Serum cytokine patterns are modulated in infants fed formula with probiotics or milk fat globule membranes: A randomized controlled trial

Background Proteins and lipids of milk fat globule membrane (MFGM) and probiotics are immunomodulatory. We hypothesized that Lactobacillus paracasei ssp. paracasei strain F19 (F19) would augment vaccine antibody and T helper 1 type immune responses whereas MFGM would produce an immune response closer to that of breastfed (BF) infants. Objective To compare the effects of supplementing formula with F19 or bovine MFGM on serum cytokine and vaccine responses of formula-fed (FF) and BF infants. Design FF infants were randomized to formula with F19 (n = 195) or MFGM (n = 192), or standard formula (SF) (n = 194) from age 21±7 days until 4 months. A BF group served as reference (n = 208). We analyzed seven cytokines (n = 398) in serum at age 4 months using magnetic bead-based multiplex technology. Using ELISA, we analyzed anti-diphtheria IgG (n = 258) and anti-poliovirus IgG (n = 309) concentrations in serum before and after the second and third immunization, respectively. Results Compared with SF, the F19 group had greater IL-2 and lower IFN-γ concentrations (p<0.05, average effect size 0.14 and 0.39). Compared with BF, the F19 group had greater IL-2, IL-4 and IL-17A concentrations (p<0.05, average effect size 0.42, 0.34 and 0.26, respectively). The MFGM group had lower IL-2 and IL-17A concentrations compared with SF (p<0.05, average effect size 0.34 and 0.31). Cytokine concentrations were comparable among the MFGM and BF groups. Vaccine responses were comparable among the formula groups. Conclusions Contrary to previous studies F19 increased IL-2 and lowered IFN-γ production, suggesting that the response to probiotics differs across populations. The cytokine profile of the MFGM group approached that of BF infants, and may be associated with the previous finding that infectious outcomes for the MFGM group in this cohort were closer to those of BF infants, as opposed to the SF group. These immunomodulatory effects support future clinical evaluation of infant formula with F19 or MFGM.

Bovine Milk-Derived Emulsifiers Increase Triglyceride Absorption in Newborn Formula-Fed Pigs

Efficient lipid digestion in formula-fed infants is required to ensure the availability of fatty acids for normal organ development. Previous studies suggest that the efficiency of lipid digestion may depend on whether lipids are emulsified with soy lecithin or fractions derived from bovine milk. This study, therefore, aimed to determine whether emulsification with bovine milk-derived emulsifiers or soy lecithin (SL) influenced lipid digestion in vitro and in vivo. Lipid digestibility was determined in vitro in oil-in-water emulsions using four different milk-derived emulsifiers or SL, and the ultrastructural appearance of the emulsions was assessed using electron microscopy. Subsequently, selected emulsions were added to a base diet and fed to preterm neonatal piglets. Initially, preterm pigs equipped with an ileostomy were fed experimental formulas for seven days and stoma output was collected quantitatively. Next, lipid absorption kinetics was studied in preterm pigs given pure emulsions. Finally, complete formulas with different emulsions were fed for four days, and the post-bolus plasma triglyceride level was determined. Milk-derived emulsifiers (containing protein and phospholipids from milk fat globule membranes and extracellular vesicles) showed increased effects on fat digestion compared to SL in an in vitro digestion model. Further, milk-derived emulsifiers significantly increased the digestion of triglyceride in the preterm piglet model compared with SL. Ultra-structural images indicated a more regular and smooth surface of fat droplets emulsified with milk-derived emulsifiers relative to SL. We conclude that, relative to SL, milk-derived emulsifiers lead to a different surface ultrastructure on the lipid droplets, and increase lipid digestion.

Milk Polar Lipids Reduce Atherogenic Lipoprotein Cholesterol and Attenuate Atherosclerosis Development in LDL-Receptor Knockout Mice Fed a Western-Type Diet (P06-051-19)

Objectives Purified milk sphingomyelin (SM), a polar lipid (PL) component of milk fat globule membranes, is protective against dyslipidemia in high-fat, diet-fed mice. However, it is unclear whether ingestion of the total milk PL fraction can have similar hypolipidemic effects as purified SM, and if this confers protection from inflammation and atherosclerosis. To determine this, LDLr-/- mice were fed high-fat, high-cholesterol diets with and without added milk PL from butter serum, a dairy co-product rich in PLs and SM. Methods Male LDLr-/- mice (age 6 weeks) were fed ad libitum either a high-fat, high-cholesterol diet (CTL; 45% kcal from fat, 0.22% cholesterol added by weight; n = 15), or the same diet supplemented with 1% milk PL (1% MPL; n = 15) or 2% milk PL (2% MPL; n = 15) added by weight from butter serum. All diets were adjusted to match in energy density, macronutrient composition, and calcium content. After 14 weeks on diets, mice were fasted for 6 hours prior to blood and tissue collection/processing at sacrifice. Results Supplementation with 2% MPL significantly reduced serum cholesterol (−51%) compared to CTL (p < 0.01), with fast protein liquid chromatography (FPLC) showing dose-dependent effects in lowering VLDL- and LDL-cholesterol. Serum CCL2 was 56% lower in 2% MPL compared to CTL (p < 0.05). No other significant differences were found in serum markers of inflammation or insulin resistance. Compared to CTL, 2% MPL-fed mice had significantly lower mRNA expression of Scd1 and Mip1b in liver, and Ccl2 in epididymal adipose tissue (p < 0.05). Notably, milk PL dose-dependently reduced atherosclerosis development in thoracic aorta, with 2% MPL significantly lowering neutral lipid plaque size by 59% (p < 0.01). Ccl2 mRNA expression was also significantly reduced by 67% in the descending aorta with 2% MPL (p < 0.05). Conclusions Milk PL strongly reduced atherogenic lipoprotein cholesterol in LDLr-/- mice fed a Western-type diet, and modestly lowered inflammatory markers in the serum, liver, adipose, and aorta. These factors likely contributed to the strong reduction in atherosclerosis development in thoracic aorta seen with 2% MPL. Milk PL content may be important to consider when choosing dairy products as food. Funding Sources This work was supported by a research grant to C. Blesso from the National Dairy Council.