Human Milk Bioactives: Future Perspective

Abstract

Human milk is a dynamic, complex fluid that offers much more than nutrition to infants. The macronutrient content of human milk has been well characterized and described. However, human milk is not a simple matrix of protein, carbohydrate, fat, and micronutrients. The Na­tional Institutes of Health have defined bioactives in food as elements that “affect biological processes or substrates and hence have an impact on body function or condition and ulti­mately health.” Bioactives are cells, anti-infectious and anti-inflammatory agents, growth factors, and prebiotics that are naturally present in human milk. They may explain the differ­ences in health outcomes observed between breastfed and non-breastfed infants. They in­fluence the development of the immune and gastrointestinal systems, gut microbiota, neu­rodevelopment, metabolic health, and protection against infection. Human milk oligosaccharides are one bioactive that have been an increasingly popular area of research. This review provides a broad overview of some bioactive components that positively affect the immune system and touches on certain well-known growth factors present in human milk. Future research will look at the interplay of the multitude of bioactive components in human milk as a biological system and beyond singular compounds.

Introduction

Human milk is a complex, dynamic fluid providing ideal nutrition for the human infant and is uniquely designed for each mother-infant dyad. Beyond providing majority of macronutrients and micronutrients at optimal levels for growth and development, it is a source of nutrition containing bioactive components that serve a variety of biological functions, including development of the immune and gastrointestinal systems, gut microbiota, neurodevelopment, metabolic health, and protection against infection. The bioactive cells, anti-infectious and anti-inflammatory agents, growth factors, and prebiotics that are naturally present in human milk may explain the differences in health outcomes observed between breastfed and non-breastfed infants throughout the ages.

Nutritive Components of Human Milk

The composition of human milk changes throughout lactation stages from colos-trum to late lactation, varying within feeds and between mothers. Despite varia-tions in maternal nutrition, macronutrient composition of human milk is remark-ably conserved across populations with the mean macronutrient composition of mature, term milk estimated to be approximately 0.9-1.2 g/dL for protein, 3.2-3.6 g/dL for fat, and 6.7-7.8 g/dL for lactose. Energy estimates range from 65 to 70 kcal/dL [1]. Lactose is the primary carbohydrate source of human milk, and the carbohydrate content of human milk is the least variable of the macronutrients. Human milk fat is more variable in human milk, even within a feed, where the fat content of hindmilk (milk expressed at the end of a feed) can be two to three times greater than that of foremilk (milk expressed at the beginning of a feed). Palmitic and oleic acids are the dominant fatty acids in human milk. The fatty acid profile of human milk varies in relation to maternal diet, particularly in the long-chain polyunsaturated fatty acids. Casein and whey are the main protein components of human milk, and the ratio of these two changes over the course of lactation where human milk is predominantly whey in early lactation and closer to a 50:50 whey: casein ratio in late lactation. Protein levels decrease over time in human milk and are less affected by maternal diet. Micronutrient content of human milk can be dependent on maternal diet and stores, including vitamins A, B1, B2, B6, B12, D, and iodine. As the maternal diet may not always be optimal, use of a multivitamin during lactation is often recommended. Even with supplementation, vitamins K and D are often present at low levels in human milk, and therefore a vitamin K injection at birth is recommended by the American Academy of Pediatrics as well as supplementation of vitamin D in the breastfed infant [1].

Bioactive Components of Human Milk

Bioactive components of food have been defined as elements that “affect bio-logical processes or substrates and hence have an impact on body function or condition and ultimately health” [2]. As defined by the NIH, bioactive food components are constituents in foods or dietary supplements, other than those needed to meet basic human nutritional needs, that are responsible for changes in health status [3]. The commonality of these definitions is that the focus is on the impact on health, not on the nutritive aspect of the food component, such as providing energy or protein.

Human milk has thousands of bioactive components coming from a variety of sources. Some are secreted by the mammary epithelium, some are produced by cells present in the milk, and others are transported across the mammary epithelium from maternal serum [1]. Milk fat globule that is present in human milk and secreted by the mammary epithelium also contains membrane-bound proteins and lipids that it brings into human milk [4].

Broad categories of bioactive components in breastmilk include immunological and growth factors. Immunological factors include immune cells such as T cells and lymphocytes, cytokines, secretory immunoglobulin A (sIgA), and human milk oligosaccharides (HMOs) [1]. Growth factors include growth- and metabolism-regulating hormones like calcitonin and adiponectin in addition to bioactive proteins such as insulin like growth factor, vascular endothelial growth factor, and neuronal growth factors [1].

