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Intestinal Dysbiosis in the Infant and the Future of Lacto-Engineering to Shape the Developing Intestinal Microbiome

      Abstract

      Purpose

      The goal of this study was to review the role of human milk in shaping the infant intestinal microbiota and the potential of human milk bioactive molecules to reverse trends of increasing intestinal dysbiosis and dysbiosis-associated diseases.

      Methods

      This narrative review was based on recent and historic literature.

      Findings

      Human milk immunoglobulins, oligosaccharides, lactoferrin, lysozyme, milk fat globule membranes, and bile salt–stimulating lipase are complex multifunctional bioactive molecules that, among other important functions, shape the composition of the infant intestinal microbiota.

      Implications

      The co-evolution of human milk components and human milk–consuming commensal anaerobes many thousands of years ago resulted in a stable low-diversity infant microbiota. Over the past century, the introduction of antibiotics and modern hygiene practices plus changes in the care of newborns have led to significant alterations in the intestinal microbiota, with associated increases in risk of dysbiosis-associated disease. A better understanding of mechanisms by which human milk shapes the intestinal microbiota of the infant during a vulnerable period of development of the immune system is needed to alter the current trajectory and decrease intestinal dysbiosis and associated diseases.

      Keywords

      Introduction

      Whether shaped by the tremendous pressures of evolution or an incredible feat of bioengineering (or both), human milk contains not only a near-perfect balance of macronutrients and micronutrients for term infants but also a dizzying array of bioactive molecules to protect the infant against infection, shape the intestinal microbiota, and tune the developing innate and adaptive immune systems of the infant. The abandonment of breastfeeding in favor of the convenience of formula feeding in the middle decades of the 20th century is one of several factors leading to an increase in intestinal dysbiosis (Table I) and its associated diseases (Table II). Attempts to improve infant formulas with the addition of components similar to those found in human milk have met with only limited success to date.
      Table IFactors contributing to intestinal dysbiosis.
      Antibiotics
      High-fat, low-fiber diet
      Cesarean delivery
      Formula feeding
      Antimicrobial soaps and cleansers
      High-heat dishwashers
      Modern hygiene practices
      Table IIDysbiosis-associated diseases (all associations are between intestinal dysbiosis and the noted disease except early-onset sepsis and Group B streptococcal [GBS] sepsis in which the association is with maternal vaginal dysbiosis or GBS colonization of the genitourinary tract).
      GroupDisease
      Preterm infantNecrotizing enterocolitis,
      • Pammi M.
      • et al.
      Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis.
      ,
      • Baranowski J.R.
      • Claud E.C.
      Necrotizing enterocolitis and the preterm infant microbiome.
      early-onset sepsis,
      • Brown R.G.
      • et al.
      Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin.
      ,
      • Bennett P.R.
      • Brown R.G.
      • MacIntyre D.A.
      Vaginal microbiome in preterm rupture of membranes.
      late-onset sepsis
      • Graspeuntner S.
      • et al.
      Gut dysbiosis with bacilli dominance and accumulation of fermentation products precedes late-onset sepsis in preterm infants.
      ,
      • El Manouni El Hassani S.
      • et al.
      Profound pathogen-specific alterations in intestinal microbiota composition precede late-onset sepsis in preterm infants: a longitudinal, multicenter, case-control study.
      Term infantGBS sepsis,
      • Zhou P.
      • et al.
      Perinatal antibiotic exposure affects the transmission between maternal and neonatal microbiota and is associated with early-onset sepsis.
      colic,
