Human Milk as a Model for Next-Generation Infant Formula: Opportunities and Challenges

Abstract

Human milk (HM) is a dynamic biological system replete with functional components that support infant growth, development, and immune protection. HM is regarded as the optimal infant food; however, many infants rely on infant formula (IF) as their sole source of nutrition or as a supplement to HM. Although many ingredients have been added to IF over the past 30 y, it is not equivalent to HM in terms of composition or infant outcomes. Potential opportunities to improve IF composition include adding components extracted from the milk of other species, synthesized by precision fermentation, or produced by cultured mammary epithelial cells. However, many hurdles remain to adding novel ingredients to IF, including determining how much to add to best align with variable HM composition, ensuring reasonable certainty of no harm, considering possible functional effects of the ingredients, establishing methods to assess the safety of the IF when the functional ingredient is added, and determining how best to assess functional outcomes. Advancing the field to improve infant health hinges on achieving consensus across a range of questions among academic, industry, and regulatory scientists.

Statements of significance.

There is a critical need to improve the quality of infant formula (IF) for the many infants and their caregivers who rely on it to meet their nutritional needs. This work presents an integrated perspective from authors and nonauthor contributors from a tripartite coalition, the Infant Nutrition Science Coalition, comprising researchers from academia, industry, and government, on the current opportunities provided by emerging technologies to enhance IF’s similarity to human milk, the challenges to the incorporation of novel ingredients, and the need for consensus building to drive improvements in IF.

Introduction

Human milk (HM) is a complex biological fluid that has evolved >250 million years to provide nourishment to the developing infant [1]. Exclusive HM feeding is recommended for the first 6 mo of life because of HM’s nutritive and functional components [[2], [3], [4]]. HM feeding is linked to reduced morbidity and mortality compared with infant formula (IF) feeding [5,6], including reduced risks of ear infections, gastrointestinal (GI) illnesses [7,8], hospitalization [9], becoming overweight or obese later in life [[10], [11], [12]], developing asthma or diabetes [7], and improved markers of cognitive development [[13], [14], [15]]. Currently, there are compositional differences between IF and HM, which contribute to observed differences in health outcomes.

Although HM feeding offers numerous benefits, only 25% of United States infants meet the guidelines for exclusive HM feeding for the first 6 mo of life [16]. The low rates of exclusive HM feeding result from a wide array of issues, including low milk supply; insufficient paid family leave and workplace policies for time and space to pump; and a lack of standardized prenatal lactation education and postpartum support [[17], [18], [19]].

When infants do not receive HM, IF must support growth and development as the sole source of nutrition until complementary foods are introduced [20]. Although HM is the gold standard, improved IF could lead to better health outcomes for infants and families that rely on IF to meet their nutritional needs. IF is required to include an array of essential nutrients [21]. Although IF remains the only safe, regulated, and clinically studied alternative to HM, the observed differences in health outcomes between HM-fed and IF-fed infants likely stem from differences in the composition, structural complexity, and functionality of milk components. Consequently, there remains a critical need to align the composition and functionality of IF with those of HM to help bring developmental outcomes for IF-fed infants closer to those of exclusively HM-fed infants. Ideally, IF would provide a composition and functionality similar enough to HM so that the differences in health outcomes between HM-fed and IF-fed infants are reduced or no longer observed. Approximately 23% to 32% of infants receive mixed feeding, consisting of both IF and HM, in the first year of life [22], and realizing improvements to IF could also lead to health benefits for these individuals.

HM contains thousands of components in a complex matrix that exert functions beyond basic nutrition. Components whose primary function is to provide energy to the growing infant are often referred to as “nutritive components” or “nutrients,” whereas components with primary roles outside the provision of energy are referred to as “bioactive components” or “functional components” [23]. Bioactive components have been defined as components that “affect biological processes or substrates and hence have an impact on body function or condition and ultimately health” [23]. Some components of milk have multiple effects on the human body, contributing to the complexity of HM. For example, the milk fat globule membrane (MFGM) serves functional roles in the infant GI tract but also contains metabolizable lipids [24], and some proteins in milk have bioactive properties as well as nutritive value [25]. Examples of bioactive components in HM include bioactive proteins [e.g., Igs, lactoferrin (Lf)], enzymes (e.g., lysozyme and bile salt-stimulated lipase), peptides, hormones, carbohydrates [including >200 HM oligosaccharides (HMOs)], lipids, extracellular vesicles, leukocytes, and bacteria that are not found in IF [26] (Figure 1). Moreover, HM components are delivered in a complex matrix of supramolecular structures that exert multifactorial functions on infant health and development [26]. For instance, lipids in HM are packaged into complex milk fat globules (MFGs) [27], casein proteins form micelles [26], and extracellular vesicles contain other components, such as microRNAs [28]. Some of these structures are normally absent from IF because they are not commercially available or are destroyed by processes used in IF production from non-HMs (e.g., heat treatment, homogenization, and drying to powdered form) [29].

FIGURE 1.

Top row left box: Human milk (HM) is a complex matrix of components and serves as the gold standard for infant nutrition and the model for the future development of IF. Top row middle box: Incorporating components from HM to improve IF is challenging due to variability in HM composition and other factors. Top row right box: New technologies have created opportunities for the incorporation of new components in IF to increase similarity with HM. Bottom row left box: The Infant Nutrition Science Coalition was developed as a tripartite organization to improve IF. Bottom row right box: Proposed strategy to realize improvements to IF through the incorporation of novel components. IF, infant formula; 2′-FL, 2′-fucosyllactose; 3′-SL, 3′-sialyllactose; 3FL, 3-fucosyllactose; 6′-SL, 6′-sialyllactose. Created in BioRender. CS (2025). https://BioRender.com/9u9hj0n.