Human Milk Oligosaccharides

HMOs represent a vast, functional component of human milk making up the third most abundant proportion of the solid components of milk surpassed in concentration only by lactose and lipids. The oligosaccharide content of human milk varies between 10-15 g per liter (g/L) of mature milk and 20-25 g/L of co-lostrum [5, 6]. They are predominantly present in free form in human milk and in this free form are customarily called HMOs. Although some of these structures are also present in the milk of other mammals, oligosaccharides are much more abundant in human milk [5].

HMOs are comprised of a linear or branched elongation of lactose by the building blocks fucose, sialic acid, and N-acetylglucosamine. HMOs are generally classified into four different categories: non-fucosylated, non-sialylated (neutral), fucosylated, non-sialylated (fucosylated, neutral), non-fucosylated, si- alylated (acidic), fucosylated, sialylated (fucosylated acidic) [5]. Over 150 unique structures have been analytically separated, but about 20 structures comprise the major portion of HMOs with the remaining glycans representing a smaller fraction [5, 7]. Individual variation of milk oligosaccharides can partially be explained by material genetics, and this is well characterized by expression of two fucosyltransferases: FUT2 (secreter gene) and FUT3 (Lewis gene) [5]. Both are polymorphic with different alleles being responsible for the non-secretor (FUT2- /-) and Lewis negative (FUT3-/-) types. Milk of secretor women is abundant in 2'-fucosyllactose (2'FL), LNFP I, and other a1-2-fucosylated HMOs. In contrast, non-secretors lack the functional FUT2 enzyme, and their milk contains very little a1-2-fucosylated HMOs but does contain other fucosylated HMOs [8]. HMO content may also be influenced by nongenetic maternal and infant factors. Lactation stage, maternal BMI, maternal age, maternal diet, mode of delivery, parity, infant gestational age, and infant sex have all been suggested as factors that determine individual variation in milk oligosaccharide content [9].

A number of biological and physiological benefits have been attributed to HMOs, and these roles are mainly characterized on the basis of protective prop-erties that comprise an innate defense system for infants [5]. HMOs are selective substrates for specific intestinal bacteria, protect against infection by blocking pathogen attachment to epithelial cells, promote immunomodulatory activity, and improve gut barrier function [10].

The majority of ingested HMOs reach the large intestine where they provide selective substrates for specific gut bacteria. This prebiotic activity of HMOs is best characterized among specific bifidobacteria species, which have the capability to transport them for internal digestion or secrete glycosidases that externally hydrolyze HMOs to byproduct constituents that can be metabolized by the bacterium [11]. Bifidobacterium longum subsp infantis has a gene that expresses not only glycosidases, but also sugar transporters and glycan-binding proteins likely linked to HMO metabolism [11]. B. breve, or B. bifidum strains have also been shown to grow in the presence of select HMOs [12, 13]. HMOs metabolize into short-chain fatty acids including butyrate, the preferred energy source for colon epithelial cells, contributing to the maintenance of the gut barrier [14].

HMOs can also inhibit pathogens by competitive binding with the host cell surface receptor [15]. HMOs have been shown to provide protection against a wide spectrum of pathogens, including those associated with diarrheal, respiratory, and urinary infections and human immunodeficiency virus [16].

An increasing number of in vitro studies suggest that HMOs also exert effects independent of microbiota, by directly modulating immune responses by affecting immune cell populations and cytokine secretion [10]. HMOs may either act locally on cells of the mucosa-associated lymphoid tissues or on a systemic level [5]. When cord blood T cells are exposed to sialylated HMOs, the resulting lymphocyte response was suggestive of lymphocyte maturation and a shift toward a more balanced Th1/Th2 cytokine production [17]. In regard to neutral oligosaccharides, significant associations between levels of 2'FL in mothers' breast milk and “any allergic disease,” IgE-associated disease, eczema, and IgE-associated eczema in C-section-born infants have been reported [18].

HMOs have been considered as potentially important components in support of brain development and cognitive function. The predominance of the research in this area has focused on the role of sialylated HMOs, an important source of sialic acid in infancy [19]. However, 2'FL may specifically have an important role in early cognitive development. Vazquez et al. [20] studied the ability of 2'FL, specifically, to affect learning in a small trial of mice and rats evaluating excitatory postsynaptic potentials, various types of learning, and behavior. The results demonstrated that dietary 2'-FL exerts a positive effect on learning and memory in rodents [21, 22].

