Human breast milk: A review on its composition and bioactivity

EHD-04166; No of Pages 7 Early Human Development xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev

Human breast milk: A review on its composition and bioactivity Nicholas J. Andreas a,⁎, Beate Kampmann a,c, Kirsty Mehring Le-Doare a,b,c a b c

Centre for International Child Health, Department of Paediatrics, Imperial College London, St. Mary’s Hospital, Praed Street, London, W2 1NY UK Wellcome Trust Centre for Global Health Research, Norfolk Place, London, UK MRC Unit-The Gambia, Vaccines & Immunity Theme, Atlantic Road, Fajara, The Gambia

a r t i c l e Available online xxxx Keywords: Human milk Child nutrition science Neonate Immunity

i n f o

a b s t r a c t Breast milk is the perfect nutrition for infants, a result of millions of years of evolution, finely attuning it to the requirements of the infant. Breast milk contains many complex proteins, lipids and carbohydrates, the concentrations of which alter dramatically over a single feed, as well as over lactation, to reflect the infant’s needs. In addition to providing a source of nutrition for infants, breast milk contains a myriad of biologically active components. These molecules possess diverse roles, both guiding the development of the infants immune system and intestinal microbiota. Orchestrating the development of the microbiota are the human milk oligosaccharides, the synthesis of which are determined by the maternal genotype. In this review, we discuss the composition of breast milk and the factors that affect it during the course of breast feeding. Understanding the components of breast milk and their functions will allow for the improvement of clinical practices, infant feeding and our understanding of immune responses to infection and vaccination in infants. © 2015 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . Lipid . . . . . . . . . . . . . . . . . . . . . . . . . . . Breast milk protein . . . . . . . . . . . . . . . . . . . . . Non-protein nitrogen . . . . . . . . . . . . . . . . . . . . Antibody in breast milk . . . . . . . . . . . . . . . . . . . 5.1. Group B Streptococcal antibody in breast milk . . . . . 6. Carbohydrate. . . . . . . . . . . . . . . . . . . . . . . . 7. Human milk oligosaccharides . . . . . . . . . . . . . . . . 7.1. Influences on breast milk composition . . . . . . . . . 8. Time-associated changes in breast milk composition . . . . . . 8.1. Length of lactation . . . . . . . . . . . . . . . . . . 8.2. Time since last feed . . . . . . . . . . . . . . . . . 9. Stage of the nursing process . . . . . . . . . . . . . . . . . 9.1. Diurnal variation . . . . . . . . . . . . . . . . . . . 9.2. Maternal characteristics altering breast milk composition 9.2.1. Age of mother . . . . . . . . . . . . . . . . 9.2.2. Diet . . . . . . . . . . . . . . . . . . . . 9.2.3. Ethnicity . . . . . . . . . . . . . . . . . . 9.2.4. Weight gain during pregnancy . . . . . . . . 9.2.5. Birth weight. . . . . . . . . . . . . . . . . 10. Summary . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest statement . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: Group-B streptococcus, GBS; human milk oligosaccharides, HMO; secretory IgA, SIgA; toll-like receptor, TLR; Transforming growth factor beta, TGF-β. ⁎ Corresponding author at: Department of Paediatrics, Imperial College London, St. Mary’s Hospital, Praed Street, London, W2 1NY, UK. Tel.: +44 207594 2063. E-mail addresses: [email protected] (N.J. Andreas), [email protected] (B. Kampmann), [email protected] (K. Mehring Le-Doare).

http://dx.doi.org/10.1016/j.earlhumdev.2015.08.013 0378-3782/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013

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1. Introduction Breast milk is an extremely complex and highly variable biofluid that has evolved over millennia to nourish infants and protect them from disease whilst their own immune system matures. The composition of human breast milk changes in response to many factors, matching the infant’s requirements according to its age and other characteristics [1,2]. Therefore, the composition of breast milk is widely believed to be specifically tailored by each mother to precisely reflect the requirements of her infant [3]. The many antimicrobial and immunomodulatory components of breast milk are suggested to compensate for the deficiencies in the neonatal immune system, and impair the translocation of infectious pathogens across the gastrointestinal tract [4]. In addition, breastfed infants are also known to possess a more stable and less diverse intestinal microbiota than formula-fed infants, but possess more than twice the number of bacterial cells [5]. This may be partially due to alterations at the level of the gut mucosa due to bioactive substances in human milk. Demonstrating the bioactivity of breast milk, a study on shed epithelial cells in the faeces of infants has shown that gene expression in the neonatal gastrointestinal tract is influenced by breastfeeding, with differential expression found between formula-fed and breast-fed infants in genes regulating intestinal cell proliferation, differentiation and barrier function [6]. Breast milk contains bioactive factors that are capable of inhibiting inflammation, as well as enhancing specific-antibody production, including the compounds PAF-acetylhydrolase, antioxidants, interleukins 1, 6, 8 and 10, transforming growth factor (TGF), secretory leukocyte protease inhibitors (SLPI) and defensin 1 [4]. Breast milk also contains factors with the potential to mediate the differentiation and growth of B cells, including high concentrations of intracellular adhesion molecule 1 and vascular adhesion molecule 1; and lower concentrations of soluble S-selectin, L-selectin and CD14 [4]. Additionally, pattern-recognition receptors, which are crucial factors in the recognition of microorganisms in the neonatal respiratory tract and gut, are present in breast milk. Factors such as the Toll-like receptors (TLR-2 and TLR-4) provide efficient microbial recognition, working in synergy with the co-receptor CD14 and soluble CD14, which are found in high quantities in breast milk [7]. Further regulation by soluble tolllike receptor 2 (sTLR-2), which regulates cell activation via cell surface TLR-2, has also been noted in breast milk but not in infant formula [8]. Similarly, an as-yet unnamed 80 kDA protein identified in breast milk appears to inhibit TLR-2–mediated but activates TLR-4–mediated transcriptional responses in human intestinal epithelial and mononuclear cells [9]. Reduced TLR-2 responsiveness at birth has been proposed to facilitate the normal establishment of beneficial microbiota such as bifidobacteria. Various studies have examined the influences of maternal characteristics on breast milk composition. Important factors known to influence breast milk composition—such as the gradual increase in fat concentrations throughout a feed, have well-defined effects. However, other potential influences, such as the mode of delivery and maternal BMI, have less high-quality evidence supporting their role. The difficulties in accurately assessing the composition of breast milk (e.g. sampling time) hinder efforts to elucidate the true value of these effects. Furthermore, there is a profound lack of knowledge regarding how alterations in breast milk composition may subsequently impact infant and later health outcomes. Metabonomics, the study of multiple metabolites in biofluids, using techniques including mass spectrometry and 1H NMR spectroscopy, is capable of measuring components in extremely low concentrations. This may assist in unravelling the factors influencing breast milk composition, as well as identifying previously unidentified components and their influence on human health [10,11]. In this review, we discuss the nutritional and non-nutritional components of breast milk and the effect of breast milk components on