To narrow gaps in infant outcomes, scientists look toward components in HM that are lacking or insufficient in IF, which may contribute to the observed differences in health and development between HM-fed and IF-fed infants. IF composition has been modified over the years through the addition of novel ingredients to bring it closer to that of HM. Examples include the addition of arachidonic and DHA for brain and eye development [30,31], bovine MFGM for cognitive development [32], carotenoids for ocular and cognitive development [33], nucleotides for immune function [34], bovine Lf for immune support [35], bovine α-lactalbumin to reduce total protein needed for growth and possibly enhance GI tolerance [36,37], HMOs and probiotics for GI and immune health [38,39]. There are additional challenges and opportunities to add components to IF to make it more similar to HM and bridge the outcome gaps [40,41] (Figure 1). Herein, known differences between HM and IF are presented by calling attention to specific components of HM that are generally absent from IF. Components were categorized based on macronutrient class (proteins, lipids, or carbohydrates), as it affects their physiological roles as well as technological considerations.

A barrier to making IF more like HM is the incomplete understanding of HM composition and the significance of individual components to its overall functionality. For instance, the diverse components of HM exhibit variability in concentration across stages of lactation. Interestingly, this challenge could also lead to an opportunity to design a range of IFs that more closely follow the lactational stage (for instance, colostrum, transitional, and mature milk). Additionally, the potential interactions or synergistic effects among functional components of HM have not been fully characterized (Figure 1). As HM composition varies among lactating parents, over time and across populations, it is challenging to determine reference ranges for the numerous components of HM, let alone optimal values for novel additions to IF [5,42]. Furthermore, there are challenges to ensure that novel additions to IF are safe. Demonstrating positive impacts of novel ingredients on infant development is particularly challenging because there is a lack of standardized methods to track infant development beyond basic anthropometrics: the ideal reference ranges for the developing immune system, microbiome, metabolism, bone, muscle, and brain have not been fully defined [20].

The objective of this manuscript was to provide the perspective of an array of both author and nonauthor contributors from a tripartite consortium of academics, industry members, and government researchers called the Infant Nutrition Science Coalition on 1) the known compositional differences between HM and IF; 2) how new components that increase similarity of IF to HM can be obtained or produced; 3) the remaining challenges to incorporating novel components into IF; and 4) key scientific questions that require further consensus-building to enable improvements to IF (Figure 1).

Section 1. Opportunities: Known Differences between HM and IF

Proteins

Although IF is designed to mimic the nutritional profile of HM, protein content and composition differ between HM and IF, and protein structure and function are sometimes affected by the processing used in IF manufacturing. IFs have different whey-to-casein ratios, which can affect protein digestibility, gastric emptying [43], and metabolism in the infant [44]. The ratio of whey-to-casein in HM is dynamic across lactation: 90:10 in early lactation to 60:40 in late lactation [45]. The dynamic changes in the total protein content and whey-to-casein ratios are often not reflected in IF.

Beyond these considerations, the composition of proteins, especially those with bioactivity, also differs between HM and other animal milks. Proteins in IF are typically obtained from bovine or caprine (goat) milk, which contain some proteins homologous to those in HM. For example, bovine milk Lf and HM Lf have ∼70% sequence homology [46]. These proteins differ in post-translational modifications, such as glycosylation [47]. These structural differences between proteins in HM and the milk of other species can result in distinct functional effects [48]. For example, researchers quantified the inhibitory effect of Lf against 2 neonatal pathogens (Staphylococcus epidermidis and Escherichia coli) and found that HM Lf exhibited significantly greater inhibitory effect on bacterial growth than bovine Lf [48]. Kim et al. [49] found that HM Lf had a greater capacity to survive hydrolysis during gastric digestion than bovine Lf. Moreover, plant-based IFs (such as those that use soy proteins) lack any homologous proteins to HM.

Osteopontin is a highly glycosylated and phosphorylated protein found in HM at a concentration of ∼130 mg/L, and the bovine form of osteopontin is typically much less abundant in IF (∼10 mg/L) [50,51]. Several bioactive effects of osteopontin have been previously described, including effects on immune activation [52], wound healing [53], angiogenesis [54], bone development [55], and effects on the microbiome [56]. Although bovine milk osteopontin is commercially available, it differs somewhat in amino acid sequence and post-translational modifications from HM osteopontin and, therefore, may have differences in bioactivity. A randomized clinical trial examining a formula supplemented with bovine osteopontin in a cohort of 80 term infants found that osteopontin modified the serum cytokine profile of infants, making it more similar to that of HM-fed infants, increased the proportion of circulating T cells, and reduced the incidence of fever relative to infants fed a nonsupplemented formula [57,58]. The processing necessary for IF production can impact the structure and function of bioactive proteins [59]. Some milk proteins are denatured or lose their native structure after the multiple heat-based pasteurization steps commonly used for IF preparation [60,61].

