Human milk shows wide variations in fat, protein and carbohydrate contents 19 . Donor milk contains significantly less protein, fat and energy than the milk, provided by the mothers of preterm infants 20 . Compared to term milk, preterm milk is richer in cholesterol, phospholipids and long chain polyunsaturated fatty acids, which are integral cellular membrane components and essential for brain cell growth and myelin development in newborn infants 21 . These components decrease in milk as lactation progresses. A review of the effects of pasteurisation on human milk concluded that Holder pasteurisation did not significantly affect its composition with regard to saccharides, protein, fat, energy or fatty acids 22 .

Bioactive components

In addition to macronutrients, breastmilk is rich in bioactive components, such as oligosaccharides, immunoglobulins, lactoferrin, cytokines, enzymes, growth factors, hormones, anti‐inflammatory agents, microbial factors and fatty acids, particularly in the first months after birth. These factors appear to play a multitude of overlapping roles in gut and general development 23, although little is presently known about their functions in very preterm neonates.

While milk proteins mainly serve as a source of amino acids for infant growth, peptides may also possess bioactive capacities, either directly or after digestion 24. Bioactive milk proteins can provide protection from infections, enhance nutrient absorption and promote immune system development and neurodevelopment. Some of these proteins are destroyed or inactivated by pasteurisation, leading to different properties in raw maternal milk and pasteurised donor milk 22. In preterm formula, the proteins are derived from bovine milk, which differs from human milk in many respects.

The functions of milk proteins and peptides have mainly been investigated in experimental studies. In vitro models have revealed that human milk proteins generate a wide variety of bioactive peptides. Beta‐casein 25 is the most abundant casein in milk and the greatest source of bioactive peptides 24. In vitro studies have shown that kappa‐casein from breastmilk inhibits Helicobacter pylori binding to human gastric mucosa 26. Pasteurisation did not appear to affect the bioactive peptides derived from the digestion of major human milk proteins 25.

The most prevalent whey protein in human milk is alpha‐lactalbumin, which accounts for 20–25% of total milk protein. It plays several physiological roles during the neonatal period, including providing a balanced supply of essential amino acids. Moreover, alpha‐lactalbumin digestion results in the transient formation of peptides with immune‐stimulatory and bactericidal properties, which may be protective against infection 24, 27. One study reported that pasteurisation did not alter the alpha‐lactalbumin and serum albumin concentrations in breastmilk 28.

Lymphocytes migrate from the mother's intestine to the mammary gland, where they are transformed to immunoglobulin‐A‐producing cells and produce the secretory immunoglobulin‐A (sIgA) found in human milk 29. Through this process, the milk contains antibodies that are directed against microbial antigens present in the mother's gut. The milk sIgA blocks the mucosal adherence of bacterial, parasitic and viral pathogens and is critical for maintaining a diversified microbiotic environment 30, 31. Thus, sIgA in breastmilk transfers maternal immunity to infectious agents and other antigens in the mother's and hence the infant's environment to the infant.

One study showed that pasteurisation of previously frozen milk reduced sIgA by 51% 32, while another reported that sIgA was 60% lower in pasteurised term donor milk compared to fresh term milk 28. Research also showed that stool samples from breastfed term infants contained large amounts of intact sIgA, with the highest concentrations during the first weeks of life 33.

The iron‐binding protein lactoferrin is a major whey protein in human milk. In the gut, it binds to lactoferrin receptors that are expressed in the small intestine. Human lactoferrin receptors have also been found in monocytes, lymphocytes, platelets, fibroblasts and bone 34. Stool samples of full‐term exclusively breastfed infants contain intact lactoferrin and the concentration decreases with age and is naturally associated with the decreasing concentrations in milk 33. It has been suggested that absorption of lactoferrin‐bound iron in milk is the main route for iron uptake during the neonatal period 35. However, neonatal lactoferrin knockout mice exhibited no evidence of reduced intestinal iron uptake 36. Lactoferrin has been reported to protect newborn infants from infection by withholding iron from bacteria 37 and by destabilising the bacterial cell surface 38. Lactoferrin has also been found to enter cell nuclei and affect the expression of genes, modulate cell proliferation and differentiation 39 and interact with the immune system 34, 40.

Studies have shown that lactoferrin has high structural homology between species and bovine lactoferrin exerts biological effects on human enteral cells 41. In addition, bovine milk‐based formulas contain very small amounts of lactoferrin without supplementation 42.