infant colonisation with potentially pathogenic bacteria and factors which are known to influence its composition.

2. Lipid Lipids are the largest source of energy in breast milk, contributing 40%–55% of the total energy of breast milk [12]. These lipids are present as an emulsion. The vast majority of lipids secreted are triacylglycerides, contributing towards 98% of the lipid fraction. The remainder predominantly consists of diacylglycerides, monoacylglycerides, free fatty acids, phospholipids and cholesterol. These components are packaged into milk fat lipid globules, with the phospholipids forming the bulk of the membrane of the globules and the triacylglycerols found in the core [13] (Fig. 1). These globules usually range from 1 to 10 μm across, with an average diameter in mature milk of 4 μm [14]. Breast milk contains over 200 fatty acids; however, many of these are present in very low concentrations, with others dominating. For example, oleic acid accounts for 30–40 g/100 g fat in breast milk [16]. De novo synthesis of fatty acids accounts for approximately 17% of the total fat in breast milk [17]. Long-chain polyunsaturated fatty acids, molecules with a chain length of more than 20 carbon atoms—plus 2 or more double bonds—constitute ~ 2% of the total fatty acids present in breast milk [18]. The positions occupied by fatty acids along the glycerol backbone are highly conserved, with the fatty acids commonly appearing in specific positions [19] (Fig. 2). For example, fatty acids present in the highest concentrations in breast milk; oleic, palmitic and linoleic acid, are commonly found at the sn-1, sn-2 and sn-3 positions, respectively [19]. Interestingly, the distribution of fatty acids along glycerol influences their availability; with palmitic acid at the sn-2 position being absorbed more readily. Significantly, this positional preference is not replicated by many artificial formulas, and has been observed to influence the infants’ plasma lipid profile, including cholesterol concentration [20]. Short-chain fatty acids (SCFA) found in breast milk are also an important source of energy [22], as well as being essential for normal maturation of the gastrointestinal tract [23]. Sphingomyelins, present in the milk fat globule membrane, are especially important for central nervous system myelinisation, and have been shown to improve the neurobehavioral development of low-birth weight infants [24]. Breast milk lipids have been shown to inactivate a number of pathogens in vitro, including Group B streptococcus (GBS). This suggests

Fig. 1. An optical microscopy image of milk fat lipid globules, displaying the structure of milk. Adapted with permission from ref. [15], American Chemical Society.

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013

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Lactocytes produce approximately 80%–90% of breast milk protein. The majority of the breast milk proteins not synthesised by lactocytes are taken up from the maternal circulation via transcytosis, passing into the lumen [32].

4. Non-protein nitrogen

Fig. 2. Structure of triacylglycerol with the sn positions annotated. Adapted with permission from ref. [21].

that lipids provide additional protection from invasive infections at the mucosal surface, particularly medium-chain monoglycerides [25].

3. Breast milk protein Breast milk contains over 400 different proteins which perform a variety of functions; providing nutrition, possessing antimicrobial and immunomodulatory activities, as well as stimulating the absorption of nutrients [26,27]. Proteins present in milk can be divided into three groups, caseins, whey and mucin proteins [28]. Whey and casein are classified according to their solubility, with the soluble whey proteins present in solution, whilst caseins are present in casein micelles, suspended in solution [29]. Mucins are present in the milk fat globule membrane [27]. Proteins present in significant quantities in the whey fraction are α-lactalbumin, lactoferrin, IgS, serum albumin and lysozyme [27]. Three types of casein are present in human milk: α-, β- and κ-casein. κ-casein stabilises the insoluble ɑ- and β-caseins, forming a colloidal suspension (the casein micelle shown in Fig. 3). Caseins do not form disulfide bonds causing the micelles to form a tangled web structure [30]. The total protein content of human breast milk consists of ~13% casein, the lowest casein concentration of any studied species, corresponding to the slow growth rate of human infants [31].

Fig. 3. Structure of a casein micelle of bovine origin, image from a scanning electron microscope. Reprinted with permission from Elsevier, International Dairy Journal, volume 14, issue 12, Dalgleish et al., 2004.

Non-protein nitrogen, consisting of molecules such as urea, creatinine, nucleotides, free amino acids and peptides, contribute towards ~ 25% of the total nitrogen present in milk [33]. This understudied fraction of breast milk contains many bioactive molecules. For example, nucleotides are considered as conditionally essential nutrients during early life, and perform key roles in various cell processes, such as altering enzymatic activities, and acting as metabolic mediators [34]. Furthermore, nucleotides are known to be beneficial for the development, maturation and repair of the gastrointestinal tract [34], as well as the development of the microbiota [35], and immune function [36].

