Key Biologically Active Components of Breast Milk and their Beneficial Effects

Abstract

Maternal breast milk is the penultimate nutritional source for term and preterm neonates. Its composition is highly complex and includes multiple factors that enhance the development of nearly every neonatal organ system leading to both short- and long-term health benefits. Intensive research is focused on identifying breast milk components that enhance infant health. However, this research is complicated by the significant impact of maternal factors and the processing of pumped breast milk on bioactive ingredients. Optimizing enteral nutrition is particularly important for preterm neonates who miss the transplacental acquisition of nutrients in the third trimester of pregnancy and are at risk for illnesses associated with gut barrier dysfunction, including sepsis and necrotizing enterocolitis. In this review, we will discuss the health benefits of breast milk and its bioactive components.

Introduction

Human breast milk is the preferred and recommended nutrition for all infants because of its diverse array of bioactive components, which provide a multitude of health benefits.¹ At birth, neonates transition from receiving transplacental nutrition to an extrauterine environment that requires their immature intestine to meet the requirements for proper growth and development while serving as a barrier to billions of gut microorganisms. Breast milk contains a complex milieu of ingredients that aid the neonate in this challenging transition (Figure 1). However, maternal factors, as well as processing, sterilization, and storage, can significantly impact breast milk composition (Figure 1). Therefore, intensive research has focused on identifying the critical components of breast milk that enhance its beneficial effects, which would allow for the enrichment of these components in donor breast milk or formula. Optimizing enteral nutrition is particularly important for preterm neonates who are highly vulnerable due to critical illness, intestinal immaturity, microbial dysbiosis, and often an inadequate maternal breast milk supply.

Figure 1: Important bioactive components in human milk and detrimental exogenous factors.

Maternal breast milk contains a complex milieu of components that positively impact infant health. These include lactoferrin, fatty acids, cytokines, lipases, microbes, human milk oligosaccharides (HMOs), antibodies, and growth factors. Breastfeeding delivers these components directly to the infant. However, infants cannot breastfeed for numerous reasons, including prematurity, critical illness, and an inadequate supply of maternal breast milk. Storage, processing, packaging, pasteurization, and delivery of breast milk via a feeding tube can negatively impact its composition and bioactivity. Figure made with Biorender.com.

In this manuscript, we will review the benefits of maternal breast milk for term and preterm neonates with a brief discussion on the role of breast milk in preventing the intestinal disease necrotizing enterocolitis (NEC). We will also provide an overview of breast milk composition and its key bioactive ingredients.

Benefits of breast milk

The American Academy of Pediatrics published an update to their policy, "Breastfeeding and the Use of Human Milk," in 2022. This policy recommended exclusive breastfeeding for six months and support for continued breastfeeding until two years of age.¹ The health benefits of an exclusive human milk diet for infants have been extensively documented and include but are not limited to a reduced risk of otitis media,² diarrheal illness,^3–5 respiratory infections,^{3, 4, 6} Sudden Infant Death Syndrome,^{7, 8} necrotizing enterocolitis (NEC), ^9–12 and mortality.^{13, 14}

Breast milk and the prevention of NEC

Necrotizing enterocolitis (NEC) is a multifactorial, potentially fatal intestinal disease that primarily occurs in very low birth weight (VLBW, <1,500 grams) preterm infants. In the United States, the incidence of NEC is ~8–9% in infants with birth weights from 400–1,000 grams born between 22–28 weeks gestation.¹⁵ The administration of breast milk feeds has been repeatedly demonstrated to protect against the development of NEC.^9–12 A 2020 meta-analysis by Altobelli et al. reviewed clinical trials and observational studies that evaluated the impact of breast milk, pasteurized donor milk, or preterm formula feedings on the risk of NEC in preterm infants.⁹ The authors concluded that there was a reduced risk of NEC for human milk (maternal and donor) versus formula (RR:0.62, 95% CI: 0.42–0.93) and for human milk (maternal and donor) versus a mixture of feeding types (formula and human milk) (RR:0.74, 95% CI: 0.63–0.91).⁹ The role of breast milk in preventing NEC is supported by the success of quality improvement initiatives demonstrating that increased provision of breast milk is associated with decreased rates of NEC.^16–18

The pathogenesis of NEC involves a complex interplay of factors that remain an area of intensive research. Breast milk feeds are a highly effective way to reduce the risk of NEC, but the specific components responsible for this protective effect remain incompletely defined. In vitro mechanistic studies and animal models support a role for epidermal growth factor (EGF)-mediated inhibition of signaling via the innate immune receptor Toll-like receptor 4 (TLR4),¹⁹ human milk oligosaccharide (HMO)-associated enhancement of intestinal perfusion,²⁰ and binding of intestinal bacteria by Immunoglobulin (Ig) A.²¹ Breast milk likely prevents NEC through a combination of these effects and others by supporting a healthy microbiome, attenuating inflammation, promoting intestinal repair, and enhancing intestinal development. Specific breast milk components and their influence on health and disease will be discussed below.

