestion and indications of higher in vivo lipid absorption in preterm piglets. Further studies are required to: I. Determine whether the observations related to the lipid digestion and absorption from the current project are reproducible and can be translated to human infants and II. Determine whether improved lipid digestion and absorption has a positive impact on the cognitive development of infants fed IF. Still, the concept of using bioinspired lipid droplets in IFs appear to have great potential to improve the lipid bioavailability in infants.