Carbohydrates

The carbohydrate content of HM and IF also differs substantially [62,63]. The most abundant nutrient in HM by weight is lactose (75 g/L), which serves as the primary energy source for infants. Although most standard IFs contain 100% lactose as the carbohydrate source, some IFs contain maltodextrins, corn syrup solids, sucrose, and reduced concentrations of lactose [64]. One study found that 59% of IFs purchased in the United States contained a nonlactose carbohydrate such as maltodextrin or sucrose [65], and better aligning the carbohydrate composition of IF with HM may involve removing such components. The use of reduced lactose formulas is increasing in the United States despite congenital lactose intolerance being a very rare condition [66,67]. It has been theorized that transient lactose intolerance underlies infantile colic, but lactose-reduced IFs have not consistently been shown to be beneficial [67]. In recent years, concerns have been raised that consumption of lactose-reduced IFs during infancy may be a risk factor for childhood obesity [66,68].

HM contains a high concentration of HMOs (a wide array of complex, soluble carbohydrate structures), most of which are absent from IF. More than 200 HMO structures have been identified in HM [69], with total concentrations ranging from 7 g/L in mature milk to 23 g/L in colostrum [[70], [71], [72]]. HMO variability between individuals is largely dependent on genetics, with the activity of 2 specific enzymes (α1-2-fucosyltransferase and α1-3/4-fucosyltransferase, encoded by Secretor and Lewis genes, respectively) influencing the biosynthesis of fucosylated HMOs in the mammary gland [73]. Maternal diet can also influence HMO composition [74,75]. HMOs are not digested by human enzymes and reach the colon intact, where they can be fermented by specific members of the GI microbiome [76]. HMOs influence infant GI microbiome development, specifically enriching beneficial GI microbes, including several Bifidobacterium species [[77], [78], [79], [80], [81]]. More recently, Clostridium spp. and other early life colonizers have also been shown to metabolize HMOs, in turn producing a range of beneficial products that can suppress pathobiont growth and pro-inflammatory cytokine concentrations [82]. Furthermore, HMOs exhibit immunomodulatory properties and can act as decoys by binding to pathogen receptors, thereby inhibiting pathogen-mediated infections [83]. Some HMOs are positively associated with neurodevelopment, especially sialylated HMOs, which are a source of sialic acid, an important component of neurotransmitters [84,85].

Manufactured HMOs, referred to as human-identical milk oligosaccharides (HiMOs), are structurally and chemically identical to their HM counterparts [2]. HiMOs were first introduced to IF in 2010, but they are typically present at lower concentrations than in HM [86] and represent only a small number of the complex array present in HM [87]. Only 15 HMOs represent >75% of the total oligosaccharide abundance in HM by concentration [71,86,88,89]. Therefore, representing their functions in IF may not require including every structure. Currently, 7 HiMOs have been approved for incorporation in IF in the United States that together represent ∼45% of the HMOs in HM by weight [89]. Numerous clinical trials have demonstrated that formulas supplemented with HiMOs are safe, well-tolerated, support normal infant growth, and promote gut microbiota profiles that more closely resemble those of HM-fed infants [[90], [91], [92], [93]]. Studies have investigated IF supplemented with a single HMO such as 2′-fucosyllactose [94,95], a binary mixture of HMOs consisting of 2′-fucosyllactose in combination with lacto-N-neotetraose [96,97], and a blend of 5 HMOs [[98], [99], [100]]. Further research is needed to define the optimal levels and combinations of HMOs necessary to improve outcomes of IF-fed infants.

Lipids

IF and HM also have some differences in their lipid structure and composition. HM lipids are packaged as MFGs, supramolecular complexes with a nonpolar triglyceride core surrounded by the MFGM. This trilamellar structure is rich in bioactive phospholipids, sphingolipids, cholesterol, and membrane-bound proteins, such as butyrophilin, mucin-1, xanthine oxidase, and lactadherin. MFGM supports lipid transport in aqueous environments and has bioactive roles [101]. Mounting evidence suggests that bioactive components of MFGM support brain development, GI maturation, and immune education [102]. MFGM delivers structural lipids and proteins that aid in membrane organization, signaling, and neurodevelopment [103]. Non-HMs also contain complex MFGM structures (albeit nonidentical to HM MFGM) that can be used to supplement IF. A recent meta-analysis found that bovine milk-derived MFGM fortification of IF improved executive function, such as attention, memory, and problem-solving, relative to unfortified IF, but had no effect on language, motor, or social-emotional domains [32]. The selective cognitive benefits may relate to bioactive lipids and proteins that influence myelination and synaptic signaling, especially in prefrontal cortical regions. Bovine MFGM-enriched IF shows potential metabolic benefits, including improved cholesterol profiles and fatty acid oxidation markers [104], lower concentrations of insulinogenic amino acids and increased markers of ketogenesis [105], resembling HM-fed infants. However, it is also common for IF to contain no MFGM, creating a very different product from HM.

Although fatty acid profiles are extremely variable and reflect, in large part, the lactating parent’s diet, genetics, and health, the overarching lipid composition of HM is likely tailored for infant absorption and metabolic needs [106]. Notably, ∼25% of the fatty acid pool within the MFG is palmitic acid, with approximately half in the stereospecifically numbered 2 (sn-2) position of the triacylglycerol, which improves absorption and reduces insoluble calcium soaps in the GI tract—a feature less well replicated in vegetable oil-based IFs [107]. Some IFs contain structured lipids with palmitic acid in the sn-2 position. Such structural differences affect calcium absorption, stool consistency, and bone mineral content [108,109], highlighting the importance of designing formulas that mimic HM lipids [110,111].

