TABLE 2.

Multifunctional bioactivities of isolated milk components supplemented to infant formula.

Milk Component and [concentration] (ref)	Functional Outcome
	Growth/Tolerance	Infection	Immune Function	Neurocognitive	Microbiome	Serum Metabolites
HMOs
•2′FL [0.2 or 1.0 g/L] + galacto-oligosaccharides (GOS) to 2.4 g/L •4 month intervention •BF reference [136,137]	Growth of 2′-FL supplemented infants not different than SF [136]	NR	•2′FL reduced circulating pro-inflammatory cytokine concentrations and their secretion by isolated PBMC stimulated with RSV compared with SF; levels not different than [137]	NR	NR	•2′FL detected in blood and urine of BF infants and those fed formula + 2′-FL [136]
•2′FL [1.0 g/L] and LNnT [0.5 g/L] •6 month intervention •Follow-up at 12 mos [138,139]	•HMO-supplemented infants had softer stool and fewer nighttime wake-ups at 2 mo of age [138]	•Reduced infectious episodes in the HMO-supplemented group [138] •Fewer parental reports of bronchitis through 4, 6, and 12 mo and LRTI through 12 mo [138] •Reduced parent-reported antipyretics use through 4 mo and antibiotics use through 6 and 12 mo [138]			•Microbial alpha and beta diversity of the HMO group was closer to that of the BF than control formula [139]
•5 HMO: LNT, 2′FL, 3′FL, 3′SL, 6′SL •Total HMO concentration of 5.75 g/L •4 month intervention [141]	•No differences in growth or tolerance between infants fed SF or formula + 5 HMO
Lactoferrin
•Term infants fed formula with native bLF [102 mg/L] or added bLF [850 mg/L] for 12 mo [146]		•Reduction of URTI and wheezing (P < 0.05)				•Higher hematocrit levels at 9 mos (37.1% vs 35.4%; P < 0.05) in bLF-supplemented than CON
•Term infants fed formula or added bLF [38 mg mg/L] for 3 mo (105)		•Lower respiratory-related illnesses and occurrences of diarrhea-related illnesses BF and bLF groups than CON (P < 0.05)
•Preterm, VLBW infants •Multicenter, randomized, double-blind, placebo-controlled trial •Orally dosed with bLF [100 mg/d, LF]; LGG [6×109 CFU/d [151] bLF+LGG]; or placebo (CON) •Birth to 30 d of life [151, 152]	•No adverse effects or intolerances to treatment occurred [151]	•Incidence of ≥ stage 2 NEC and of death-and/or ≥ stage 2 NEC was lower in LF (p =0.055) and LF+LGG (p<0.001) vs. CON •Incidence of LOS due to bacterial or fungal infection was lower in LF (p=0.002) and LF+LGG (p<0.001) vs. CON (109)		•
•Preterm, VLBW infants <32 wk GA •Multicenter (37 centers), randomized, double-blind, placebo-controlled trial •Orally dosed with bLF [150 mg/kg/d, LF]; or sucrose [CON] •≤ 72 hours postpartum to 34 wk PMA [153]		•316 (29%) of 1093 LF infants acquired a LOS vs 334 (31%) of 1089 CON infants •Risk ratio adjusted for minimization factors was 0.95 (95% CI 0.86-1.04; p=0·233).
•Preterm infants weighing 500-2000 g •Multicenter (3 centers), randomized, double-blind, placebo-controlled trial •Orally dosed with bLF [200 mg/kg/d, LF]; or sucrose [CON] •8 week intervention •Follow-up at 24 mo [154]	•Growth outcomes and rehospitalization rates during the 2-year follow-up were similar in both groups	•LOS or sepsis-associated death occurred in 22 LF infants (10.5%) vs 30 (14.6%) CON •No difference after adjusting for hospital and birth weight; hazard ratio 0.73 (95% CI, 0.42-1.26). •At 24 mo, LF infants had less bronchiolitis than CON (rate ratio, 0.34; 95% CI, 0.14-0.86).		•Mean age-adjusted normalized Mullen composite score at 24 mo was 83.3 ± 13.6 in the LF group vs. 82.6 ± 13.1 in CON (N.S.).
•LF (0.6 g/L +MFGM (5 g/L) for 1 y study •Follow-up at 1.5 y [156]	•No difference in growth vs. CON	•Respiratory-associated adverse events and diarrhea were significantly lower for the MFGM + LF group through 1.5 y (p<0.05)		•Bayley II mean cognitive (+8.7), language (+12.3), and motor (+12.6) scores were higher (P< 0.001) for the MFGM + LF group vs. CON at 1 y •Differences no longer present at 1.5 y
MFGM
•MFGM protein fraction twice daily added to weaning food •6- to 11-month-old infants [158]		•Global prevalence of diarrhea was 3.84% in MFGM vs 4.37% in CON (P < 0.05). •MFGM reduced episodes of bloody diarrhea by 46% (P = 0.025)
