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

Premature birth affects roughly 1 in 10 infants globally and 8.7% in Europe. While breast milk (BM) is the preferred enteral feed for preterm infants, very preterm and very low-birthweight infants often require fortification to meet gestation-specific nutritional needs. Historically, fortification used bovine-derived multinutrient fortifiers (CMDF), but concerns about nutritional limitations and risks (e.g., increased necrotizing enterocolitis, NEC) have driven interest in human milk-derived fortifiers (HMDF) as part of an exclusive human milk diet (EHMD). MOM is preferred; when insufficient, DHM is recommended by AAP and WHO, though processing of DHM (freezing, pasteurization) can attenuate bioactive proteins. This study aimed to analyze biochemical and bioactive characteristics of fresh and frozen MOM and pasteurized DHM, and to compare the effects of HMDF versus CMDF on macronutrients and key bioactive components (a-lactalbumin, lactoferrin, lysozyme, caseins, antioxidant activity), with a focus on feeding extremely preterm/ELBW infants.

- EHMD in NICUs has been associated with reductions in NEC, late-onset sepsis, bronchopulmonary dysplasia, retinopathy of prematurity, parenteral nutrition use, and length of stay compared with diets including bovine products. - When MOM is unavailable, DHM is recommended by AAP and WHO and is used by an estimated 800,000 infants annually; DHM offers benefits over bovine-origin preterm formula but exhibits reduced bioactive components due to processing (freezing, Holder pasteurization) which decreases lactoferrin, lysozyme, secretory IgA, and antioxidant capacity. - Freezing tends to better preserve bioactive proteins than pasteurization; Holder pasteurization retains macronutrients but reduces immunological bioactivity. - a-Lactalbumin comprises 20–25% of HM protein but is low in bovine milk; lactoferrin is abundant in colostrum and preterm milk and provides immunoprotective functions; lysozyme is part of innate defense facilitating protective microbiota. - Prior reports indicate DHM has lower macronutrients and bioactive proteins than MOM; fortification strategies, pasteurization methods (Vat vs UHT/retort), and handling can influence bioactivity. - Emerging work suggests milk source (MOM vs DHM) may have greater impact on the preterm microbiome than fortifier type (human vs bovine).

Design: Observational feasibility study assessing biochemical and immunochemical characteristics of HM with and without fortification. Specimens: 25 total samples comprising 22 HM-based and 3 commercial formulas. HM categories included: - Fresh MOM (FreMOM) from mothers of infants born at 24, 26, and 28 weeks’ gestation; collected at 7–10 days postnatal age; analyzed unfortified and fortified with HMDF or CMDF. - Frozen MOM (FroMOM) paired from the same mothers (stored 2–4 weeks at −20°C); analyzed unfortified and fortified with HMDF or CMDF. - Donor HM (DHM): one preterm (PTDHM) and one term (FTDHM) sample from a single milk bank, both Holder-pasteurized; analyzed unfortified and fortified with HMDF or CMDF. - Comparators: three commercial bovine-based formulas (two preterm formulas, one term formula). Sample procurement and handling: FreMOM and FroMOM collected across morning, afternoon, and night; triplicate analyses to account for diurnal variation. MOM expressed using hospital-grade electric pumps at the lactation center; 1:1 foremilk/hindmilk mixtures analyzed. DHM supplied by Western Trust Donor Milk Bank (Northern Ireland), transported frozen with cold-chain maintenance, stored at −20°C, thawed and warmed to 37°C prior to feeding. Holder pasteurization at the bank (62.5°C for 30 min). FreMOM transported to the laboratory within 2 hours or stored at 4°C overnight for morning transport. Fortifiers and mixing: - CMDF: bovine-origin powdered sachets; SMA (2 × 5 g to 100 mL HM) and C&G (2 g to 25 mL HM), added to fresh or thawed HM per manufacturers’ instructions. - HMDF: Humavant +6 (Prolacta Bioscience); produced from pooled donor BM, transported at −70°C and thawed before use; mixed at 15 mL HMDF to 35 mL HM. Analytical methods (all in triplicate): - Chemical composition: Total nitrogen by Kjeldahl (conversion factor 6.25) to compute protein; total solids by SMART-6; fat by ORACLE Universal Fat Analyzer; pH by digital pH meter. - Antioxidant activity (AA): DPPH radical scavenging assay; diluted milk (1:10) mixed 1:1 with DPPH reagent, incubated 30 min at 37°C, absorbance at 517 nm; results reported as percent AA. - Protein profiling: SDS-PAGE under reducing conditions using stain-free precast gels; bands stained with Coomassie and compared with molecular weight markers to identify major proteins (a-lactalbumin, lactoferrin, lysozyme, caseins). - Quantification of a-lactalbumin (a-LA) and lactoferrin (LF): RP-HPLC (Agilent 1260 Infinity) on Zorbax 300SB-C18 column; gradient elution with TFA/water and TFA/acetonitrile/water solvents at 0.8 mL/min, detection at 215 nm. Calibration with human a-LA and LF standards (0.156–5 mg/mL; R2 ≥ 0.992). Statistics: ANOVA using general linear model with Tukey’s test for pairwise comparisons; results as mean ± SD; significance at p < 0.05. Ethics and PPI: Approved by University of Limerick Hospital Group REC (No: 10/21). Written informed consent obtained. Patient and public involvement through the Irish Neonatal Health Alliance; DHM bank participated as collaborator.