5. Antibody in breast milk Immunoglobulins, present in particularly high concentrations early in lactation, are found in breast milk as secretory IgA (SIgA), the most predominant form, followed by SIgG. These provide immunological protection to the infant, whilst its own immune system matures [37]. The decrease in antibody reflects the infants’ decreased requirement as their immune system becomes more functional. Also, this reflects the increasing inability of the infant gut to absorb whole proteins, as gut permeability to macromolecules decreases over the first few days of life [38]. Protection from invasive pathogens at the mucosal surface relies heavily on breast milk antibodies, as neonatal secretions only contain trace amounts of SIgA and SIgM [39]. In concordance with this, IgA is found in breast-fed infants’ faeces on the second day of life, compared to 30% of formula-fed infants (formula does not contain IgA), whose faeces only contains IgA at one month post-partum [40]. The antibodies found in breast milk occur as a result of antigenic stimulation of maternal mucosa-associated lymphoid tissue (MALT) and bronchial tree (bronchomammary pathway) [41]. Therefore, these antibodies target the infectious agents encountered by the mother during the perinatal period, meaning that they also target the infectious agents most likely to be encountered by the infant. For example, maternal immunization with a Neisseria meningococcal vaccine demonstrated elevated N. meningitidis-specific IgA antibodies in breast milk, up to six months post-partum [42]. SIgA is hypothesised to function as the primary protective agent of breast milk [43,44]. In colostrum, SIgA concentrations are around 12 mg/ml whilst mature milk contains only ~ 1 mg/ml, highlighting the protective role of colostrum. Breast-fed infants ingest approximately 0.5–1.0 g of SIgA per day [45]. SIgA protects against mucosal pathogens via a number of mechanisms, both immobilizing pathogens, and thereby preventing adherence to epithelial cell surfaces, as well as neutralizing toxins and virulence factors. SIgA antibodies against bacterial adhesion sites like pili are also found in breast milk [4,46]. As SIgA is relatively resistant to proteolysis, it is able to provide protection against pathogens in the gastrointestinal tract [4]. Breast milk contains SIgA antibodies specific for many different enteric and respiratory pathogens. For example, breast milk contains antibodies protective against Vibrio cholerae, Campylobacter, Shigella, Giardia lamblia and respiratory tract infections [47–49]. SIgA antibodies against bacterial adhesion sites like pili have been found in breast milk [4,46]. For example, adherence of S. pneumoniae and Haemophilus influenza to human retropharyngeal cells is blocked by SIgA antibody in breast milk [46].

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013

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5.1. Group B Streptococcal antibody in breast milk Several antibody classes present in breast milk appear to protect against neonatal GBS infection [50]. The administration of GBS-specific IgM antibodies via breast milk have been shown to protect against GBS infection in animal models [51]. A similar ability to protect against GBS may be obtained from breast milk SIgA; however, SIgA does not appear to be taken up into the neonatal circulation [52], except in preterm infants [53], suggesting that SIgA’s effectiveness is limited to the mucosal surfaces of the gastrointestinal tract in term infants. However, even if SIgA does not cross into the neonatal circulation, these antibodies may still afford protection to neonates via other mechanisms. SIgA may interfere with the carbohydrate-mediated attachment of GBS to nasopharyngeal epithelial cells, reducing the colonizing organism load, and therefore reducing the morbidity and mortality caused by GBS [54]. IgA antibodies to capsular polysaccharide (CPS) type III GBS have been detected in 63% of a cohort of 70 Swedish mothers [55], whilst IgG antibody concentrations to type Ia, II or III have been found in concentrations approximately 10% of those found in maternal serum [54]. To date, no human studies have demonstrated a correlation between GBS-antibody levels in breast milk and infant colonization. However, using a rodent model, maternal immunization with GBS CPS-II and CPS-III antibody was shown to increase pup survival when pups were exposed to breast milk containing high titers of antibody in comparison to low titers [51,56]. 6. Carbohydrate A huge variety of different and complex carbohydrates are present in milk with lactose, a disaccharide consisting of glucose covalently bound to galactose, being the most abundant by far. Indeed, lactose is present in the highest concentration in humans compared to any other species, corresponding to the high energy demands of the human brain. Human milk oligosaccharides (HMO) also make up a significant fraction of breast milk carbohydrate, but are indigestible by the infant, their function instead is to nourish the gastrointestinal microbiota [57]. 7. Human milk oligosaccharides Human milk oligosaccharides (HMO) are an important component of human milk carbohydrate, and are the third largest component in breast milk, totalling on average 12.9 g/L in mature milk and 20.9 g/L at 4 days post-partum [57]. HMO contain between 3 to 22 saccharide units per molecule, and are made up of 5 different sugars, found in varying different sequences and orientations. The monosaccharides which make up the oligosaccharides are L-fucose, D-glucose, D-galactose, N-acetylglucosamine and N-acetylneuraminic acid. There are over 200 different types of oligosaccharides known to be in human milk, all of which feature lactose at the reducing end [58].