HM contains both arachidonic acid (ARA), a long-chain omega-6 fatty acid, and DHA, a long-chain ω-3 fatty acid, with ARA typically exceeding the concentrations of DHA, with a worldwide mean ratio of 1.5:1, ARA:DHA [112,113]. ARA (and its derivatives) and DHA are the most predominant long-chain fatty acids in the infant brain [31,114,115]. Early studies showed that over the first 6 mo of life, HM-fed infants accumulate twice as much DHA in their brains compared with infants fed IF without DHA and that HM-fed infants accumulate DHA in the rest of the body, whereas infants fed IF without DHA lose total body DHA [[114], [115], [116]].

Although humans can synthesize ARA from its precursor, linoleic acid, and poorly synthesize DHA from its precursor, α-linolenic acid, the conversion rates are even more limited during infancy [117]. Several clinical trials have demonstrated that the addition of ARA and DHA to IF improves visual acuity and neurodevelopmental outcomes, especially when they are included at HM levels and ratios [30,115,[118], [119], [120], [121], [122], [123]]. DHA is a mandatory ingredient in IF in the European Union (European Food Safety Authority, 2014). It remains an optional IF ingredient in the United States and other regions, but when DHA is added, at least an equal amount of ARA must also be added [21,124]. Many groups of experts recommend the mandatory addition of both ARA and DHA to IFs to help close the neurodevelopmental gaps observed between HM-fed and IF-fed infants [31,117,125].

Opportunities to reduce differences between HM and IF

In summary, despite improvements in IF, many differences in IF and HM composition remain, particularly in bioactive components. Improving the similarity of IF and HM composition by incorporating components homologous or identical to HM proteins, HMOs, MFGM, and fatty acids, including palmitic acid in the sn-2 position, could likely improve health outcomes for IF-fed infants.

Section 2. Opportunities: Technologies to Improve IF

Milk source

The application of several innovative technologies has the potential to improve IF by addressing compositional and functional differences between IF and HM. Bovine milk inherently contains components that, if isolated, would be considered bioactive novel ingredients with relatively high similarity to HM, and therefore, bovine milk has traditionally been the primary source of bioactive ingredients for IF. Additionally, a growing body of research highlights the potential of other non-HMs, including caprine (goat), camel, and donkey, as sources of bioactive components. A recent analysis of market trends reported that bovine milk-based IFs comprised 78% of the global market, with soy-based formulas holding 10% and caprine milk-based formulas comprising 5% [126]. Emerging research has identified a range of promising bioactive ingredients in various milks that could offer functional and health benefits in IF. For example, caprine milk is used as a milk base for IF and, depending on the breed, has a casein composition more similar to HM than bovine milk [127]. HM casein is predominantly (∼90%) composed of β-casein, which makes up ∼30% of bovine milk casein but ∼60% of caprine milk casein [128]. A caprine milk-based IF was shown to have more similar protein digestive kinetics to HM than a bovine milk-based IF [129,130] in part because it produces a softer curd from β-casein compared with α_s1-casein [127,131]. Additional ingredients that could be extracted from caprine milk in the future include bioactives like Lf, which has a similar extent of sequence homology with human Lf as bovine Lf (both 70%) and an N-glycan profile with fewer glycan compositions that differ from HM compared with bovine milk [132]. Compared with other milks, camel milk contains a wide variety of oligosaccharide species structurally identical to those present in HM, which could serve as a source for IF [133]. Additionally, camel milk could serve as a source of bioactive milk proteins, as it is high in α-lactalbumin, Lf, and thermostable Igs [134,135], although it should be noted that the Ig profile of non-HMs differs substantially from that of HMs [136]. Because of the lack of β-lactoglobulin and lower concentrations of α_s1-casein in camel milk, it may be an option for children with bovine milk protein allergy specific to these proteins [137]. Donkey milk has also been studied for bovine milk food protein-induced enterocolitis syndrome and was found to be well-tolerated in a small study of children [138], although more research is needed. Donkey milk also contains high concentrations of lysozyme and Lf [135]. Extracting bioactive components from bovine and other milks could help bridge the gap between IF and the functional properties of HM [126].

Milk processing

Although opportunities exist to develop IF with components extracted from bovine and other species’ milk, milk-based ingredients for IF typically require several processing steps to ensure safety, make their composition more like HM, and enhance shelf life [139]. IF ingredients are typically exposed to heat treatments to enhance safety prior to the IF production process. After individual ingredients are combined to create IF, the mixture is pasteurized to reduce the microbial load by ≥5-log CFU/mL, followed by homogenization to form a stable emulsion. This emulsion may be evaporated to lower water content before spray drying [140]. These heating steps, both before and during the IF production process, may lead to modifications including denaturation, aggregation, and glycation of milk components, especially proteins [[141], [142], [143]].