Breast milk components

Breast milk composition is neither static nor immutable. It varies between feeds, time of day, and stage of lactation (colostrum, transitional, and mature). Maternal and environmental factors, geographical variance, and gestational age also affect breast milk composition.^22–24 Finally, storage, handling, and method of milk delivery to the infant can directly impact its stability and bioactivity (Figure 1). Below, we will discuss some of the most abundant and well-studied breast milk components.

Lipids

Lipids, in the form of fat globules, are the most abundant component of human milk, representing ~4% of milk content and 50% of its energy provision.^{25, 26} However, content is highly variable even within a single feeding, with foremilk having a lower lipid concentration than hindmilk.^{26, 27} Human milk also contains lipase, which aids in the digestion of these lipids.²⁸ This is particularly important for neonates who have a relative pancreatic insufficiency and inadequate lipase production, which gradually improves with age.^{29, 30}

Breast milk lipids are important in numerous biologic processes, including cell membrane synthesis, intracellular signaling, organ development, and the regulation of inflammation.²⁶ Critical components of the breast milk lipidome are the essential fatty acids linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3 n-3). These fatty acids are precursors to the highly bioactive long-chain polyunsaturated fatty acids (LC-PUFAs), including arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3). Most of these LC-PUFAs are acquired during the third trimester of pregnancy, leading to the rapid development of a deficit in preterm neonates.^{31, 32} LC-PUFAs are particularly important in brain, retinal, and immunologic function and development.^{33, 34} Clinical trials providing enteral supplementation of LC-PUFAs have had mixed results.³⁵ For example, a clinical trial of enteral DHA supplementation found an increased risk of the lung disease bronchopulmonary dysplasia (BPD) in preterm neonates that received DHA supplementation versus placebo.³⁶ In contrast, a trial supplementing ARA and DHA found a decreased risk of severe retinopathy of prematurity and no changes to the risk of BPD with the supplemented versus the control group.³⁷ This is a complex area of research with more studies needed to determine the optimal ratios, quantities, timing, and duration of LC-PUFA provisions to maximize clinical benefits.

Human milk oligosaccharides

Human milk oligosaccharides (HMO) are a large family of structurally diverse glycans consisting of greater than 100 different members.³⁸ They are highly abundant in breast milk, with levels only exceeded by lipids and lactose.³⁹ HMOs are prebiotics that are not digested by human intestinal enzymes. Instead, they are metabolized by intestinal bacteria, particularly Bifidobacterium spp.^40–42 HMOs aid in establishing the neonatal microbiome by acting as a nutritional source for these bacteria and protecting against potential pathogen invasion. For example, HMOs enhance intestinal epithelial barrier function⁴³ and bind to potentially harmful enteric pathogens.⁴⁴ In addition, HMOs are metabolized by bacteria that produce short-chain fatty acids (SCFAs), highly bioactive lipids that may serve a beneficial role in neonatal health.^{45, 46}

Numerous factors, including lactational stage and maternal genetics, influence the abundance of HMOs in breast milk.⁴⁷ For example, the activity of the maternal "secretor" gene, fucosyltransferase 2 (FUT2), influences the abundance of 2′-fucosylated (2-FL) HMOs, which can then have a significant impact on the abundance of Bifidobacterium spp. in the stool of breastfeeding infants.⁴⁸ This likely has a direct influence on neonatal health as HMO composition in maternal breast milk has been associated with the risk of NEC in preterm neonates,⁴⁹ and 2-FL is one of the HMOs that has been shown to decrease the severity of NEC in an animal model.²⁰ HMO supplementation of preterm formula and breast milk is under active investigation.