HMO function as prebiotics, encouraging the growth of certain strains of beneficial bacteria, such as Bifidobacterium infantis, within the infant gastrointestinal tract, protecting the infant from colonisation by pathogenic bacteria [59]. HMO play an important role in preventing neonatal diarrhoeal and respiratory tract infections [60,61]. The production of HMO is genetically determined, different profiles of milk oligosaccharide occur as a result of specific transferase enzymes expressed in the lactocytes. Two such genes, important for determining the HMO profile a mother produces, are the Secretor and Lewis blood group genes. The Secretor gene encodes for the enzyme α[1,2]fucosyltransferase (FUT2), responsible for linking fucose in a α1-2 linkage to elongate the HMO chain. The enzyme FUT3 is encoded for by the Lewis blood group gene; this enzyme catalyses the reaction between fucose in a α1-3/4 linkage, creating further fucosylated oligosaccharides (Fig. 4). As a result of the different expressions of these enzymes, there are four main phenotypes in relation to HMO profile: Se+/Le+, Se−/Le+, Se+/Le− and Se−/Le− [62]. Furthermore, HMO have been observed to modulate intestinal epithelial cell responses, as well as acting as immune modulators, altering both the environment of the intestine, by reducing cell growth, and inducing differentiation and apoptosis [63], as well as immune responses, potentially shifting T-cell responses to a balanced Th1/Th2-cytokine production [64]. One study investigating breast milk HMO profile demonstrated that Se+/Le+ mothers produced all types of fucosylated oligosaccharides, whilst Se−/Le+ mothers did not produce α1,2-fucosylated structures, such as 2’-fucosyllactose. Se+/Le− mothers secreted α1,2- and α1,3fucosylated oligosaccharides, but not HMO containing α1,4-fucose residues [65]. However, it was noted that in Se−/Le+ mothers, α1,3fucosylated oligosaccharides, such as 3’-fucosyllactose, were between two and fivefold higher than in Se+/Le+ mother’s breast milk. This suggests that there is an increase in FucT3 activity in non-secretor mothers, meaning that the total oligosaccharide production is relatively equal between the different groups [65]. One mechanism by which HMO protect infants against gastrointestinal infection is by acting as receptor decoys. A crucial step in the initiation of infection is the binding of pathogens to carbohydrates present on intestinal epithelial cells. HMO inhibit this process due to their analogous shapes to cell surface carbohydrates: pathogens recognise and bind to HMOs anchoring the bacteria in the mucosal layer and prevent cell adhesion to epithelial cells. Once bound, pathogens pass harmlessly from the gastrointestinal tract. An observational study found a significant association between levels of specific 2-linked fucosylated oligosaccharides in human milk and rates of Campylobacter diarrhoea infection in breast-fed infants. Furthermore, infants who received milk containing a low concentration of lacto-N-difucohexaose had an increased incidence of calicivirus diarrhoea [66]. HMO also prevent the adherence of S. pneumonia [67] and Escherichia coli [68], suggesting that HMO are capable of delivering protection against many bacterial and viral infections. GBS type Ib and II polysaccharides are virtually identical to certain HMO present in breast milk [56,69,70], raising the possibility of cross-reactivity with HMO [71].

Fig. 4. Structure of 2′- and 3′-fucosyllactose. Reproduced from ref. [73].

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013

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Different pathogen receptors have different affinities for specific carbohydrate structures, as the structures of the HMO produced are genetically determined: mothers possessing different genotypes, and therefore different HMO profiles, may protect their infants against certain infections to a greater or lesser extent, depending on the presence of specific HMOs. Likewise, the different HMO produced alters the types of microbiota colonising infants, as well as the timing of the establishment of the microbiota [72]. 7.1. Influences on breast milk composition Breast milk composition is extremely complex, varying with the time of day, stage of the nursing process and many other factors, with the lipid being most variable in terms of concentration [74]. 8. Time-associated changes in breast milk composition 8.1. Length of lactation Milk is commonly classified into colostrum, transitional milk and mature milk; however, these are not distinct classes of milk, but refer to the gradual alteration in the content of milk throughout lactation [33]. Colostrum, the first milk produced, is significantly different from mature milk, containing high concentrations of whey protein, whilst the caseins are almost undetectable [27]. The average content of protein in breast milk gradually decreases from the second month to the seventh month, after which the speed of reduction of protein content levels off. Colostrum contains low concentrations of both lactose and fat in comparison to mature milk [33,75]. Lactose production is highest in the fourth to seventh month, after which it decreases, whilst a gradual increase in the concentration of lipid occurs over lactation [76]. Colostrum is dramatically different from mature breast milk in terms of its bioactive properties, containing high concentrations of secretory immunoglobulin [77]. These qualities suggest that the primary role of colostrum is not nutritional, but immunologic, protecting the baby as it emerges from the relatively sterile environment of the womb, to being exposed to many environmental pathogens. In agreement with this, the concentration of HMO in colostrum is particularly high, being approximately double that of mature milk, with concentrations reducing from ~21 g/L to ~13 g/L from day 4 to day 120 post-partum [78]. As well as its immunologic and nutritional roles, colostrum appears to also act as a growth promoter. Colostrum contains many growth factors, again often in greater concentrations than in mature milk, for example, epidermal growth factor [79], TGF-β [80] and colony-stimulating factor1 [81] are all found in higher concentrations in colostrum than in mature breast milk. 8.2. Time since last feed One of the most significant predictors of milk fat concentration is the length of time since the last feed; the longer this interval is, the lower the concentration of fat in the milk. In keeping with this, fat concentrations at the end of the previous feed, as well as the volume of milk received at the previous feed, have been found to be particularly important predictors of milk fat concentration [82].