Immunological Function of Bioactives in Human Milk

The level of immunological components of breastmilk varies in the first year of life. For example, a-lactalbumin declines gradually over time with significantly lower levels in mature milk at 4-8 months of life compared to the first few days of life. Other immune factors such as lactoferrin, immunoglobulins, and transforming growth factor p1 (TGF"P1) follow a similar pattern with significantly higher levels in very early milk compared to mature milk at 2-4 weeks of life, and then remain relatively stable over the first year of life. TGF-^2 follows a slightly different pattern, declining significantly over the first month of life but then fluctuating throughout the first year [23]. The high levels of immunological components and relatively low levels of lactose and some micronutrients in colostrum suggest that its evolutionary function is more immunological than nutritive [1].

Human milk contains many immune cells such as T cells, stem cells, lympho-cytes, and macrophages that originate from the mother's mature immune system, migrating from the bloodstream to the mammary epithelium. Although the quantity of these cells differs between individuals, infants consume roughly ten billion immune cells daily in early lactation; roughly 80% of which are macro-phages [1]. In addition to pathogen phagocytosis, breastmilk macrophages have unique functions including the ability to convert into dendritic cells which then stimulate the infant's immune system via T-cell activity [1, 24]. About 6% of cells in human milk are stem cells which can differentiate into multiple tissue types and may be involved in the development of the infant's immune cells [25]. During infection of either the mother or infant, an increase in leukocytes has been observed indicating a protective function of leukocytes for the infant [24].

Immunoglobulins are central to the passive transfer of immunity from mother to infant [26]. sIgA is the most abundant immunoglobulin in human milk making up over 90% of total immunoglobulins, although levels decline over time [24, 27]. Other immunoglobulins, such as immunoglobulin M follows a similar pattern whereas immunoglobulin G is higher in mature milk compared to transitional milk [27]. Maternal sIgA provides passive immunity by binding to pathogens and blocking infection as well as facilitating their removal by entrapping them in mucus [24]. Not only does maternal sIgA provide passive immunity, it stimulates the infant immune system via dendritic cell activation without stimulating an inflammatory response [1, 28].

Human milk is the primary source of lactoferrin in the infant gut [29]. It is known for having antibacterial, antimicrobial, antiviral, antiparasitic, antifungal, antioxidant, anti-inflammatory, and even anticancerous properties [29]. Since a major role of lactoferrin is to transport iron in the plasma, it has a strong iron binding capability enabling it to withhold iron from iron dependent pathogens [29, 30]. Lactoferrin receptors on the mucosal surface of intestinal cells allow for lactoferrin to enter the intestinal microvilli where it stimulates the immune response by inducing leukocyte activity such as increased natural killer cell and phagocyte activity. Lactoferrin has also been shown to increase and decrease production of proinflammatory cytokines, dendritic cells, T cells, and B cells [29].

Osteopontin is present in most human tissues and fluids but is most abundant in human milk. Osteopontin is involved in several biological functions including bone remodeling, inhibiting calcification of soft tissues, and plays an important role in the development of immune response and modulation. It has been hypothesized that osteopontin attaches to monocytes directing them to sites acting as a transporter. It influences the function of other immune cells such as macrophages, T cells, and dendritic cells. It also has antibacterial properties, binding itself to foreign microorganisms making them more susceptible to phagocytosis [31].

Gangliosides are present in almost all human tissues and play a pivotal role in infant growth and immune response. Total ganglioside concentration is sig-nificantly higher in mature milk compared to colostrum but is highly variable between individuals. Gangliosides follow different trajectories over time with GD2 decreasing and GM3 increasing over the course of lactation [32]. They act as decoy receptors for pathogens preventing binding to intestinal cells. They also play a role in immune system development such as influencing the production of IgA and cytokines as well as promoting maturation of dendritic cells [33].

Reactive oxygen species (ROS) are molecules that participate in cellular sig-naling pathways, but in excess can cause cellular damage. To combat this, the body has a variety of antioxidant systems to maintain the balance between ROS and antioxidants. If excessive ROS production occurs or insufficient antioxidant systems persist, the result is oxidative stress. Examples of endogenous antioxidants are enzymatic proteins like superoxide dismutase or glutathione peroxidase. Other examples are those antioxidants found in foods such as vitamins and carotenoids. All these examples plus more are present in human milk to help the infant combat excessive ROS and protect against disease. Levels of antioxidants range, depending on time of collection and geographic site of collection. The total antioxidant capacity of human milk seems to be higher in colostrum compared to mature milk [25].