Microbes

Given its rich nutritional substrate and direct fuel sources for bacteria such as HMOs, it is not surprising that breast milk contains its own microbiome. A comprehensive review of studies analyzing the microbial composition of breast milk determined that 820 bacterial species have been detected in breast milk thus far, with a predominance of Proteobacteria and Firmicutes.⁵⁰ The bacterial species present in breast milk are impacted by maternal factors, including body mass index (BMI),^{51, 52} atopic status,^{53, 54} mode of delivery,^{52, 55–58} geographical location,⁵⁷ HMO levels, ^59–61and diet.⁶² The source of these microbes is thought to be a combination of maternal skin flora and intestinal microbiome as well as the breastfeeding infant’s oral cavity.⁶² These bacteria are among the first to colonize the infant’s intestinal tract and thus can have significant short- and long-term health effects by shaping the establishment of the intestinal microbiome.^63–66 For preterm infants, the composition of the intestinal microbiome is particularly important, given that microbial dysbiosis precedes the development of NEC.^{67, 68} Thus, research focused on modulating the microbial composition of human milk has the potential to directly impact neonatal health.

Exosomes

Exosomes are membrane-lined vesicles of endosomal origin released from a diverse array of from 30–150 nm in size.⁶⁹ Exosomes are present in human breast milk, including colostrum, transitional, and mature milk.^{70, 71} They contain a complex array of cellular components, including proteins, microRNA, and lipids.⁷² Breast milk exosomal content and activity are influenced by maternal factors, including the length of pregnancy^{73, 74}, lactational stage⁷¹, and overall health.^{75, 76} Expoloration of the biological activity of breast milk-derived exosomes is an ongoing area of research. In vitro data indicate that exosomes act directly on intestinal epithelial cells to promote epithelial cell growth^{77, 78} and prevent apoptosis.⁷⁸ They also reduce epithelial injury upon exposure of organoids to the Gram-negative bacterial byproduct lipopolysaccharide (LPS).⁷¹ Animal studies have found that intraperitoneal (i.p.) administration of exosomes derived from stem cells reduces the incidence and severity of intestinal injury in a rat model of NEC.⁷⁹ Interestingly, the route of administration of exosomes likely influences their protective effect, with oral administration found to be more protective than i.p. administration in a rat model of NEC.⁷⁸ These data provide support for further research into exosomes as a preventative and therapeutic approach for NEC.

Lactoferrin

Lactoferrin is the most abundant protein in the whey fraction of breast milk and has robust antimicrobial properties.⁸⁰ This glycoprotein is in the transferrin family and has a high affinity for binding iron, which confers antioxidant and bacteriostatic properties.⁸¹ This iron-binding activity subsequently provides a competitive advantage for potentially beneficial bacteria that have a low iron requirement, such as Lactobacillus and Bifidobacterium spp.^{82, 83} In addition to lactoferrin’s modulation of bacterial growth, its protective effects include the promotion of intestinal cell proliferation, differentiation, and maturation, as well as regulation of the host’s immune response.⁸⁴ Animal studies demonstrate that lactoferrin suppresses inflammation, increases immunoglobulin levels, and enhances intestinal cell proliferation.^85–89

Despite theoretical benefits and animal model data, trials of enteral lactoferrin supplementation with human or bovine lactoferrin have had varied outcomes. A 2020 Cochrane review analyzed clinical trials studying enteral lactoferrin supplementation for the prevention of sepsis and NEC in preterm infants.⁹⁰ With low-certainty evidence, they reported a decrease in suspected or confirmed late-onset sepsis (RR:0.80, 95% CI 0.72 to 0.89) but did not detect differences in NEC (RR:1.10, 95% CI, 0.86 to 1.41) or overall mortality (RR: 0.90, 95% CI 0.69 to 1.17).⁹⁰ Similarly, the Enteral Lactoferrin in Neonates (ELFIN) and NEOLACTO studies did not find benefits for very preterm or low birth weight infants who received supplemental bovine lactoferrin.^{91, 92} The Lactoferrin Infant Feeding Trial (LIFT_Canada) is a randomized control trial studying the hypothesis that oral bovine lactoferrin will reduce the rate of mortality or major morbidity (defined as brain injury, late-onset sepsis, NEC, or retinopathy of prematurity) in VLBW preterm infants and is expected to be three years in duration.⁹³ One of the secondary aims of this study is to evaluate the presence of major neurodevelopmental outcomes at 24 months corrected age. This trial will help clarify if there are clear short- or long-term benefits of lactoferrin supplementation on outcomes for preterm infants.