      • Dubois N.E.
      • Gregory K.E.
      Characterizing the intestinal microbiome in infantile colic: findings based on an integrative review of the literature.
      ,
      • Ouald Chaib A.
      • Levy E.I.
      • Ouald Chaib M.
      • Vandenplas Y.
      The influence of the gastrointestinal microbiome on infant colic.
      antibiotic-associated diarrhea
      Child/adultClostridium difficile colitis, antibiotic-associated diarrhea, atopic dermatitis,
      • Pothmann A.
      • Illing T.
      • Wiegand C.
      • Hartmann A.A.
      • Elsner P.
      The microbiome and atopic dermatitis: a review.
      food allergy,
      • Shu S.A.
      • et al.
      Microbiota and food allergy.
      asthma,
      • Noval Rivas M.
      • Crother T.R.
      • Arditi M
      The microbiome in asthma.
      diabetes,
      • Vatanen T.
      • et al.
      The human gut microbiome in early-onset type 1 diabetes from the TEDDY study.
      ,
      • Arora A.
      • et al.
      Unravelling the involvement of gut microbiota in type 2 diabetes mellitus.
      obesity,
      • Vallianou N.
      • Dalamaga M.
      • Stratigou T.
      • Karampela I.
      • Tsigalou C.
      Do antibiotics cause obesity through long-term alterations in the gut microbiome? A review of current evidence.
      breast cancer,
      • Parida S.
      • Sharma D.
      The microbiome-estrogen connection and breast cancer risk.
      colon cancer,
      • Foegeding N.J.
      • Jones Z.S.
      • Byndloss M.X.
      Western lifestyle as a driver of dysbiosis in colorectal cancer.
      inflammatory bowel disease,
      • Matsuoka K.
      • Kanai T.
      The gut microbiota and inflammatory bowel disease.
      NASH/NAFLD,
      • Hrncir T.
      • et al.
      Gut microbiota and NAFLD: pathogenetic mechanisms, microbiota signatures, and therapeutic interventions.
      Parkinson disease,
      • Dumitrescu L.
      • et al.
      Serum and fecal markers of intestinal inflammation and intestinal barrier permeability are elevated in Parkinson's disease.
      Alzheimer disease,
      • Ivakhniuk T.
      • Ivakhniuk Y.
      Intestinal microbiota in Alzheimer's disease.
      anxiety/depression,
      • Chinna Meyyappan A.
      • Forth E.
      • Wallace C.J.K.
      • Milev R.
      Effect of fecal microbiota transplant on symptoms of psychiatric disorders: a systematic review.
      ,
      • Settanni C.R.
      • Ianiro G.
      • Bibbo S.
      • Cammarota G.
      • Gasbarrini A.
      Gut microbiota alteration and modulation in psychiatric disorders: current evidence on fecal microbiota transplantation.
      graft-versus-host disease,
      • Stein-Thoeringer C.K.
      • et al.
      Lactose drives Enterococcus expansion to promote graft-versus-host disease.
      lupus,
      • Vieira J.R.P.
      • Rezende A.T.O.
      • Fernandes M.R.
      • da Silva N.A.
      Intestinal microbiota and active systemic lupus erythematosus: a systematic review.
      rheumatoid arthritis,
      • Li M.
      • Wang F.
      Role of intestinal microbiota on gut homeostasis and rheumatoid arthritis.
      multiple sclerosis
      • Wang X.
      • et al.
      Role of gut microbiota in multiple sclerosis and potential therapeutic implications.
      NASH/NAFLD = nonalcoholic steatohepatitis/nonalcoholic fatty liver disease.
      The purpose of the present review was to briefly summarize the evolution of human milk and the evidence that intestinal dysbiosis is part of the pathogenesis of several inflammatory diseases. We also describe how milk components have the potential to restore the infant microbiome, as well as the progress and future directions of bioengineering infant nutrition to decrease intestinal dysbiosis and dysbiosis-associated diseases. It is worth nothing that this is a narrative rather than a systematic review and does not touch on all bioactive aspects of human milk. The focus is on those milk molecules for which strong evidence suggests a role in shaping the microbiota; thus, human milk hormones, cytokines, growth factors, exosomes, and microRNAs are not discussed.