•Formula with or without added MFGM fed for 4 mos •MFGM proteins constituted 4% (wt:wt) of the total protein content •∼2-6 month-old infants •Follow-up at 12 mo •BF reference [63, 64, [159], [160], [161], [162], [163]]	•No effect of MFGM on growth or tolerance vs. CON [169] •At 6.5 y of age, no differences between MFGM and CON in weight, length, or head or abdominal circumference [163]	•Lower incidence of diarrhea, otitis media and anti-pyretic use vs. CON [159, 160]		•At 12 mo of age, Bayley cognitive score was higher (P = 0.008) in the MFGM (105.8 ± 9.2) than CON (101.8 ± 8.0), but was not different than BF (106.4 ± 9.5; P = 0.73) [159]. •At 6.5 y of age, no differences between MFGN and CON in any measure of cognitive or adaptive functioning [163]	•Little effect on fecal microbiome between MFGM and CON [63] •MFGM fed infants had a lower abundance of oral Moraxella catarrhalis than CON [161].	•MFGM reduced fecal lactate, succinate, amino acids and their derivatives vs. CON [63] •Infants fed MFGM had higher levels of fatty acid oxidation products in serum than CON [64] •Plasma lipidome of infants fed MFGM differed at 4 mo (SM and PCs) and 6 mos (SM, PCs, ceramides) vs. CON [162] •Erythrocyte SMs, PEs and PCs differed between MFGM and CON at 6 mo [162] •At 6.5 y of age, no differences between MFGM and CON plasma concentrations of homocysteine, lipids, insulin, or glucose [163]
Osteopontin
•Double-blind RCT •Formula with 0 (CON), 65 (F65) or 130 (F130) mg/L bovine OPN for 6 mos •BF reference [165,167,168]	•Growth patterns, formula intake, sleep patterns and adverse events were similar in all formula-fed groups [167]	•Infants fed OPN had significantly fewer days with fever than CON [167]	•Infants fed OPN had serum TNF-α and higher IL-10 than CON (124) •Infants fed F130 had higher T-cell proportions than F0 or F65 [168]			•At 4 and 6 mo, plasma human OPN was higher in BF, F65, and F130 than CON [165] •Plasma bovine OPN in F130 was greater than F65 [165]
LC-PUFA
•Double-blind RCT (DIAMOND STUDY) •Term infants, n=∼40 per group •Compared 4 formula containing 0.64% AA and either 0% (CON), 0.32%, 0.64% or 0.96% DHA for 12 mo [180]				•Any level of DHA compared with CON (0% DHA) improved: •Visual acuity in infants fed at 12 mo, but not 3 mo [180] • Cognitive development through 6 y of age [181] •Brain ERP responses and synchronization during a task requiring response inhibition at 5.5 y [182] •Brain structure, function and metabolism at 9 y [183]
•RCT •Preterm infants <33 wk GA and 750-1800 g) •Compared CON formula with formula containing 0.26% DHA and 0.42% AA from either fish/fungal or egg/fish oil sources •Body composition measured by DXA [185]	•No significant differences among the 3 groups at any time point in weight, length, or head circumference or bone mineral content or density. •Greater (p<0.05) lean mass and reduced fat mass in infants fed formula with LC-PUFA at 12 mo
•Subset of term infants in the DIAMOND STUDY •Measured growth outcomes from 6 y. •A limitation is the small sample size at 6-year follow-up (n=18-24/group) [186]	•Compared CON formula, children who consumed LC-PUFA supplemented formula had higher length-/stature- and weight-for-age percentiles, but not BMI percentile from birth to 6 y •Maternal smoking predicted lower stature (2-6 y), higher weight-for-length (birth-18 mos) and BMI percentile (2-6 y) independent of LC-PUFA effects. •Gender interacted with the effect of LC-PUFA on stature, and the relationship between smoking and BMI, with a larger effect for boys.

2′FL, 2′-fucosyllactose; 3′SL, 3′-sialyllactose; 3′FL, 3′-fucosyllactose; 6′SL, 6′-sialyllactose; BF, breastfed; CON, control; GOS, galacto-oligosaccharides; GA, gestational age; HMOs, human milk oligosaccharides; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LOS, late onset sepsis; LC-PUFA, long-chain PUFA; LRTI, lower respiratory tract infection; MFGM, milk fat globule membrane; NR, not reported; OPN, osteopontin; PC, phosphatidylcholines; PMA, postmenstrual age; RSV, respiratory syncytial virus; SM, sphingomyelins; SF, standard formula; URTI, upper respiratory tract infection.