IF component modifications could impact the digestion and ensuing physiological outcomes of the resulting formula. For example, glycation reactions reduce protein digestibility and amino acid absorption [144]. Using in vitro digestion, researchers found that the digestibility of proteins in heat-sterilized liquid IF (treatment temperature >110°C) was 4 to 11% lower than the same products exposed to a milder heat treatment (60°C) [145]. Cattaneo et al. [60] found that ∼90% of advanced glycation end products in IF were attributed to the high-temperature sterilization process. Additionally, many bioactive proteins are heat sensitive, so pasteurization may induce their denaturation or aggregation, reducing their functional potential [146]. Reducing processing-induced modifications while preserving the nutritional quality and achieving product safety is challenging [147]. Short-term improvement in the quality of IF can thus be achieved through 1) using ingredients that have undergone as minimal heat processing as possible and 2) limiting heating during production to the minimum required to achieve a safe product. In addition to optimizing heat treatment processes, there are opportunities to use new technologies to isolate components to improve IF composition. Emerging technologies such as high-pressure processing and ultraviolet light processing are promising alternatives to thermal treatments that could ensure the safety of IF while preserving the activity of bioactive components [[148], [149], [150]].

Milk component concentration

Several components present in HM are also present in bovine and other types of milk, but do not end up in IF in the same quantity as present in HM, such as specific proteins (e.g., α-lactalbumin, β-casein, and Lf). Different methods exist to isolate these proteins, including membrane filtration and chromatographic separation [[151], [152], [153]], which can facilitate their addition to IF. Another more complex structure that can be isolated from bovine milk is the MFGM. MFGM can be obtained from various dairy streams (e.g., whey, buttermilk, and β-serum) via several unit processes, including precipitation and filtration [154,155]. MFGM, Lf, and other bioactive proteins that have functional roles in IF are typically heat sensitive. For these heat-sensitive bovine milk-based ingredients, dry blending may be applied to avoid further heat treatment during IF production [139]. By combining different isolation techniques with fine-tuning of heating processes, IF can be further optimized using technologies already available.

Novel ingredient production systems

Beyond extracting compounds from non-HMs, bioactive components can now be produced via cell culture systems and recombinant production in microbial, plant, or animal hosts. Recombinant technology can be used to produce proteins matching the amino acid sequence of HM proteins, such as Lf, in nonhuman species (i.e., microorganisms, plants, and other animals) that could be incorporated into IF [156]. When this process applies to microorganisms, it is often referred to as precision fermentation, defined as the use of engineered microorganisms to produce food ingredients, such as proteins, fats, flavors, pigments, and vitamins [157,158]. To date, recombinant technology has been used to produce a select variety of HMOs (including 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3′-sialyllactose, and 6′-sialyllactose) approved for use in IF. Although yeast and bacteria [159] are the most common hosts for recombinant production, numerous other host species have been explored, including rice [160,161], transgenic cows [162], transgenic pigs [163], chicken eggs [164], and goats [165].

Mammary epithelial cell-based HM production is an emerging field, with steadily improving in vitro models of lactation. Traditionally reliant on 2D cultures, the field has evolved to include mammary epithelial cell organoids, which are 3D culture systems that have key features of mammary epithelial organization and function [[166], [167], [168], [169], [170]]. In addition to primary mammary epithelial cells isolated from reduction mammoplasties, cells isolated noninvasively from HM have also emerged as a promising alternative source [171,172]. In parallel, a range of engineered platforms such as hydrogels, decellularized extracellular matrices, and bioprinted scaffolds have been introduced to support secretory activity in vitro [168,170,171]. Currently, there is no direct evidence that any of the existing mammalian cell culture models secrete milk proteins, lipids, HMOs, or immune components at physiologically relevant amounts. Accurately reconstructing the multicellular architecture of the mammary epithelium, including the spatial coordination of luminal and basal epithelium with surrounding matrix and stromal cells, will be essential for mimicking directional secretion and hormonal responsiveness. Sustained secretion will also require advances in long-term maintenance and cell expansion, ideally in serum-free media. Finally, to scale this technology into viable tools for milk production, it will be essential to develop integrated systems that enable perfusion, real-time collection, and external modulation of lactogenic stimuli.

Opportunities in technologies overall

In summary, various technological advancements, such as approaches to reduce thermal load during processing, novel extraction techniques from various non-HMs, mammary epithelial cell culture, and recombinant technology, can be used to translate discovery science on the composition and systems biology of milk into meaningful improvements to IF.

Section 3. Challenges: Implementing Changes in IF Composition

Implementing changes to IF remains challenging despite advances in understanding HM and in novel technologies to improve IF with components more like HM. Many challenges can prevent or delay novel IF ingredients from reaching the market, including determining the optimal concentration for inclusion, verifying the safety of the ingredient alone and in the IF, and addressing scientific uncertainty about how to evaluate the safety of components with functional effects. Beyond what is needed to bring products to market, there is a need for further research from the academic community evaluating the functional impact of these added ingredients on infant health outcomes.

Selecting novel components for IF

One major challenge is selecting which components present in HM to add to IF. Components selected for inclusion in IF should be present in HM and have research demonstrating possible functional benefits to the infant. Although milk is the product of millions of years of evolution, this suggests that every component must benefit the infant; some components may not benefit the infant and thus may not be useful additions to IF. Some components of HM may be present primarily to benefit the lactating parent rather than the infant [173]. Some components of HM that benefit the lactating parent include serotonin [174] and a bioactive whey peptide (feedback inhibitor of lactation) that prevents oversecretion of milk [175], parathyroid hormone-related protein, which modulates calcium resorption in the lactating individual [176,177], and leukocytes that help protect the mammary gland from infections [172]. Of course, a demonstrated function in the mammary gland does not preclude it from having additional functions in the infant. Regardless, it cannot be assumed that the primary function of every component in HM will lead to a primary and/or measurable benefit in the infant; selection of novel ingredients for inclusion in IF should be guided by studies demonstrating the functional benefits of these molecules in the infant or infant models.