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9.1. Diurnal variation A diurnal variation in milk fat concentration occurs, with a peak fat content occurring at midmorning, and a low overnight, varying from ~5 g/100 ml to ~3 g/100 ml [33]. 9.2. Maternal characteristics altering breast milk composition 9.2.1. Age of mother Protein concentration is highest in breast milk of mothers aged 20–30; however, maternal age does not seem to influence either lipid or lactose concentrations [76], and maternal age does not have a large impact on breast milk composition. 9.2.2. Diet The influence of maternal diet on breast milk composition is complex. Depending on the type of nutrient, maternal diet can have virtually no impact on a nutrient’s concentration, whilst for other nutrients, maternal diet can result in large variations [84]. Previous research on the macronutrient content of breast milk from mothers of different ethnicities found little variation based on diet [85], and the variation in milk lipid concentration appears to be independent of maternal diet [86]. However, the specific fatty acids which form the lipid fraction are sensitive to maternal diet. These fatty acids are either endogenously synthesised by the mammary gland, or taken up from the maternal plasma, and both of these fatty acid sources are influenced by maternal diet [87]. Numerous studies investigating the fatty acid profile of breast milk have noted that it can be altered by manipulating the maternal diet [87–89], especially the monounsaturated omega-6 and omega-3 fatty acids. Dietary fatty acids are transferred rapidly to breast milk, and within 2 to 3 days, breast milk changes to mimic that of dietary fat [90]. The mammary gland is capable of synthesizing the medium-chain fatty acids (MCFAs) 10:0, 12:0 and 14:0. Women receiving a highcarbohydrate, low-fat diet have been observed to increase MCFA synthesis in order to maintain the quantity of triacylglycerides in breast milk [91]. 9.2.3. Ethnicity An analysis summarising research on the composition of milk of mothers from seven countries suggests breast milk composition is relatively consistent across different ethnicities. Among the variations that were observed, fat content was seen to vary by the greatest amount. Importantly, the magnitude of inter-individual variation between mothers of the same ethnicity was as great as that observed between mothers of different ethnicities [33]. 9.2.4. Weight gain during pregnancy A correlation between maternal weight gain during pregnancy and breast milk fat content has been reported; however, this was only observed to be significant at four months post-partum. The authors hypothesise that this phenomenon may be due to the laying down of fat stores during pregnancy, which are used as an energy reserve during lactation and subsequently more quickly diminished in the low weight gain group of mothers [92]. Despite this finding, two further studies were unable to identify an association between maternal weight gain during pregnancy and breast milk fat content [93,94].

9. Stage of the nursing process The stage of the nursing process results in a large alteration in the composition of breast milk, responsible for some of the largest variabilities seen in milk composition. There is a gradual increase in the fat content from the beginning, known as fore milk, to the end of a feed, hind milk, whilst lactose shows an inverse correlation to the change in fat content [83].

9.2.5. Birth weight Milk fat concentration increases when a deviation from normal birth weight occurs, i.e. there is a u-shaped association between fat content and infant birth weight, with a 20%–30% increase observed at the lowest and highest infant birth weights. Protein and carbohydrate concentrations do not appear to change significantly in relation to infant birth weight [2]. However, this study did not collect information on length

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of gestation; therefore, this influence may simply be a marker of the maturity of the infant. 10. Summary Studying the composition of breast milk can be challenging, in such a dynamic fluid without a benchmark against which to compare. However, if we are to improve the understanding of the biology of the lactating mother and her infant, as well as improving the quality of formula milks produced, investigating this is a necessity. Also, exactly how the composition of breast milk alters, and the downstream effects this may have on subsequent adult health will be of great interest in regard to the programming of the human metabolism during this early period. Many unknowns remain. Although some preliminary data exists, exactly how different profiles of HMO influence the species and types of bacteria which colonise the infant’s gastrointestinal tract, and how these microbiota subsequently influence the biology of the host are all questions of great interest. Likewise, just how infant genotype influences the environment of the intestine, and how this influences the species of microbiota present is yet to be delineated. Furthermore, many components of breast milk are altered during digestion, taking on new properties, and the consequences of this for infant immunity from infection and infant growth have not been sufficiently examined. Breast milk is vital in protecting infants from neonatal sepsis and for the promotion of infant growth and development. Its role in the mediation of potentially pathogenic gut organisms is just emerging and components such as HMO may prove useful adjuncts to antimicrobial therapy. Conflicts of interest statement NJA has received support from Medela and Danone to attend an educational conference, but declared no other conflicts of interest. KLD has received support from the Wellcome Trust and Thrasher Research Fund for her work. BK is funded by the MRC and has received support from other funders, such as the Wellcome Trust, the BMGF and the Thrasher Foundation. Acknowledgements We acknowledge the support of the Imperial College Biomedical Research Centre and the Wellcome Trust for our work. Also, we would like to acknowledge Jessica Birt, Amadou Faal, Asmaa Al-Khalidi, and Mustapha Jaiteh. References [1] Fujita M, Roth E, Lo YJ, Hurst C, Vollner J, Kendell A. In poor families, mothers' milk is richer for daughters than sons: a test of Trivers-Willard hypothesis in agropastoral settlements in Northern Kenya. Am J Phys Anthropol 2012;149(1):52–9. [2] Michaelsen KF, Skafte L, Badsberg JH, Jorgensen M. Variation in macronutrients in human bank milk: influencing factors and implications for human milk banking. J Pediatr Gastroenterol Nutr 1990;11(2):229–39. [3] The Surgeon General's Call to Action to Support Breastfeeding. Rockville (MD): Publications and Reports of the Surgeon General; 2011. [4] Hanson LA, Korotkova M. The role of breastfeeding in prevention of neonatal infection. Semin Neonatol 2002;7(4):275–81. [5] Bezirtzoglou E, Tsiotsias A, Welling GW. Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH). Anaerobe 2011;17(6):478–82. [6] Donovan SM, Wang M, Monaco MH, Martin CR, Davidson LA, Ivanov I, et al. Noninvasive molecular fingerprinting of host-microbiome interactions in neonates. FEBS Lett 2014;588(22):4112–9. [7] Labeta MO, Vidal K, Nores JE, Arias M, Vita N, Morgan BP, et al. Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14. J Exp Med 2000;191(10):1807–12. [8] Levy O. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nat Rev Immunol 2007;7(5):379–90. [9] LeBouder E, Rey-Nores JE, Raby AC, Affolter M, Vidal K, Thornton CA, et al. Modulation of neonatal microbial recognition: TLR-mediated innate immune responses are specifically and differentially modulated by human milk. J Immunol 2006;176(6):3742–52.