Bioactives Affecting Growth

In addition to the immunological impact human milk bioactives impart on the infant, human milk contains a variety of growth factors. Growth factors such as epidermal growth factor, growth hormone and insulin-like growth factor-1 are present in human milk and affect the morphology and function of the gastroin-testinal tract [34, 35]. Insulin-like growth factor 1 and its binding proteins also play a role in programming infant growth trajectories due to their well-established roles in linear growth, and body composition [36]. Brain-derived neurotrophic factor and glial cell-line derived neurotrophic factor are neuronal growth factors found in human milk that play a role in the maturation of the enteric nervous system [37]. Adipokines are cytokines from adipocytes that modify weight gain and body composition in infants that have long-term effects on metabolic programming and are involved in regulation of food intake and energy balance. Examples include adiponectin, ghrelin, and leptin. Adiponectin tends to be the most abundant and is a multifunctional hormone that regulates metabolism and suppresses inflammation. Human milk adiponectin also seems to have an influence on infant growth [25]. Human milk contains many other growth factors beyond those mentioned here affecting the vascular, neuronal, and other organ systems of the infant [1].

Factors That Influence Bioactives in Human Milk

There are several maternal factors that influence the quantity, quality, and com-position of bioactives in human milk. These would include physical factors such as genotype, body mass index, physiologic state, and underlying medical conditions, environmental factors such diet, mode of delivery, medication use, and other factors such as infant gestational age and stage of lactation [9, 38]. Influencing modifiable factors may optimize human milk such that it improves infant outcomes ranging from infectious and allergic diseases to cognitive, gastrointes-tinal, and immune system development. 

Conflict of Interest Statement

K.L.F., B.D.K., L.A.C. and R.S.C. are employees of Nestlé Nutrition.

