Recent analysis of the peptides generated during HM digestion has redefined our understanding of the neonatal digestive process. It has shifted from a model focused solely on amino acid release to one that accounts for the programmed generation of AMPs and immunomodulators [70]. Peptidomic and functional studies demonstrate that HM and IF exhibit vastly different immunological and peptide profiles despite similar macronutrient contents [71]. HM displays higher levels of IgA, IgG, and LF, and a predominance of β-casein-derived peptides with known bioactivity, whereas IF often exhibits heterogeneous bovine fragments altered by industrial processing [72]. In vivo characterization of the gastric peptidome in preterm infants has identified thousands of peptides, with a significant increase in the number of sequences showing AMP homology following digestion. This suggests that peptide generation depends on specific proteolytic kinetics and the unique neonatal enzymatic environment [73]. Key fragments of LF, caseins, and lysozyme have been shown to persist in the intestine, where they inhibit pathogens like E. coli and S. aureus while simultaneously promoting the growth of Bifidobacterium [74]. Furthermore, the digestion of colostrum releases bacteriostatic fragments effective against pathogens associated with NEC, such as Klebsiella aerogenes (K. aerogenes) and Citrobacter freundii (C. freundii) [8]. These findings highlight that neonatal digestion drives the digestion-microbiota-immunity axis, underscoring the need for formula innovation to consider structural architecture and release kinetics, not just total protein content (Figure 1).

The stability and safety of powdered infant formula (PIF) have historically been addressed through a microbiological control approach focused on intensive thermal processing. However, this paradigm has evolved into a more comprehensive perspective that simultaneously considers the nutritional quality, chemical stability, and biological functionality of the added components. In this context, storage conditions, industrial processing, and microbiological control systems remain key determinants of the safety of the final product. In formulations enriched with polyunsaturated fatty acids (PUFAs), lipid oxidation poses a significant risk after opening the package and exposure to oxygen, as evidenced by increased malondialdehyde levels at specific storage temperatures [75]. This phenomenon not only deteriorates nutritional quality but can also compromise the structural integrity and functional activity of the incorporated bioactive peptides.

In parallel, pathogen control remains a critical challenge. Traditional methods, such as ISO/TS 22964, have been instrumental in detecting C. sakazakii, but their limited sensitivity has prompted the adoption of molecular techniques, such as real-time PCR, which can detect low bacterial loads more quickly and accurately [76]. Despite these advances, the presence of microorganisms such as Mycobacterium avium subsp. paratuberculosis in commercial formulations highlights the need for more robust safety frameworks, especially due to their possible association with neonatal gut dysbiosis [77]. In addition, the persistence of resistant bacteria such as Bacillus cereus (B. cereus), even after reconstitution at temperatures above 70°C, reinforces the idea that safety cannot depend solely on thermal removal, but must also explain the intrinsic ability of the matrix to resist microbial colonization [76, 78].

In response to these limitations, innovative strategies based on biological barriers have emerged. In this regard, although bacteriophage therapy has demonstrated efficacy against specific pathogens and has received regulatory approval, AMPs derived from milk proteins such as LF and casein stand out as more stable and versatile alternatives [79, 80]. These AMPs are highly compatible with complex food matrices and align with current “clean label” trends. However, their functionality is highly dependent on processing conditions, as intensive heat treatments and chemical reactions such as the Maillard reaction can induce protein aggregation and reduce immune activity [81–83]. Although some degree of denaturation may facilitate initial digestion, the formation of pepsin-resistant aggregates may limit the effective release of bioactive sequences [82].

To address these challenges, the industry has begun to implement gentler technologies, such as membrane filtration and moderate heat treatments, which preserve key native proteins, including secretory IgA, lysozyme, and LF [84]. Semi-industrial pilot studies have shown that these approaches minimize protein denaturation without compromising microbiological safety, thereby optimizing the preservation of bioactive protein structures [85]. Overall, current evidence indicates that, although macronutrients remain stable during storage, precise control of processing parameters is essential to preserve the structural integrity and psychochemical pH stability of the incorporated AMPs throughout manufacturing and storage [86].

Within this framework, the integration of AMPs together with management systems such as hazard analysis and critical control points (HACCP) represents a dual strategy that not only provides direct antimicrobial action but also contributes to the ecological modulation of intestinal colonization. This approach reflects a shift from models focused exclusively on sterility to more complex systems that seek to balance safety, biological functionality, and nutritional quality in modern IFs (Figure 2).

The evolution of the IF market reflects a broader global dietary transition, in which sustained growth, particularly in middle- and high-income countries, has contributed to the displacement of breastfeeding [87]. This expansion has been accompanied by increasingly sophisticated marketing strategies that frequently employ terms such as “protection” and “immunity” to create a perception of functional equivalence with HM, often without comparable biological evidence [88]. While research on the cost-effectiveness of specialized formulas has demonstrated clear benefits in specific clinical contexts, these advantages remain context-dependent. For example, partially hydrolyzed whey-based formula (PHF-W) has been shown to reduce the risk of atopic dermatitis in high-risk infants, and extensively hydrolyzed formulas supplemented with Lacticaseibacillus rhamnosus (L. rhamnosus) GG (EHCF+LGG) are cost-effective in managing cow’s milk protein allergy [89–91]. However, these targeted applications do not justify their use as substitutes for breastfeeding in general.

In response to these trends, public health initiatives grounded in the International Code of Marketing of Breast-milk Substitutes emphasize that specialized nutrition should complement, not replace, breastfeeding, which remains the primary and irreplaceable strategy for neonatal immune protection. Despite these efforts, documented violations of the NetCode protocol persist across multiple countries, highlighting ongoing gaps between policy and practice [92].