[10] Andreas NJ, Hyde MJ, Gomez-Romero M, Lopez-Gonzalvez MA, Villasenor A, Wijeyesekera A, et al. Multiplatform characterization of dynamic changes in breast milk during lactation. Electrophoresis 2015. http://dx.doi.org/10.1002/elps. 201500011 [Epub ahead of print]. [11] Villasenor A, Garcia-Perez I, Garcia A, Posma JM, Fernandez-Lopez M, Nicholas AJ, et al. Breast milk metabolome characterization in a single-phase extraction, multiplatform analytical approach. Anal Chem 2014;86(16):8245–52. [12] Koletzko B, Rodriguez-Palmero M, Demmelmair H, Fidler N, Jensen R, Sauerwald T. Physiological aspects of human milk lipids. Early Hum Dev 2001;65(Suppl.): S3–S18. [13] Lopez C, Menard O. Human milk fat globules: polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane. Colloids Surf B Biointerfaces 2011;83(1):29–41. [14] Michalski MC, Briard V, Michel F, Tasson F, Poulain P. Size distribution of fat globules in human colostrum, breast milk, and infant formula. J Dairy Sci 2005;88(6):1927–40. [15] Zou XQ, Guo Z, Huang JH, Jin QZ, Cheong LZ, Wang XG, et al. Human milk fat globules from different stages of lactation: a lipid composition analysis and microstructure characterization. J Agric Food Chem 2012;60(29):7158–67. [16] Koletzko B, Mrotzek M, Bremer HJ. Fatty acid composition of mature human milk in Germany. Am J Clin Nutr 1988;47(6):954–9. [17] Prentice A, Jarjou LM, Drury PJ, Dewit O, Crawford MA. Breast-milk fatty acids of rural Gambian mothers: effects of diet and maternal parity. J Pediatr Gastroenterol Nutr 1989;8(4):486–90. [18] Laryea MD, Leichsenring M, Mrotzek M, el-Amin EO, el Kharib AO, Ahmed HM, et al. Fatty acid composition of the milk of well-nourished Sudanese women. Int J Food Sci Nutr 1995;46(3):205–14. [19] Martin JC, Bougnoux P, Antoine JM, Lanson M, Couet C. Triacylglycerol structure of human colostrum and mature milk. Lipids 1993;28(7):637–43. [20] Innis SM, Quinlan P, Diersen-Schade D. Saturated fatty acid chain length and positional distribution in infant formula: effects on growth and plasma lipids and ketones in piglets. Am J Clin Nutr 1993;57(3):382–90. [21] Alvarez HM, Steinbuchel A. Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 2002;60(4):367–76. [22] Donohoe DR, Garge N, Zhang X, Sun W, O'Connell TM, Bunger MK, et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab 2011;13(5):517–26. [23] Peng L, Li ZR, Green RS, Holzman IR, Lin J. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. J Nutr 2009;139(9):1619–25. [24] Tanaka K, Hosozawa M, Kudo N, Yoshikawa N, Hisata K, Shoji H, et al. The pilot study: sphingomyelin-fortified milk has a positive association with the neurobehavioural development of very low birth weight infants during infancy, randomized control trial. Brain Dev Jpn 2013;35(1):45–52. [25] Isaacs CE, Litov RE, Thormar H. Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk. J Nutr Biochem 1995;6(7):362–6. [26] Molinari CE, Casadio YS, Hartmann BT, Livk A, Bringans S, Arthur PG, et al. Proteome mapping of human skim milk proteins in term and preterm milk. J Proteome Res 2012;11(3):1696–714. [27] Lonnerdal B. Human milk proteins: key components for the biological activity of human milk. Adv Exp Med Biol 2004;554:11–25. [28] Lonnerdal B. Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 2003;77(6):1537S–43S. [29] Jensen R. Handbook of Milk Composition. San Diego: Academic Press, Inc.; 1995. [30] Holt C, Horne DS. The hairy casein micelle: evolution of the concept and its implications for dairy technology. Neth Milk Dairy J 1996;50(2):85–111. [31] Lonnerdal B, Woodhouse LR, Glazier C. Compartmentalization and quantitation of protein in human milk. J Nutr 1987;117(8):1385–95. [32] Neville M, AJWC. Lactation: Physiology, Nutrition and Breastfeeding. New York: Plenum Press; 1983. [33] Jenness R. The composition of human milk. Semin Perinatol 1979;3(3):225–39. [34] Uauy R, Quan R, Gil A. Role of nucleotides in intestinal development and repair: implications for infant nutrition. J Nutr 1994;124(8 Suppl):1436S–41S. [35] Singhal A, Macfarlane G, Macfarlane S, Lanigan J, Kennedy K, Elias-Jones A, et al. Dietary nucleotides and fecal microbiota in formula-fed infants: a randomized controlled trial. Am J Clin Nutr 2008;87(6):1785–92. [36] Gutierrez-Castrellon P, Mora-Magana I, Diaz-Garcia L, Jimenez-Gutierrez C, RamirezMayans J, Solomon-Santibanez GA. Immune response to nucleotide-supplemented infant formulae: systematic review and meta-analysis. Br J Nutr 2007;98(Suppl. 1):S64–7. [37] Hurley WL, Theil PK. Perspectives on immunoglobulins in colostrum and milk. Nutrients 2011;3(4):442–74. [38] Vukavic T. Timing of the gut closure. J Pediatr Gastroenterol Nutr 1984;3(5):700–3. [39] Brandtzaeg P. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 2007;25(30):5467–84. [40] Jatsyk GV, Kuvaeva IB, Gribakin SG. Immunological protection of the neonatal gastrointestinal tract: the importance of breast feeding. Acta Paediatr Scand 1985; 74(2):246–9. [41] Goldman AS. Modulation of the gastrointestinal tract of infants by human milk. Interfaces and interactions. An evolutionary perspective. J Nutr 2000;130(2S Suppl): 426S–31S. [42] Shahid NS, Steinhoff MC, Roy E, Begum T, Thompson CM, Siber GR. Placental and breast transfer of antibodies after maternal immunization with polysaccharide meningococcal vaccine: a randomized, controlled evaluation. Vaccine 2002;20(17-18): 2404–9. [43] Krakauer R, Zinneman HH, Hong R. Deficiency of secretory Ig-A and intestinal malabsorption. Am J Gastroenterol 1975;64(4):319–23.

