Infant Milk Formula Enriched in Dairy Cream Brings Its Digestibility Closer to Human Milk and Supports Intestinal Health in Pre-Clinical Studies

Abstract

Human breast milk (HBM) is the "gold standard" for infant nutrition. When breast milk is insufficient or unavailable, infant milk formula (IMF) can provide a safe and nutritious alternative. However, IMFs differ considerably from HBM in composition and health function. We compared the digestibility and potential health functions of IMF containing low cream (LC-) or high cream (HC-) with pooled HBM. After simulated infant digestion of these samples, the bioavailability of key nutrients and immunomodulatory activities were determined via cell-based in vitro assays. A Caenorhabditis elegans leaky gut model was established to investigate cream effects on gut health. Distinct differences were observed in peptide diversity and sequences released from HC-IMF compared with LC-IMF during simulated digestion (p < 0.05). Higher levels of free fatty acids were absorbed through 21-day differentiated Caco-2/HT-29MTX monolayers from HC-IMF, compared with LC-IMF and HBM (p < 0.05). Furthermore, the immune-modulating properties of HC-IMF appeared to be more similar to HBM than LC-IMF, as observed by comparable secretion of cytokines IL-10 and IL-1β from THP-1 macrophages (p > 0.05). HC-IMF also supported intestinal recovery in C. elegans following distortion versus LC-IMF (p < 0.05). These observations suggest that cream as a lipid source in IMF may provide added nutritional and functional benefits more aligned with HBM.

Keywords: cream; dairy lipids; infant formula; infant nutrition; milk fat globule.

Figure 1

Schematic representation of the C. elegans model of intestinal damage: preventive (A) and recovery (B) experimental setup for intestinal damage in the C. elegans model. Concentrations used were as follows: IMF (10 μL/mL); MTX (0.5 μg/mL); Nile red (0.05 μg/mL). Sixty worms were used per treatment, and experiments were performed in duplicates. C. elegans, Caenorhabditis elegans; MTX, methotrexate.

Figure 2

Peptide release and absorption from HBM, HC-IMF, and LC-IMF in infant gastrointestinal digestion. Molecular weight distribution of proteins and peptides by SE-HPLC prior to the digestion (G0), after the gastric phase (G60), and after the intestinal phase (I60) (A). Number of released individual peptides in stages of simulated infant digestion and absorption by LC–MS/MS in LC-IMF, HC-IMF, and HBM. Different letters indicate significant differences in number of individual unique peptides, p < 0.05 (B). Relative abundance of released individual peptides from different parental proteins in stages of simulated infant digestion and absorption by LC–MS/MS in LC-IMF and HC-IMF (C). N = 3. HC-IMF, high-cream IMF; LC-IMF, low-cream IMF.

Figure 3

Free AAs and free FAs release and absorption from HBM, LC-IMF, and HC-IMF. Profile of free AAs release (A,B) and free FAs release (C,D) in simulated gastrointestinal digesta and after the simulated absorption with 21-day-old Caco-2/HT-29MTX monolayers over 4 h incubation with I60 samples at 500 μg protein/cm² in HBSS buffer. N = 3. Different letters within a time point indicate significant differences in total AAs or FAs concentrations, p < 0.05. Raw data on free AA release are provided in Supplementary Table S2.

Figure 4

Mineral release and absorption in HBM, LC-IMF, and HC-IMF. The concentration of Ca (A) and Mg (B) in IMF and HBM prior to the digestion (G0), after the gastric phase (G60), after the intestinal phase (I60), and after absorption across 21-day-old Caco-2/HT29-MTX monolayers at 500 μg protein/cm². Values are represented as mean with SD as error bars, N = 3. Different letters at each time point indicate significant differences (p < 0.05). HC-IMF, high-cream IMF; LC-IMF, low-cream IMF; SD, standard deviation.

Figure 5

Functional effects. Tight junction (TJ) protein expression in 21-day differentiated monolayers after 4 h incubation with IMF I60 samples (at 500 μg protein/cm² in HBSS) (A), and quantification of the bands by densitometry (B), N = 3. Intestinal barrier monolayer integrity by TEER in 21-day differentiated monolayers after 4-h incubation with IMF samples (C). Monolayers in the HBSS buffer had an average TEER of 664 ± 72 Ω×cm² and were assigned a value of 100%, N = 18. Release of satiety hormone GLP-1 (active) over 4 h incubation with 0.5 × 10⁶ cells/well in 12-well plates STC-1 cells treated with 500 μg protein/cm² of IMF I60 digesta in Krebs buffer (D), N = 9. Different letters for each treatment indicate significant differences (p < 0.05). HC-IMF, high-cream IMF; LC-IMF, low-cream IMF.

Figure 6

Immunomodulatory properties. THP-1 cells (5 × 10⁵ cells/well in 12 well plates) were differentiated for 3 days and then treated with digested IMF samples (at 500 μg protein/cm² in HBSS) or HBM in the same dilution for 4 h. Concentrations of secreted IFN-ɣ (A), IL-10 (B), IL1-β (C), IL-6 (D), IL-8 (E), and TNF-α (F) are represented as mean with SD as error bar, N = 6. Different letters at each time point indicate significant differences (p < 0.05). HC-IMF, high-cream IMF; LC-IMF, low-cream IMF; SD, standard deviation.

Figure 7

Intestinal damage. Effect of LC-IMF and HC-IMF on the recovery (A) and prevention (B) of intestinal barrier damage by MTX (positive control) in a C. elegans model. Prevention and recovery effects were determined by the intensity of Nile red staining of the body after leakage from the intestinal cavity, which was expressed as a percentage of fluorescence for each treatment group compared to the MTX-treated group (positive control). Concentrations used were as follows: IMF (10 μL/mL); MTX (0.5 μg/mL); Nile red (0.05 μg/mL). Sixty worms were used per treatment, and experiments were completed in duplicate. Values are represented as mean with SD as error bar, N = 3. Values without common letters differ significantly from each other (p < 0.05). C. elegans, Caenorhabditis elegans; HC-IMF, high-cream IMF; LC-IMF, low-cream IMF; MTX, methotrexate; NGM, normal growth medium; SD, standard deviation.