Selecting a concentration for novel components in IF

Another challenge in adding specific components to IF is determining the ideal concentration to match HM, as HM composition can vary widely whereas IF has a static recipe with a defined concentration of all ingredients. HM component concentrations change over time postpartum (including across stages of lactation) [23,178], time within a day (including with circadian rhythm), and time within a single feed [179,180]. HM composition also varies across individuals, geographical regions, environmental factors (including diet), genetics, disease status, and body composition of the lactating parent [42,181]. For instance, concentrations of vitamins (e.g., thiamin, riboflavin, niacin, vitamin B6, vitamin C, and vitamin A) can be influenced by their concentrations in the lactating parent’s diet [27,182]. Recent efforts have constructed comprehensive databases for each HM nutrient and how they vary across these factors (e.g., time of lactation, diets, and parent genetics) [183]. Aside from cases of lactating parent malnutrition or dietary exclusion (e.g., veganism) resulting in milk with insufficient concentrations of essential nutrients, there are gaps in our knowledge regarding whether this variation is evolutionarily important and whether it impacts infant health.

In the United States, IF must provide essential nutrients at or above appropriate minimum levels to meet the needs of infants from 0 mo to 12 mo [21]. These levels have been established by the scientific community through a comprehensive evaluation of the nutritional needs of this population. Updated evaluations of nutritional needs for IF are likely needed based on more recent literature. Similar to essential nutrients, the appropriate concentration of bioactive ingredients to be supplemented into IF should also be determined through a comprehensive evaluation of scientific evidence, including taking into account the concentrations in HM and the amount needed to achieve the intended functional effect [124].

Determining whether a component is beneficial alone or in conjunction with other components

Another challenge is determining whether a bioactive ingredient will be beneficial on its own or requires the presence of 1 or more other components to be functional. The addition of a single HM-like component to IF may not result in functional benefits because the bioactive component may only function, or function best, in synergy with other HM components [26,181]. Some HM components interact with each other and are only beneficial when other components are present. For example, studies show that IF supplemented with DHA, but not ARA, may result in decreased brain accumulation of ARA, leading to suboptimal neurodevelopment and potentially undesirable effects on immune system development and growth [31]. However, fortification of IF with both DHA and ARA to more closely resemble levels and ratios in HM led to improved outcomes of cognitive development, visual acuity, and immune response [184]. Many interactions between HM components and their functional consequences likely remain unknown. Researchers should continue to identify how multiple HM components interact, which can inform IF manufacturers on the design of IF.

Safety testing

Evaluation of the safety of novel ingredients for IF involves both consideration of the safety of the ingredient on its own and when incorporated into the IF product. The safety of the ingredient is assessed using established approaches for evaluating food ingredient safety, including characterization of the ingredient composition and information from relevant safety studies. Additionally, IF products that contain a novel ingredient must be evaluated in a clinical study prior to being sold to consumers, and the evaluation must include a growth-monitoring study to ensure the absence of adverse events.

Verifying the safety of the ingredient

The safety of each novel ingredient must be verified prior to inclusion in IF [185]. FDA has established guidance on the evaluation of the safety of food ingredients [185,186], but because of the unique nature of the infant population, additional studies may also be required to verify the safety of novel HM-like ingredients intended for use in IF. In 2004, the Institute of Medicine (IOM, now the National Academy of Medicine) published guidance on how to evaluate the safety of IF ingredients [187]. Many elements of safety assessments recommended by the IOM for evaluating novel ingredients intended to be added to IF are standard for all food ingredients, such as characterizing the ingredient and the standard, validated safety studies referenced in the FDA guidance. The IOM guidance establishes a tiered approach to evaluate the safety of IF ingredients, where any safety concerns identified in the initial tier of safety studies must be investigated by a second tier of studies that provide more specific evaluations of that endpoint, and, if questions remain, clinical studies may be required.

The evaluation of the safety of IF ingredients requires special consideration of the infant population, and the IOM guidance could serve as a basis for discussion on how to evaluate ingredient safety. It may also be helpful to re-evaluate the relevance of these recommendations in the context of how the science of HM, IF, and food ingredient safety assessment has evolved since its publication in 2004. For example, traditional toxicological studies have primarily utilized animal models, but United States- and European Union-based regulatory agencies have prioritized developing nonanimal methods to help evaluate safety. These new approach methodologies include in silico (based on computer simulations) and in vitro (cell models) methods to predict the effects of substances on organisms. An evaluation of how the safety of IF ingredients can be assessed should consider whether new approach methodologies can be leveraged. Novel approaches are continually evolving, suggesting a need for periodic reassessment of their uses in the overall context of IF safety testing [188].

Considerations for safety specific to bioactive compounds

Novel bioactive ingredients that provide functional effects and may ultimately bring health outcomes for IF-fed infants closer to those of HM-fed infants are being evaluated by researchers globally. However, the current regulatory frameworks for food ingredients (including IF ingredients) cannot consider these potential benefits when evaluating safety. Although efficacy and risk-benefit analyses are not part of the safety determination for ingredients intended for use in food, many novel ingredients for consideration for addition to IF are being considered because of their bioactivity/functionality. The IOM panel recommended that the safety dossier for novel ingredients for IF include at least a rationale for the addition of the ingredient [187]. They also note that many novel IF ingredients would be considered bioactive.