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013

N.J. Andreas et al. / Early Human Development xxx (2015) xxx–xxx [44] Dickinson EC, Gorga JC, Garrett M, Tuncer R, Boyle P, Watkins SC, et al. Immunoglobulin A supplementation abrogates bacterial translocation and preserves the architecture of the intestinal epithelium. Surgery 1998;124(2):284–90. [45] Lawrence RM, Lawrence RA. Breast milk and infection. Clin Perinatol 2004;31(3): 501–28. [46] Eden Svanborg, CB C, Hanson LÅ, et al. Anti-pili antibodies in breast milk. Lancet 1979:1235 [ii]. [47] Walterspiel JN, Morrow AL, Guerrero ML, Ruiz-Palacios GM, Pickering LK. Secretory anti-Giardia lamblia antibodies in human milk: protective effect against diarrhea. Pediatrics 1994;93(1):28–31. [48] Edmond K, Zaidi A. New approaches to preventing, diagnosing, and treating neonatal sepsis. PLoS Med 2010;7(3), e1000213. [49] Cruz JR, Gil L, Cano F, Caceres P, Pareja G. Breast milk anti-Escherichia coli heat-labile toxin IgA antibodies protect against toxin-induced infantile diarrhea. Acta Paediatr Scand 1988;77(5):658–62. [50] Le Doare K, Kampmann B. Breast milk and Group B streptococcal infection: Vector of transmission or vehicle for protection? Vaccine 2014;32(26):3128–32. [51] Heiman HSWL. Transplacental or enteral transfer of maternal immunizationinduced antibody protects suckling rats from type III group B streptococcal infection. Pediatr Res 1989;26(6):629–32. [52] Stephens S, Kennedy CR, Lakhani PK, Brenner MK. In-vivo immune responses of breast- and bottle-fed infants to tetanus toxoid antigen and to normal gut flora. Acta Paediatr Scand 1984;73(4):426–32. [53] Weaver LTWN, Taylor CE, Greenwell J, Toms GL. The ontogeny of serum IgA in the newborn. Pediatr Allergy Immunol 1991;2:2–75. [54] Weisman LE, Dobson FM. The potential impact of group B streptococcal antibodies in breast milk. Adv Exp Med Biol 1991;310:345–51. [55] Lagergard T, Thiringer K, Wassen L, Schneerson R, Trollfors B. Isotype composition of antibodies to streptococcus group B type III polysaccharide and to tetanus toxoid in maternal, cord blood sera and in breast milk. Eur J Pediatr 1992;151(2):98–102. [56] Gray BM, Egan ML, Pritchard DG. Specificity of monoclonal antibodies against group B streptococcus type II and inhibition of their binding by human secretions. Pediatr Res 1988;24(1):68–72. [57] Coppa GV, Gabrielli O, Pierani P, Catassi C, Carlucci A, Giorgi PL. Changes in carbohydrate composition in human milk over 4 months of lactation. Pediatrics 1993;91(3):637–41. [58] German JB, Freeman SL, Lebrilla CB, Mills DA. Human milk oligosaccharides: evolution, structures and bioselectivity as substrates for intestinal bacteria. Nestle Nutr Workshop Ser Pediatr Program 2008;62:205–18 [discussion 18–22]. [59] Ward RE, Ninonuevo M, Mills DA, Lebrilla CB, German JB. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl Environ Microbiol 2006;72(6):4497–9. [60] Newburg DS, Walker WA. Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res 2007;61(1):2–8. [61] Morrow AL, Ruiz-Palacios GM, Altaye M, Jiang X, Guerrero ML, Meinzen-Derr JK, et al. Human milk oligosaccharide blood group epitopes and innate immune protection against campylobacter and calicivirus diarrhea in breastfed infants. Adv Exp Med Biol 2004;554:443–6. [62] Thurl S, Henker J, Siegel M, Tovar K, Sawatzki G. Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides. Glycoconj J 1997;14(7):795–9. [63] Kuntz S, Kunz C, Rudloff S. Oligosaccharides from human milk induce growth arrest via G2/M by influencing growth-related cell cycle genes in intestinal epithelial cells. Br J Nutr 2009;101(9):1306–15. [64] Eiwegger T, Stahl B, Haidl P, Schmitt J, Boehm G, Dehlink E, et al. Prebiotic oligosaccharides: in vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties. Pediatr Allergy Immunol 2010;21(8):1179–88. [65] Pratico G, Capuani G, Tomassini A, Baldassarre ME, Delfini M, Miccheli A. Exploring human breast milk composition by NMR-based metabolomics. Nat Prod Res 2014; 28(2):95–101. [66] Morrow AL, Ruiz-Palacios GM, Altaye M, Jiang X, Guerrero ML, Meinzen-Derr JK, et al. Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants. J Pediatr 2004;145(3):297–303. [67] Andersson B, Porras O, Hanson LA, Lagergard T, Svanborg-Eden C. Inhibition of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human milk and receptor oligosaccharides. J Infect Dis 1986;153(2):232–7.