The process of freezing, thawing, pasteurising and freezing and thawing again reportedly decreases the lactoferrin concentration in human milk by 70% 43. Other studies have demonstrated that 91% of lactoferrin was lost after the pasteurisation of previously frozen milk 32 and that pasteurised donor milk had a 44% lower lactoferrin concentration than fresh milk 28. In preterm infants, enteral supplementation with bovine lactoferrin decreased invasive fungal infections 44, late‐onset sepsis 45-47 and NEC 48. In some countries, bovine lactoferrin is added to commercial formulas, and the US Food and Drug Administration has classified bovine lactoferrin as generally recognised as safe 49.

A randomised controlled trial investigated enteral administration of human recombinant lactoferrin (talactoferrin) to infants with birthweights of 750–1500 g It reduced hospital‐acquired infections by nearly 50%, along with a significant reduction in coagulase‐negative Staphylococcus and, or, line infections. However, the authors stated that talactoferrin was no longer available in the United States 50, 51. A Cochrane review from 2017 concluded that low‐quality evidence suggested that supplementing oral feeds with lactoferrin, bovine or talactoferrin, with or without probiotics, decreased late‐onset sepsis and NEC stage II or III in preterm infants, without adverse effects. Ongoing trials will provide evidence from over 6000 infants 52.

Lysozyme is a host defence protein that is secreted in high concentrations in human milk. It is part of the innate immune system and causes lysis of bacteria via degradation of their outer wall. The presence of lysozyme may stimulate overgrowth of intestinal bifidobacteria, which are apparently resistant to lysozyme 53. Lysozyme is excreted in the faeces of preterm infants with a gestational age of 28–30 weeks 54 but not in healthy term infants 33. Pasteurisation reportedly reduces lysozyme by 59% 32 and pasteurised donor milk showed 60% lower lysozyme activity than raw milk 28. Similarly, concentrations of another host defence protein, lactoperoxidase, were 82% lower in donor milk than in the breastmilk of mothers delivering at term 28.

Osteopontin is a multifunctional protein that is present in most tissues and body fluids. It is found in high concentrations in human milk 55, 56 and the highest levels have been found three to seven days’ post‐partum 57. Experimental studies have revealed that milk osteopontin plays essential roles in immunological, intestinal and early cognitive development 55. It can bind to various receptors of enterocytes or be absorbed into systemic circulation. Osteopontin concentrations have been found at lower levels in bovine milk than in human milk and even lower levels have been found in bovine milk‐based formula 42, 56. Infants born at term who received formula with added bovine osteopontin had lower tumour necrosis factor‐alpha serum concentrations than infants on nonsupplemented formula and similar concentrations to breastfed infants. They also had fewer fevers, increased proportions of T cells and upregulated pathways of cell proliferation and cell to cell adhesion. The effects of osteopontin supplements appeared to be dose dependent in one study, with some functions promoted by lower osteopontin concentrations and others by higher concentrations 58. Osteopontin has been found to have high affinity for lactoferrin and may act as a carrier protein for lactoferrin in milk and as a modulator of lactoferrin's anti‐microbial and immune‐stimulatory activities 59. Little is known about the biological functions of milk osteopontin in preterm infants and we were unable to find any studies on the effect of pasteurisation on osteopontin concentrations. In a preterm animal model that used piglets, formula supplemented with osteopontin reduced the severity, but not the incidence, of NEC 60.

Bile salt‐stimulated lipase in milk facilitates lipid digestion and absorption, especially in immature infants with low endogenous capacity to digest fat early in life (S61 in Appendix S1). Bile salt‐stimulated lipase is inactivated by pasteurisation (S62) and one study showed that feeding infants with pasteurised maternal milk reduced fat absorption and growth (S63). When recombinant human bile salt‐stimulated lipase was used to supplement pasteurised human milk or formula, the absorption of docosahexaenoic acid and arachidonic acid was increased, but not the total fat absorption (S64). Treatment with recombinant human bile salt‐stimulated lipase significantly improved growth in infants born small for gestational age, but not in infants born appropriate for gestational age and was associated with increased risk of infections and gastrointestinal intolerability (S65).

Milk fat globules comprise a core of mainly triglycerides, surrounded by a membrane composed of phospholipids, cholesterol, proteins and glycoproteins, including several proteins with known or suggested bio‐activities. Free fatty acids and monoglycerides, which are digestion products of the milk fat globule triglyceride core, have been shown to act as detergents and have lytic functions against viruses, bacteria and protozoa (S66). Human milk fat globule membranes contain a multitude of proteins: in one study, 20% were shown to be related to immune responses and 19% to cell communication and signal transduction (S67). In addition, milk fat globule membranes also contain compounds involved in central nervous system development, such as choline and sphingomyelin. Filaments on the surface of human milk fat globules have been reported to contain mucin‐1, which inhibits bacterial adhesion to other cells (S68), and lactadherin, which protects against the rotavirus infection (S69). Both mucin‐1 and lactadherin survive and maintain their integrity in the stomachs of preterm infants. Their concentrations in human milk have been found to be highest during the first weeks of lactation and to decline thereafter (S70). The average milk fat globule size in mature milk increases with the lactation progress, and thereby, the proportion of milk fat globule membrane decreases. One study showed that, human milk fat globules significantly decreased in size during pasteurisation and showed a greater globule size distribution (S71). Another study reported that storage and cooling affected the stability and membrane composition of bovine milk fat globules and that pasteurisation drastically modified milk fat globule membrane composition and functionality (S72).

