Exclusive Human Milk Diet for Extremely Premature Infants: A Novel Fortification Strategy That Enhances the Bioactive Properties of Fresh, Frozen, and Pasteurized Milk Specimens

**Background:** Human milk (HM) fortification is recommended for very low-birthweight infants, but traditional cow's milk-derived fortifier (CMDF) lacks bioactive and immune components. An exclusive human milk diet (EHMD) using human milk-derived fortifier (HMDF) may reduce necrotizing enterocolitis (NEC), late-onset sepsis, and other complications. This study analyzed the bioactive components of fresh and frozen mother's own milk (MOM) and pasteurized donor human milk (DHM), each supplemented with either HMDF or CMDF.

**Methods:** An observational feasibility study analyzed 25 milk specimens: 3 fresh MOM samples from mothers of infants born at 24, 26, and 28 weeks gestational age (collected 7–10 days postnatal); paired frozen MOM specimens from the same mothers; 2 DHM specimens (one preterm, one term); and 3 commercial bovine-based formulas. Each HM specimen was analyzed unfortified and with CMDF or HMDF. HMDF (Prolacta Bioscience) was added at 15 mL HMDF to 35 mL HM. CMDF from two Irish manufacturers was added per manufacturer instructions. Specimens were analyzed for pH, fat, protein, total solids, antioxidant activity (DPPH method), α-lactalbumin, lactoferrin, lysozyme, and α- and β-caseins using Kjeldahl method, SMART-6/ORACLE analyzers, RP-HPLC, and SDS-PAGE electrophoresis. Statistical analysis used general linear model and Tukey's test for pairwise comparison.

**Key Results:** DHM exhibited significantly lower (p<0.05) lactoferrin (0.03–0.04 mg/mL) and α-lactalbumin concentrations compared with fresh and frozen MOM. HMDF-fortified specimens showed significantly higher protein (3.49–4.50%), fat (4.01–5.96%), and total solids (14.89–16.81%) compared with unfortified and CMDF-supplemented specimens. HMDF addition increased antioxidant activity by almost twice that of unfortified specimens in samples with low baseline AA (e.g., FreMOM 24 weeks: 26.90% unfortified vs. 63.07% with HMDF; FroMOM 28 weeks: 37.74% unfortified vs. 61.15% with HMDF). HMDF reinstated lactoferrin and α-lactalbumin in DHM, whereas CMDF did not add measurable bioactive proteins. CMDF-fortified specimens showed various new peptides in the hydrophilic region (protein hydrolysates) and complete absence of α-LA and LF. None of the bovine-based formulas showed identifiable lactoferrin. MOM from 24-week gestation infants had the highest lactoferrin content (1.22–1.38 mg/mL), significantly decreasing with increasing gestational age (p<0.05). Lysozyme was only detected in MOM (fresh and frozen) and HMDF samples, not in DHM or CMDF. The pH values ranged from 6.38 to 6.87, with freezing showing no significant effect (p>0.05).

**Clinical Implications:** Pasteurized DHM has significantly reduced bioactive properties compared with fresh and frozen MOM. CMDF confers the least additional bioactive components. HMDF can reinstate bioactive proteins (lactoferrin, α-lactalbumin, lysozyme) that are attenuated through pasteurization of DHM and can augment these levels in MOM. The study's corroborating clinical observation of a very low NEC rate at the University Maternity Hospital Limerick (3 NEC cases among 49 ELBW infants in the 4-year pre-HMDF period vs. 1 case among 44 ELBW infants in the 4-year post-HMDF period) supports potential clinical relevance. The authors recommend freshly expressed MOM fortified with HMDF given early, enterally, and exclusively (3E) as the optimal nutritional choice for extremely premature infants. Limitations include small sample size, uncontrolled maternal characteristics, and lack of in-vivo bioactivity measurements.