Within this evolving landscape, the transition from the discovery of bioactive peptides to their implementation in IF represents a complex challenge that must balance industrial feasibility, clinical safety, and public health ethics. As outlined in previous sections, the bioactivity of milk-derived AMPs depends critically on their structural integrity, which is often compromised by conventional manufacturing and commercialization processes [22, 93].

The incorporation of advanced bioactives into IFs is therefore occurring within a market context that is not only expanding but also increasingly shaped by commercial and informational influences. IF sales have risen steadily in recent decades, often displacing breastfeeding, even though HM remains the biological gold standard due to its dynamic, synergistic composition of endogenous AMPs and immunomodulatory factors [87]. This contrast underscores a key limitation of current formulations: while individual components, such as recombinant LF or selected AMP sequences, can be added, they do not replicate the complex, multilayered matrix of peptides, oligosaccharides, and bioactive molecules present in HM.

Marketing dynamics further complicate this scenario. Evidence indicates that strategies integrated into healthcare systems, including academic sponsorship and the promotion of specialized formulas within medical settings, can significantly influence both professional recommendations and maternal decision-making, even when breastfeeding is a viable option [88]. From the consumer perspective, brand preference is shaped not only by product composition but also by digital marketing, social media, and medical endorsements [94]. For the infant as the end-user, this translates into an amplified exposure to claims centered on immune protection, concepts that are indirectly linked to the antimicrobial mechanisms discussed throughout this review.

The convergence of rapid market expansion and the strategic use of bioactivity narratives highlights the urgent need for robust regulatory oversight. Recent initiatives, such as the FDA’s Operation Stork Speed, launched in March 2025, aim to modernize IF regulation by increasing transparency and ensuring that claims of nutritional adequacy and functionality are supported by rigorous scientific evidence rather than marketing-driven assertions. Ultimately, regulatory frameworks must ensure that the incorporation of AMPs into IFs is positioned as a targeted clinical tool for vulnerable populations, rather than as a commercial strategy that contributes to the displacement of breastfeeding, which remains the cornerstone of neonatal health and immune protection.

The ultimate success of fortification strategies and technological innovations in IF depends not only on the preservation of AMPs during manufacturing and storage but also on their ability to remain biologically active after ingestion. While section Perspectives on AMP implementation and safety addressed the preservation of AMPs’ integrity during processing, storage, and microbiological control, their clinical efficacy ultimately depends on surviving gastrointestinal digestion and reaching biologically relevant target sites. In this context, bioavailability refers to the capacity of these molecules to resist premature degradation and exert local or systemic biological effects within the neonatal host [95].

Building upon the digestive kinetics discussed in section In vivo biosynthesis and digestive kinetics, current evidence confirms that milk-derived peptides can remain functionally active within the infant intestinal lumen. Research using direct peptide extraction from neonatal intestinal samples, coupled with high-resolution peptidomics, has identified specific fragments that exert a dual biological effect: direct inhibition of pathogens such as E. coli and S. aureus, and simultaneous stimulation of beneficial commensals such as Bifidobacterium longum (B. longum) subsp. infantis [74]. This selective ecological modulation is a hallmark of HM that researchers are now seeking to reproduce in IF matrices to reduce the risk of dysbiosis-associated complications, including NEC. For these peptides to exert systemic immunomodulatory or protective effects, they must overcome the proteolytic barriers of the stomach and small intestine and reach the epithelial surface intact [95, 96].

Importantly, experimental evidence indicates that a fraction of milk-derived peptides remains biologically available after digestion. In vitro models have demonstrated that naturally digested HM generates β-casein-derived fragments capable of crossing Caco-2 human intestinal epithelial cell monolayers, suggesting that a limited but physiologically relevant proportion of the milk peptidome can be absorbed transepithelially and enter systemic circulation [96]. Furthermore, experimental studies suggest that certain digestion-resistant fragments, particularly those derived from αS1-casein, may cross both the intestinal and blood–brain barriers, potentially contributing to neurodevelopmental signalling during critical stages of infant growth [97].

Translating these molecular findings into clinically meaningful outcomes remains a major priority. Recent real-world evidence (RWE) from 2025 has begun to bridge the gap between in silico peptide design and pediatric application. Longitudinal studies involving infants aged 6–12 months have demonstrated that formulas fortified with LF at concentrations resembling those found in HM are associated with significantly improved linear growth and feeding tolerance. Microbiota analyses revealed enrichment of Bifidobacterium breve and butyrate-producing taxa, including Faecalibacterium and members of the Ruminococcaceae family, resulting in a microbial profile that more closely resembles that of exclusively breastfed infants. These findings suggest that the incorporation of bioactive peptides into IF may contribute to a partial functional approximation of selected components of the HM ecosystem [98].

By ensuring that these molecules are not only present but also bioavailable and biologically active after ingestion, next-generation IFs may provide a complementary biological support system capable of promoting immune maturation, microbial homeostasis, and healthy growth in formula-fed infants (Figure 3).

Key unresolved issues include the long-term safety of chronic exposure to bioactive peptides during early life, particularly regarding immune tolerance and unintended immunostimulation [99]. Dose standardization remains a major challenge, as peptide activity is highly dependent on concentration, stability, and delivery matrix. Additionally, potential off-target effects, including disruption of commensal microbiota, require careful evaluation.

Regulatory frameworks also differ significantly between regions. While the U.S. FDA has initiated modernization efforts (e.g., Operation Stork Speed), the European Food Safety Authority (EFSA) maintains stricter requirements for functional claims and safety validation. Harmonization of these regulatory pathways will be essential for global implementation [100].