Choline is an essential nutrient in breastmilk and it is critical for nervous system development. It is a precursor of membrane constituents such as phosphatidylcholine and sphingomyelin and of the neurotransmitter acetylcholine. A study in rats showed that brain function was positively impacted by oral choline supplementation during neurogenesis and synaptogenesis, corresponding to a period from gestation to four years of age in humans (S73). Notably, phosphatidylcholine is a main component of pulmonary surfactant and the major transporter for arachidonic acid and docosahexaenoic acid (S74). During pregnancy, choline has been shown to selectively transfer from the mother to the foetus and plasma choline concentrations after preterm birth have been reported to decrease to approximately 50% of those in cord blood within 48 h. It has been suggested that choline undernourishment may contribute to adverse neurodevelopmental outcomes after preterm birth (S75). While some authors have reported that preterm human milk contained less choline than term milk during the first weeks of life (S76), others found more phosphocholine in preterm than term milk (S77). Bovine and human milk have a nearly identical phospholipid content, which was not altered by the pasteurisation of bovine milk (S78). An ongoing trial is investigating the effects of choline and docosahexaenoic acid supplementation in preterm infants (NCT02509728).

Human milk oligosaccharides (HMOs) are a group of complex sugars that are highly abundant in human milk. More than 120 different HMO structures have been identified, and it has been shown that each mother produces a unique composition (S79,S80). Most HMOs contain the sugar fucose and a mother's ability to fucosylate HMOs is genetically determined (S81). Some fucosylated HMOs prevent adhesion of enteric pathogens to the intestinal mucosa (S81). In vitro digestion studies have demonstrated that HMOs were relatively resistant to human digestion (S82,S83), with a small fraction absorbed into the infants’ systemic circulation and excreted intact in the urine (S84). Another study reported that most HMOs were either metabolised by intestinal microorganisms or excreted with faeces (S85). Within the gastrointestinal tract, different HMO categories apparently serve as metabolic substrates for specific bacteria, selectively promoting bifidobacterial growth in particular (S86,S87).

Experimental studies have shown that HMOs prevented adhesion to epithelial cells of pathogens, such as bacteria (S88), viruses (S89) and parasites (S90). In addition, HMOs inhibited proliferation of Group B Streptococcus, a leading cause of invasive infection in newborn infants (S91) and dose‐dependently reduced Candida albicans invasion of human premature intestinal cells (S92). A study of formula‐fed piglets reported that HMOs increased the abundance of various immune cells in both infected and noninfected animals (S93). Interestingly, some HMOs made urogenital tract epithelial cells more resistant to uropathogenic Escherichia coli (S94). Human milk oligosaccharides contain large amounts of sialic acid. A review summarised the evidence that supported a role for dietary sialic acid as an essential nutrient for optimal brain development (S95).

Similar HMO concentrations have been found in preterm and term milk (S96,S97), but preterm milk contained a much wider variation in the percentage of HMOs that contained fucose or sialic acid than term milk. This may have been due to the immaturity of the fucosylation process, which may influence the developing microbiota (S98). It has been suggested that preterm infants may benefit from supplementation with fucosylated HMOs, for example, from pooled donor human milk (S99). Human milk has been shown to contain 100‐fold to 1,000‐fold higher concentrations of oligosaccharides than bovine milk (S100). Pasteurisation or freeze drying of donor milk reportedly did not influence HMO content (S101).

Glycosaminoglycans are long un‐branched polysaccharides that are found on cell surfaces and cell–extracellular matrix interfaces. They regulate many processes, including cell growth and differentiation, cell to cell and cell to matrix interactions and anti‐infective and anti‐inflammatory processes (S102). Studies have found that, approximately 55% of the glycosaminoglycans in human milk are chondroitin sulphate and 40% are heparin. The concentrations of these glycosaminoglycans were several times higher in human milk than in bovine milk, which showed a different glycosaminoglycan profile. During the first month of lactation, preterm milk was reported to contain significantly more glycosaminoglycans than term milk (S103) and they were not affected by pasteurisation (S104).