References

1    Ballard O, Morrow AL. Human milk composition: nutrition and bioactive factors. Pediatr Clin North Am. 2013;60(1):49-74.
2    Schrezenmeir J, Korhonen H, Williams C, et al. Foreword. Br J Nutr. 2000;84(1):S1.
3    NIH, Office of Dietary Supplements. Federal Reg-ister Vol. 69 No. 179 FR Dec 04-20892, Sept 16, 2004 [cited 2021 Sept 6]. Available from: ods. od.nih/gov/Research/Bioactive_Food_Compo- nents_Initiative.aspx.
4    Cavaletto M, Giuffrida MG, Conti A. The pro- teomic approach to analysis of human fat globule membrane. Clin Chim Acta. 2004;347(1-2):41-8.
5    Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012;22:1147-62.
6    Kunz C, Kuntz S, Rudloff S. Bioactivity of human milk oligosaccharides. In: Moreno FM, Sanz ML, editors. Food Oligosaccharides: Production, Analysis and Bioactivity. Chichester: John Wiley & Sons; 2014. pp 5-20.
7    Thurl S, Munzert M, Henker J, et al. Variation of human milk oligosaccharides in relation to milk groups and lactational periods. Br J Nutr. 2010;104:1261-71.
8    Thurl S, Henker J, Siegel M, et al. Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides. Glyco- conj J. 1997;14:795-9.
9    Han SM, Derraik J, Binia A, et al. Maternal and infant factors influencing human milk oligosaccharide composition: beyond maternal genetics. J Nutr. 2021;151(6):1383-93.
10    Donovan SM, Comstock SS. Human milk oligo-saccharides influence neonatal mucosal and sys-temic immunity. Ann Nutr Metab. 2016;69(Suppl 2):42-51.
11    Milani C, Duranti S, Bottacini F, et al. The first microbial colonizers of the human gut: composi-tion, activities, and health implications of the infant gut microbiota. Microbiol Mol Biol Rev. 2017;81(4):e00036-17.
12    Marcobal A, Barboza M, Froehlich JW, et al. Con-sumption of human milk oligosaccharides by gut-related microbes. J Agric Food Chem. 2010;58:5334-40.
13    Asakuma S, Hatakeyama E, Urashima T, et al. Physiology of consumption of human milk oligo-saccharides by infant gut-associated bifidobacteria. J Biol Chem. 2011;286:34583-92.
14    Rivière A, Selak M, Lantin D, et al. Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut. Front Microbiol. 2016;7:979.
15    Newburg DS. Oligosaccharides in human milk and bacterial colonization. J Pediatr Gastroenterol Nutr. 2000;30(Suppl 2):S8-S17.
16    Asadpoor M, Peeters C, Henricks PA, et al. Anti- pathogenic functions of non-digestible oligosac-charides in vitro. Nutrients. 2020;12(6):1789.
17    Eiwegger T, Stahl B, Schmitt J, et al. Human milk-derived oligosaccharides and plant-derived oligosaccharides stimulate cytokine production of cord blood T-cells in vitro. Pediatr Res.
2004;56:536-40.
18    Sprenger N, Odenwald H, Kukkonen AK, et al. FUT2-dependent breast milk oligosaccharides and allergy at 2 and 5 years of age in infants with high hereditary allergy risk. Eur J Nutr. 2017;56:1293-301.
19    ten Bruggencate SJ, Bovee-Oudenhoven IM, Feitsma AL, et al. Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides. Nutr Rev. 2014;72:377-89.
20    Vazquez E, Barranco A, Ramirez M, et al. Effects of a human milk oligosaccharide, 2'-fucosyllac- tose, on hippocampal long-term potentiation and learning capabilities in rodents. J Nutr Biochem. 2015;26(5):455-65.
21    Berger PK, Plows JF, Jones RB, et al. Human milk oligosaccharide 2'-fucosyllactose links feedings at 1 month to cognitive development at 24 months in infants of normal and overweight mothers. PLoS One. 2020;15(2):e0228323.
22    Oliveros E, Martin MJ, Torres-Espinola FJ, et al. Human milk levels of 2'-fucosyllactose and 6'-si- alyllactose are positively associated with infant neurodevelopment and are not impacted by ma-ternal BMI or diabetic status. J Nutr Food Sci. 2021;4:024.
23    Affolter M, Garcia-Rodenas CL, Vinyes-Pares G, et al. Temporal changes of protein composition in breast milk of Chinese urban mothers and impact of caesarean section delivery. Nutrients. 2016;8:504.
24    Cacho NT, Lawrence RM. Innate immunity and breast milk. Front Immunol. 2017;8:584.
25    Gila-Diaz A, Arribas SM, Algara A, et al. A review of bioactive factors in human breastmilk: a focus on prematurity. Nutrients. 2019;11:1307.
26    Hurley WL, Theil PK. Perspectives on immuno-globulins in colostrum and milk. Nutrients. 2011;3(4):442-74.
27    Gao X, McMahon RJ, Woo JG, et al. Temporal changes in milk proteomes reveal developing milk functions. J Proteome Res. 2012;11(7):3897- 907.
28    Kadaoui KA, Corthésy. Secretory IgA mediates bacterial translocation to dendritic cells in mouse Peyer's patches with restriction to mucosal com-partment. J Immunol. 2007;179(11):7751-7.
29    Kanwar JR, Roy K, Patel Y, et al. Multifunctional iron bound lactoferrin and nanomedicinal ap-proaches to enhance its bioactive functions. Mol-ecules. 2015;20:9703-31.
30    Lonnerdal B. Bioactive proteins in human milk: health, nutrition, and implications for infant for-mulas. J Pediatr. 2016;173S:S4-S9.
31    Schack L, Lange A, Kelsen J, et al. Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas. J Diet Sci. 2009;92(11):578-85.
32    Giuffrida F, Cruz-Hernandez C, Bertschy E, et al. Temporal changes of human breast milk lipids of Chinese mothers. Nutrients. 2016;8:17.
33    Rueda R. The role of dietary gangliosides on im-munity and the prevention of infection. Br J Nutr. 2007;98(Suppl 1):S68-S73.
34    Hirai C, Ichiba H, Saito M, et al. Trophic effect of multiple growth factors in amniotic fluid or human milk on cultured human fetal small intestinal cells. J Pediatr Gastroenterol Nutr. 2002;34(5):524-8.
35    Dvorak B. Milk epidermal growth factor and gut protection. J Pediatr. 2010;156(2 Suppl):S31-S35.
36    Hoeflich A, Meyer Z. Functional analysis of the IGF-system in milk. Best Pract Res Clin Endocrinol Metab. 2017(31):409-18.
37    Rodrigues D, Li A, Nair D, Blennerhassett M. Glial cell line-derived neurotrophic factor is a key neu- rotrophin in the postnatal enteric nervous system. Neurogastroenterol Motil. 2011;23:e44-e56.
38    Samuel TM, Zhou Q, Giuffrida F, et al. Nutritional and non-nutritional composition of human milk is modulated by maternal, infant, and methodological factors. Front Nutr. 2020;7:576133.
39    Christian P, Smith ER, Lee SE, et al. The need to study human milk as a biological system. Am J Clin Nutr. 2021;113:1063-1072.