Immunoglobulins

Immunoglobulins in breast milk enhance neonatal immunity and promote immune development. IgA is the most abundant immunoglobulin in breast milk (90–95%), followed by IgM (2–5%) and IgG (< 1%).^{23, 94} Levels of immunoglobulins change over the course of lactation, although IgA remains the predominant immunoglobulin regardless of lactational stage.²³ Breast milk IgA undergoes proteolytic cleavage to release secretory IgA. Colostrum, the breast milk produced in the first days of life, contains high concentrations of these secretory immunoglobulins.²⁴ Secretory IgA exhibits direct antimicrobial effects in the intestine by binding to microbial toxins, bacteria, and viruses. Secretory IgA also supports the development of the gut microbiome by acting as a carbohydrate source and promoting the growth of intestinal bacteria such as Bacteroides spp.^{95, 96} In addition, data indicate that binding intestinal bacterial by secretory IgA may be protective against NEC as fecal analysis found that preterm infants with NEC had higher levels of IgA-unbound Enterobacteriaceae in their stool compared to those without NEC.²¹ This study also found that preterm infants fed breast milk had more IgA-bound bacteria than formula-fed preterm infants, providing another potential mechanism for breast milk’s protective effects against NEC.²¹ This effect of IgA is likely primarily localized in the neonatal intestine, as data indicated that IgA is not absorbed from the infant’s intestine into systemic circulation in significant quantities.⁹⁷

Cytokines and Growth Factors

Breast milk contains a variety of cytokines and growth factors, which are powerful proteins that serve a central role in numerous biologic processes, including inflammation, growth, and development (Figure 1). ⁹⁵ Research in the field of NEC has demonstrated that enteral administration of the growth factors found in breast milk, including epidermal growth factor (EGF) and Heparin-binding EGF-like growth factor, are associated with protection against NEC in animal models.^{19, 98} In addition, mechanistic studies in mice determined that activation of EGF receptor signaling via dam’s milk is protective against NEC-like intestinal inflammation via downregulation of TLR4 signaling.¹⁹ Lower levels of EGF in the saliva of preterm neonates have also been associated with an increased risk of NEC.⁹⁹

Detection of cytokines in breast milk does not necessarily translate into direct bioactivity in the neonate, given their exposure to the harsh conditions of the digestive tract, including digestive enzymes, acidity, and elevated temperatures, as well as the presence of potential inactivating agents in breast milk. Further research is needed to determine the impact of the cytokines present in breast milk on infant health.

Pasteurized Donor Breast Milk

Donor breast milk is an alternative to maternal breast milk when exclusive maternal breast milk feedings are not feasible. The steps of donor breast milk processing include the donation of breast milk to a milk bank by lactating women, followed by screening, testing, pooling, and pasteurization.¹⁰⁰ Storage, packaging, and pasteurization differentially impact the donor breast milk’s nutritional and bioactive components.^{22, 101–103} For example, compared to fresh breast milk, pasteurized donor breast milk has lower concentrations of lactoferrin and secretory IgA.^{104, 105} Pasteurization does not affect the HMOs; however, it does have varying effects on cytokine levels.¹⁰⁶ In addition, mothers that provide donor milk are at a different lactational stage than mothers providing breast milk for their newborns. For these reasons, donor and maternal breast milk are inherently different products, and benefits from maternal breast milk feeding cannot be assumed to apply to donor milk.

While pasteurized donor milk has decreased amounts and effectiveness of beneficial bioactive components when compared to maternal breast milk, preterm infants likely benefit from its use. A 2018 Cochrane review found that compared to formula, donor milk was associated with lower rates of in-hospital growth but not long-term measures of growth, including weight, length, and head circumference for preterm or low birth weight infants.¹⁰⁷ They also found an increased risk of NEC (RR:1.87, 95% CI 1.23–2.85) but no differences in overall mortality (RR:1.11, 95% CI 0.81–1.53) or neurodevelopmental disability (RR:1.21, 95% CI 0.62–2.35) for infants receiving formula compared to donor breast milk.¹⁰⁷ Donor milk is recommended by the American Academy of Pediatrics when a mother's milk is not available or insufficient.¹⁰⁸ Preterm infants, especially those with a birth weight of less than 1,500 grams, are the most frequent recipients of pasteurized donor milk.¹⁰⁹ The use of pasteurized donor milk in neonatal intensive care units (NICUs) in the United States has been increasing for over a decade.¹⁰⁶

Conclusions

Breast milk provides nutritional and bioactive components that support growth, development, and host defense. Maternal breast milk is the optimal nutritional source in early life, particularly for fragile preterm neonates. While pasteurized donor milk is an alternative, it significantly differs from maternal breast milk. The clear health benefits of breast milk motivate the ongoing research into breast milk composition, mechanistic studies of bioactive ingredients, and additional clinical trials on the various ingredients in breast milk. This ongoing research needs to be complemented by programs aiming to increase support for breastfeeding mothers and improve breast milk provision to medically fragile neonates. The overarching goal of these efforts is to improve neonatal health by providing the best enteral nutrition possible to all infants.