Understanding the mechanisms through which an ingredient elicits a physiological effect is important for evaluating safety. Traditional toxicology studies include endpoints to assess the impact on a broad range of physiological effects, which have been determined to be relevant for both infant and noninfant populations [189]. Guidance exists to identify cases where additional studies may be necessary to address the infant population [190,191]. However, some areas of scientific uncertainty remain regarding whether and what type of additional safety testing would be recommended for bioactive IF ingredients.

Safety of ingredients within the IF matrix

IF with added novel ingredients are required by regulation to be evaluated in an infant clinical study, prior to being placed on the market [185,187]. The standard growth-monitoring study used to evaluate IF clinically includes anthropometric growth measures (e.g., weight, length, and head circumference) [20,192], and the study must demonstrate noninferiority of the IF with the novel ingredient.

Considerations for IF with bioactive ingredients

Beyond standard growth-monitoring studies, further clinical studies and/or endpoints may be needed to assess the safety and the purpose (suitability) of the IF with a bioactive ingredient. For example, the addition of a functional component could have impacts beyond the standard anthropometric measures when added to an IF. Although assessing the benefit of that function within the context of the IF is not currently part of the regulatory process, assuring that function does not lead to harm would need to be evaluated as part of that process. Beyond that, assessing the variety of benefits a compound has within an IF has great interest from the perspective of gaining insights into infant nutrition. However, given the variety of potential functional effects of HM components, safety considerations must be made on a case-by-case basis to ensure that the appropriate outcomes are being considered. Herein, it is recommended that the function of the novel ingredient should guide the endpoints selected for these additional studies. For example, if a bioactive is antimicrobial, preclinical or clinical endpoints should be designed to evaluate the impact of the IF with that ingredient on the GI microbiome. Likewise, if the functional impact of the ingredient is on the immune system, then the effect of that component within the IF should be measured using suitable markers of immune development [193]. There is currently no consensus on what testing would be needed for the specific functional category, including intermediate and clinical endpoints. Likely, the selection of endpoints to measure should also be guided by endpoints observed in preclinical models. Scientists need to come together to create a consensus on these questions.

Moreover, there is currently no consensus on how long the effect of an IF with a novel ingredient should be monitored for effects across the lifetime. The IOM recommends a 3-stage in-market surveillance approach [187], and IF manufacturers are required to maintain records of any complaints associated with an IF [185]. Along with the IOM’s recommendation, in-market surveillance should be a required validation process, but experts need to reach a consensus on how such a program would be executed and whether it would supplement or replace the requirement for monitoring complaints.

Assessing ingredients for function beyond safety

Current regulations require demonstration of safety (including growth outcomes) for novel ingredients to be added to IF, not functional outcomes. Demonstration of benefits, however, is important to inform the consumer or infant caregiver of the value of the added ingredient and to help close the functional gap between HM and IF. Researchers are currently investigating bioactive compounds that could be incorporated into IF. It is the role of academic experts to guide industry on which beneficial HM components are lacking in IF, and it is the role of industry to develop HM-identical components and to add to IF with consideration of both safety and feasibility.

The functional impact of many novel HM-like ingredients added to IF may not be identifiable via growth studies. For example, prebiotics are not digested by the infant but rather are important for the growth of certain bacteria that produce key essential micronutrients [194] and short-chain fatty acids [195]. As these components would not necessarily demonstrate benefits in growth-monitoring studies, other study designs or expanded outcomes within such studies would be needed to establish functional benefits in infants.

As some added components may exhibit synergistic interactions that modulate their biological effects, academic scientists may need to design studies to determine the impact of each novel ingredient added to IF alone and in combination with other components.

Additional considerations

Scaling novel technologies

Some challenges are unique to novel production technologies. Precision fermentation, for example, could accelerate the development of functional candidate proteins for inclusion in IF, but scaling production systems from laboratory to commercial levels can be challenging [196]. Commercial-scale production of novel components must be feasible and at a cost point that allows inclusion in an IF that infant caregivers can afford.

Acceptability of novel formula components by infant caregivers

Another challenge for the inclusion of HM-like components produced via precision fermentation or recombinant technology is that infant caregivers may be hesitant toward adopting these products [197]. Caregivers may hold unfavorable perceptions of novel foods if the innovation is perceived as risky or if there is not a clear benefit [198]. Using a cross-sectional online survey of United States caregivers of infants (n = 436), researchers found that 32% of the modified IF purchased (compared with standard IF) was labeled “non-genetically modified organism (GMO)” or “organic” [199]. Recent research highlighted how framing and representative heuristics may overcome acceptability concerns or negative perceptions of novel foods by shifting innate risk perception [198].

In conclusion, enhancing infant health and nutrition by improving IF requires a global, interdisciplinary effort. In this work, we have proposed a roadmap for realizing improvements to IF that consists of more in-depth characterization of HM across diverse populations, improved translational frameworks to evaluate how candidate bioactives influence clinically relevant outcomes, innovations in ingredient manufacturing and processing technologies, and collaboration between scientists in clinical and preclinical research, industry, and regulatory roles. More fundamental scientific research on HM composition and function is needed. More research is needed to identify the functional components of HM and how they support normal infant functional development beyond simple anthropometric growth indicators, as well as to assess the variability between individuals and across different stages of lactation. Additionally, it is imperative to continue advocating for greater lactation support to prolong HM-feeding. Insights from studies characterizing the beneficial aspects of HM are necessary to identify components that could be targeted for inclusion in IF, as well as to establish reference ranges for their level of inclusion. Furthermore, novel technologies, such as specialized extractions of compounds from bovine and nonbovine milk sources and production of HM milk components via precision fermentation or mammary epithelial cell culture, should be used to produce IF that is more like HM, both compositionally and functionally. However, there are challenges in ensuring that novel additions to IF are safe, present in the appropriate quantities, and retain their functionality during processing. Within safety assessments, there are unresolved questions about how a potentially functional component should be evaluated for safety. The specific functional category of the ingredient may help guide which additional preclinical and clinical studies are needed to evaluate safety. Although not required for regulatory approval, the scientific community should assess the functional impact of added novel ingredients on infant health and development.