7

[68] Cravioto A, Tello A, Villafan H, Ruiz J, del Vedovo S, Neeser JR. Inhibition of localized adhesion of enteropathogenic Escherichia coli to HEp-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk. J Infect Dis 1991; 163(6):1247–55. [69] Wilkinson HW. Immunochemistry of purified polysaccharide type antigens of group B streptococcal types Ia, Ib, and Ic. Infect Immun 1975;11(4):845–52. [70] Kobata A. Structures and application of oligosaccharides in human milk. Proc Jpn Acad Ser B Phys Biol Sci 2010;86(7):731–47. [71] Pritchard DG, Gray BM, Egan ML. Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides. Infect Immun 1992;60(4):1598–602. [72] Lewis ZT, Totten SM, Smilowitz JT, Popovic M, Parker E, Lemay DG, et al. Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants. Microbiome 2015;3:13. [73] Pereira CL, McDonald FE. Synthesis of human milk oligosaccharides: 2′- and 3′fucosyllactose. Heterocycles 2012;84(1):637–55. [74] Lawrence RA, Lawrence RM. Breastfeeding: A Guide for the Medical Profession6th ed. ; 2005. [75] Saint L, Smith M, Hartmann PE. The yield and nutrient content of colostrum and milk of women from giving birth to 1 month post-partum. Br J Nutr 1984;52(1):87–95. [76] Kader MM, Bahgat R, Aziz MT, Hefnawi F, Badraoui MH, Younis N, et al. Lactation patterns in Egyptian women. II. Chemical composition of milk during the first year of lactation. J Biosoc Sci 1972;4(4):403–9. [77] Castellote C, Casillas R, Ramirez-Santana C, Perez-Cano FJ, Castell M, Moretones MG, et al. Premature delivery influences the immunological composition of colostrum and transitional and mature human milk. J Nutr 2011;141(6):1181–7. [78] Coppa GV, Pierani P, Zampini L, Carloni I, Carlucci A, Gabrielli O. Oligosaccharides in human milk during different phases of lactation. Acta Paediatr Suppl 1999;88(430): 89–94. [79] Okada M, Ohmura E, Kamiya Y, Murakami H, Onoda N, Iwashita M, et al. Transforming growth factor (TGF)-alpha in human milk. Life Sci 1991;48(12):1151–6. [80] Saito S, Yoshida M, Ichijo M, Ishizaka S, Tsujii T. Transforming growth factor-beta (TGF-beta) in human milk. Clin Exp Immunol 1993;94(1):220–4. [81] Flidel-Rimon O, Roth P. Effects of milk-borne colony stimulating factor-1 on circulating growth factor levels in the newborn infant. J Pediatr 1997;131(5):748–50. [82] Jackson DA, Imong SM, Silprasert A, Ruckphaopunt S, Woolridge MW, Baum JD, et al. Circadian variation in fat concentration of breast-milk in a rural northern Thai population. Br J Nutr 1988;59(3):349–63. [83] Hytten FE. Clinical and chemical studies in human lactation. Br Med J 1954;1(4855): 175–82. [84] Innis SM. Impact of maternal diet on human milk composition and neurological development of infants. Am J Clin Nutr 2014;99(3):734S–41S. [85] Lonnerdal B, Forsum E, Gebre-Medhin M, Hambraeus L. Breast milk composition in Ethiopian and Swedish mothers. II. Lactose, nitrogen, and protein contents. Am J Clin Nutr 1976;29(10):1134–41. [86] Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am 2013;60(1):49–74. [87] Innis SM. Human milk and formula fatty acids. J Pediatr 1992;120(4 Pt 2):S56–61. [88] Brenna JT, Varamini B, Jensen RG, Diersen-Schade DA, Boettcher JA, Arterburn LM. Docosahexaenoic and arachidonic acid concentrations in human breast milk worldwide. Am J Clin Nutr 2007;85(6):1457–64. [89] Koletzko B, Thiel I, Abiodun PO. The fatty acid composition of human milk in Europe and Africa. J Pediatr 1992;120(4 Pt 2):S62–70. [90] Insull Jr W, Hirsch J, James T, Ahrens Jr EH. The fatty acids of human milk. II. Alterations produced by manipulation of caloric balance and exchange of dietary fats. J Clin Invest 1959;38(2):443–50. [91] Novak EM, Innis SM. Impact of maternal dietary n-3 and n-6 fatty acids on milk medium-chain fatty acids and the implications for neonatal liver metabolism. Am J Physiol Endocrinol Metab 2011;301(5):E807–17. [92] Michaelsen KF. Nutrition and growth during infancy. The Copenhagen Cohort Study. Acta Paediatr Suppl 1997;420:1–36. [93] Butte NF, Garza C, Stuff JE, Smith EO, Nichols BL. Effect of maternal diet and body composition on lactational performance. Am J Clin Nutr 1984;39(2):296–306. [94] Dewey KG FD, Strode MA, Lonnerdal B. Human Lactation II Maternal and Environmental Factors. New York: Plenum Press; 1986.

Please cite this article as: Andreas NJ, et al, Human breast milk: A review on its composition and bioactivity, Early Hum Dev (2015), http:// dx.doi.org/10.1016/j.earlhumdev.2015.08.013