- DHM vs MOM bioactivity: Pasteurized DHM (PTDHM, FTDHM) exhibited significantly lower lactoferrin and a-lactalbumin than fresh or frozen MOM (p < 0.05). LF in DHM was barely detectable (~0.03–0.04 mg/mL), whereas MOM at 24 weeks had the highest LF (fresh 1.38 mg/mL; frozen 1.22 mg/mL), decreasing with increasing gestational age. - Effect of HMDF fortification: HMDF reinstated and significantly increased a-LA and LF across MOM and DHM samples (p < 0.05) and yielded higher protein, fat, and total solids than unfortified or CMDF-fortified counterparts (p < 0.05). SDS-PAGE showed increased bands for a-LA, LF, lysozyme, and a- and b-caseins with HMDF addition. - Effect of CMDF fortification: CMDF did not contribute measurable amounts of key human bioactive proteins; it decreased net a-LA concentration (consistent with absence of a-LA in CMDF chromatograms and gels). CMDF modestly increased AA relative to unfortified samples, likely due to protein hydrolysates, but remained significantly lower than HMDF-fortified samples. - Antioxidant activity (AA): HMDF-fortified samples had the highest AA among HM groups (e.g., PTDHM+HMDF 67.66% ±1.28; FTDHM+HMDF 79.08% ±1.28; FreMOM-28w+HMDF 79.34% ±1.44; FroMOM-26w+HMDF 75.64% ±1.34). Unfortified MOM AA varied by gestation and processing: FreMOM-24w 26.90% ±1.7 (lowest), FreMOM-28w 62.92% ±1.19; freezing and pasteurization generally lowered AA compared with fresh MOM. Among formulas, AA varied widely: CBPTF-1 84.87% ±1.92 (highest overall), CBTF 34.87% ±1.74 (lowest among formulas). - Macronutrients and pH: Across specimens, pH ranged 6.38–6.87; freezing had no significant effect on pH (p > 0.05). HMDF significantly increased fat, protein, and total solids versus unfortified and often versus CMDF-fortified HM (p < 0.05). Examples: FTDHM+HMDF fat 5.87% ±0.06, protein 3.54% ±0.18, total solids 16.31% ±0.10 vs FTDHM unfortified fat 4.20% ±0.10, protein 2.30% ±0.10, total solids 12.43% ±0.20. FroMOM-26w+HMDF protein 4.50% ±0.10 and fat 5.96% ±0.13 with total solids 16.81% ±0.12. - Freezing vs pasteurization: Freezing (FroMOM) did not significantly reduce fat, protein, or total solids (p > 0.05) and preserved more bioactive proteins than pasteurized DHM; lysozyme was detectable in MOM and HMDF-fortified samples but not in DHM or CMDF. - Commercial formulas: HPLC and SDS-PAGE revealed absence of LF and a-LA in bovine-based preterm formulas; one term formula (CBTF) had a trace a-LA (1.28 mg/mL) but significantly lower than HM specimens; numerous small hydrophilic peptides in CMDF-fortified HM and formulas suggested protein hydrolysates. - Contextual clinical observation (unit-level): NEC cases in ELBW infants at the study hospital decreased from 3/49 over four pre-HMDF years to 1/44 over four post-HMDF years; the single post-HMDF NEC occurred after transition to CMDF at 34 weeks, not during HMDF exposure.

The study demonstrates that pasteurization of DHM substantially attenuates key bioactive proteins (lactoferrin, a-lactalbumin, lysozyme) and antioxidant capacity relative to MOM, while freezing preserves these components better than pasteurization. Adding HMDF to MOM or DHM reinstates and augments bioactive proteins and increases antioxidant activity and macronutrient content, aligning with the goal of optimizing nutrition and immunoprotection for ELBW/VLBW infants. In contrast, CMDF lacks major human-specific bioactive proteins and can dilute a-LA in fortified HM, offering only modest improvements in AA, likely via hydrolysate peptides. These findings address the research question by identifying a fortification strategy (HMDF) that counteracts the bioactivity losses introduced by DHM processing and enhances MOM’s protective properties. Clinically, this supports EHMD strategies—particularly early, enteral, exclusive use of HM fortified with HMDF—for extremely premature infants, potentially contributing to reductions in NEC and other morbidities reported in prior literature. The data underscore the importance of milk source and processing on bioactivity, and the potential for HMDF to restore functional proteins critical for gut immunity, oxidative stress mitigation, and overall host defense in the most vulnerable neonatal populations.

Pasteurized DHM has significantly reduced bioactive properties compared with fresh and frozen MOM. HMDF, unlike CMDF, contributes additional human bioactive proteins and enhances antioxidant capacity and macronutrients, effectively reinstating or augmenting bioactivity attenuated by DHM pasteurization. Frozen MOM retains more bioactivity than pasteurized DHM. For ELBW infants, freshly expressed MOM fortified with HMDF and delivered early, enterally, and exclusively (3E) appears optimal. Future work should pursue personalized fortification approaches, mechanistic studies using microbiome analyses, organoids, and artificial intestinal models to elucidate how MOM-derived bioactive components mediate immunological programming and clinical outcomes.

- Small number of gestation- and age-specific specimens. - Maternal characteristics (e.g., ethnicity, diet) that could influence MOM composition were not controlled. - Exact gestation and collection timing for individual DHM donations were not determined. - No in vivo or functional bioactivity assays were performed; analyses were qualitative/biochemical. - Alternative pasteurization methods and extended freezing durations were not evaluated. - The same MOM samples could not be paired across frozen and pasteurized states. - The set of bioactive proteins assessed was not exhaustive. - Human milk oligosaccharides, free fatty acids, and milk fat globules were not analyzed.