Our perspective is that scientists, government officials, and industry members should collaborate to determine a consensus on an array of questions to make progress in the use of novel ingredients and technologies to further improve IF, including:

• 1.What are the key compositional and functional differences in bioactive components between IF and HM?
• 2.How do you establish normative or reference ranges for functional HM components responsible for healthy infant development, given the interindividual variability in HM composition?
• 3.Given the potential synergistic interactions among HM components, how can IF be designed in the context of the knowledge that HM is a complex biological system?
• 4.Should novel HM-like ingredients with possible function be examined via additional tests to verify safety beyond traditional toxicology methods, and if so, how?
• 5.Should IF with added novel ingredients with possible function be evaluated for safety metrics beyond traditional growth noninferiority studies? If so, what additional measurements/metrics are needed to determine the impact of novel IF additions appropriately?

Innovation in IF has led to improved products and tangible improvements in infant health; however, continued development is needed. Panels of interdisciplinary experts should reach consensus on the questions enumerated above to ensure that scientific research drives the development of future IF products. Uniting academic, industry, and regulatory experts is necessary to resolve the health outcome differences between HM-fed and IF-fed infants.

Author contributions

The authors’ responsibilities were as follows – MRBR, CS, SMD, JTS, JHK, CJS, DK, GR, KH, EDK, KKS, SMR, GPR, MKM, CR, DCD: wrote the paper. MRBR, CS, CR, DCD: had primary responsibility for final content; and all authors: read and approved the final manuscript.

Declaration of Generative AI and AI-assisted technologies in the writing process

The author(s) declare that no generative AI or AI-assisted technologies were used in the writing of this manuscript.

Funding

This work was partially funded by the Infant Nutrition Science Coalition (INSC), which includes financial contributions from Abbott, Arla Foods Ingredients, Bobbie, ByHeart, Cargill Inc., DSM-Firmenich, Exponent, Fonterra, FrieslandCampina, Harmony Baby Nutrition, Helaina, Hilmar, Kabrita North America, Mead Johnson Nutrition, Munchkin, Nara Organics, Novonesis, and Spherix Consulting Group. These specific sponsors did not have a role in writing the report or place any restrictions on its final content. Some members of the writing team have positions in companies that sponsor the INSC, including ByHeart, Kabrita North America, and Nara Organics. These authors did not have undue control of the report writing, and all authors fully approved of the final report prior to submission for publication. The end result of this report reflects the opinions of all authors. No academic authors were compensated for their contribution to this manuscript. The Oregon State University authors were funded solely to facilitate the writing process. This work is supported, in part, by the Oregon Agricultural Experiment Station with funding from the Hatch Act capacity funding program, award numbers NI25HFPXXXXXG022 and NI25HMFPXXXXG029, from the USDA National Institute of Food and Agriculture (CS); salary support from NIH R01 DK138032 and R01 HD112396 (SMD); and salary support from R01HD097367, R01HD109193, and R01HD106140 (DCD).

Conflict of interest

DCD reports financial support, administrative support, article publishing charges, travel, and writing assistance were provided by Infant Nutrition Science Coalition. SMD reports a relationship with Danone North America that includes: board membership; reports a relationship with National Dairy Council that includes: board membership; reports a relationship with Arla Food Ingredients that includes: consulting or advisory; reports a relationship with DSM that includes: speaking and lecture fees; reports a relationship with Nestlé Nutrition Institute that includes: speaking and lecture fees; and reports a relationship with Perrigo that includes: speaking and lecture fees. JTS reports a relationship with International Milk Genomics Consortium that includes: board membership. CJS reports a relationship with Nestlé Nutrition Institute that includes: speaking and lecture fees. DK reports a relationship with ByHeart that includes: employment. KH reports a relationship with Tamarack Biotics that includes: board membership; reports a relationship with NewMilkLab NV that includes: board membership; reports relationship with Kate Farms that includes: board membership; and reports a relationship with Ausnutria B.V. that includes: speaking and lecture fees. EDK reports a relationship with Kabrita North America that includes: employment. SMR reports a relationship with Rev Bioscience that includes: equity or stocks. GPR reports a relationship with Nara Organics that includes: employment. MKM reports a relationship with Danone North America that includes: consulting or advisory. JHK reports a relationship with Fresenius Kabi that includes: funding grants; reports a relationship with Medela that includes: consulting or advisory; reports a relationship with Infant Biotherapeutics that includes: consulting or advisory; reports a relationship with Carag that includes: consulting or advisory; reports a relationship with Mother’s Milk is Best that includes: consulting or advisory; reports a relationship with Alcresta that includes: consulting or advisory; and reports a relationship with Nicolette that includes: equity or stocks. All other authors report no conflicts of interest.