Premature birth affects a significant global population, and while breast milk is ideal, fortification is often necessary for optimal nutrition in very low-birth-weight infants. Historically, cow's milk-derived fortifier (CMDF) was standard, but concerns about its limitations and potential risks have led to the exploration of human milk-derived fortifier (HMDF) and exclusive human milk diets (EHMD). EHMD, comprising MOM or pasteurized DHM fortified with HMDF, is associated with reduced risks of NEC, sepsis, and other complications. However, the specific bioactive components responsible for these benefits remain unclear. DHM, while a valuable alternative to MOM, undergoes processing that may attenuate its bioactive properties. This study aimed to analyze the biochemical and bioactive characteristics of various HM feeding options used for extremely preterm and extremely low-birth-weight infants at the University Maternity Hospital Limerick (UMHL), the first NICU in Ireland to establish an EHMD, to understand the differences in bioactivity between MOM and DHM and evaluate HMDF's role in enhancing these components.

The literature highlights the benefits of human milk (HM) for preterm infants, emphasizing the importance of early and exclusive HM feeding. However, the need for fortification in extremely low-birth-weight (ELBW) infants has led to the use of both CMDF and, more recently, HMDF. Studies have shown that EHMD offers several clinical advantages, including a reduction in NEC, sepsis, and other morbidities. However, the processing of DHM, such as pasteurization, can reduce the concentration of essential bioactive proteins and immunomodulatory components. Existing research indicates variations in bioactive proteins in HM due to maternal factors, collection methods, and processing techniques. This study builds upon previous findings by investigating the effect of HMDF on the bioactivity of HM in various forms.

This observational feasibility study analyzed the biochemical and immunochemical characteristics of HM specimens with and without fortification. Researchers analyzed FreMOM (freshly expressed mothers' own milk), FroMOM (frozen mothers' own milk), and DHM (both preterm and full-term) supplemented with either HMDF or CMDF. Samples were collected from UMHL, with FreMOM collected at various times of day to account for diurnal variation, and DHM obtained from a single human milk bank. Analyses included macronutrients, pH, total solids, antioxidant activity (AA), α-lactalbumin (α-LA), lactoferrin (LF), lysozyme, and α- and β-caseins. The study included 25 milk specimens representing various combinations of milk source and fortifier, along with commercial preterm and term formulas for comparison. Analyses used techniques such as the Kjeldahl method, DPPH assay for AA, RP-HPLC for protein analysis, and SDS-PAGE for protein profiling. Statistical analysis involved ANOVA and Tukey's test.

DHM showed significantly lower concentrations of lactoferrin and α-lactalbumin compared to fresh and frozen MOM. HMDF supplementation significantly increased protein, fat, and total solids in all HM specimens. HMDF significantly increased antioxidant activity (AA) compared to unfortified and CMDF-supplemented samples. RP-HPLC and SDS-PAGE analysis revealed that HMDF significantly increased the levels of α-LA, LF, and lysozyme in both MOM and DHM, while CMDF had little to no impact on these bioactive proteins. Frozen MOM retained better bioactivity compared to pasteurized DHM. CMDF showed some increase in AA, likely due to its protein hydrolysate content, but significantly less than HMDF.

The findings highlight the superior bioactive properties of HMDF over CMDF in fortifying HM for extremely premature infants. The significant increase in key bioactive proteins (α-LA, LF, and lysozyme) and antioxidant activity with HMDF supplementation demonstrates its potential to improve the nutritional and immunological benefits of HM, particularly DHM, which loses bioactivity during processing. The results support the notion that freshly expressed MOM fortified with HMDF provides the most optimal nutritional composition for ELBW infants, especially during the early postnatal period. The lower bioactivity observed in pasteurized DHM emphasizes the need for fortification strategies to enhance the immunological protection provided.

Pasteurized DHM has reduced bioactive properties compared to fresh and frozen MOM. HMDF, unlike CMDF, significantly enhances the bioactive components in HM, reinstating those lost during pasteurization. Freshly expressed MOM fortified with HMDF is recommended as an optimal EHMD for very premature infants. Future research could focus on personalized fortification strategies and investigate the detailed mechanisms through which specific HM components influence infant health.

The relatively small sample size, lack of control over maternal characteristics, limited information on DHM donation details, absence of in-vivo bioactivity measurements, and the exclusion of certain HM components (e.g., oligosaccharides) are acknowledged limitations. The use of only one type of HMDF and one brand of CMDF could also limit the generalizability of findings.