      The History of Human Milk

      Providing nutrition to the immature offspring is essential to the survival of a species. In the human, the placenta is the primary source of both nutrition and immunoglobulins for the fetus, complemented by swallowed amniotic fluid, which is rich in nutrients and bioactive molecules. Following birth, there is a paradigm shift in both nutrition and immune protection, with milk filling both roles for the first months of life (Figure 1). Depending on your viewpoint, human milk has either evolved over millions of years to maximize chances of survival of the infant at significant cost to the mother or was the most complex and complete nutritional creation of all time.
      Figure 1
      Figure 1Prenatal (A) and postnatal (B) transfer from mother to offspring of nutrients; immunologic, antimicrobial, and growth factors; and microbes. Panel A displays transplacental passage of nutrients and immunoglobulin G (IgG; blue) providing nutrition and immunity to the fetus. Swallowed amniotic fluid is rich in growth factors promoting fetal intestinal development and contains nutrients. During vaginal birth, the infant is exposed to maternal microbes. Panel B, Postnatally, breast milk provides secretory immunoglobulin A (SIgA), nutrients, microbes, and several immune, antimicrobial, and growth factors. Skin-to-skin care exposes the infant to maternal skin microbes. Copyright Satyan Lakshminrusimha.
      Lactation and the consumption of milk are the defining characteristics of mammals; clues to its origin can be found by studying the evolution of mammary glands,
      • Schep R.
      • et al.
      Control of Hoxd gene transcription in the mammary bud by hijacking a preexisting regulatory landscape.
      prolactin,
      • Dobolyi A.
      • et al.
      Secretion and function of pituitary prolactin in evolutionary perspective.
      casein and whey proteins, and oligosaccharides.
      • Oftedal O.T.
      The evolution of lactation in mammalian species.
      ,
      • Oftedal O.T.
      The evolution of milk secretion and its ancient origins.
      The predecessors to mammals likely appeared >300 million years ago, with mammals, hominids, and Homo sapiens appearing about 178 million, 4 million, and 150 thousand years ago, respectively. Comparisons between human milk and the milk of non-human primates exhibit dramatic differences in oligosaccharide composition
      • Tao N.
      • et al.
      Evolutionary glycomics: characterization of milk oligosaccharides in primates.
      and protein content,
      • Beck K.L.
      • et al.
      Comparative proteomics of human and macaque milk reveals species-specific nutrition during postnatal development.
      suggesting that changes in lactation were central to the emergence and survival of our species with its large brains and vulnerable offspring. Unfortunately, the fossil record provides no information regarding differences in milk composition between Homo sapiens and earlier hominids.
      Lacto-engineering refers to tailoring milk to the needs of the infant. It is most commonly used to describe interventions to alter the composition of human milk such as feeding of hindmilk to increase fat intake or addition of fortifiers rich in macronutrients and micronutrients. In this paper, we broaden the term to include tailoring milk, either in the breast or after expression, to alter the intestinal microbiota of the infant.

      Intestinal Dysbiosis

      Bacteria were among the first living species on earth and have existed and evolved for several hundred millions to several billions of years. The primary drivers of bacterial evolution include the capacity to survive in a given location or niche; examples include defense mechanisms, virulence factors, and, perhaps most importantly, competition for nutrients. The emergence of a subspecies of Bifidobacterium with a unique set of genes encoding transport proteins and glycosidases necessary for the complete digestion of the dozens of unique human milk oligosaccharides at roughly the same time as the appearance of Homo sapiens is a compelling example of co-evolution of 2 interdependent species.
      • Duranti S.
      • et al.
      Bifidobacterium bifidum and the infant gut microbiota: an intriguing case of microbe-host co-evolution.
      • Sakanaka M.
      • et al.
      Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis.
      • Yamada C.
      • et al.
      Molecular insight into evolution of symbiosis between breast-fed infants and a member of the human gut microbiome Bifidobacterium longum.
      For most of human history, mothers have provided an array of human milk oligosaccharides and other bioactive molecules to shape the intestinal microbiota of their infants. The few bacteria capable of consuming human milk oligosaccharides (select Bifidobacterium and Bacteroides species) thus have had a distinct advantage in colonizing the neonatal intestinal tract. This pattern of a stable infant community with low diversity and anti-inflammatory properties differs dramatically from the intestinal microbiota of the adult and plays an important role in instructing and shaping both the innate and the adaptive immune systems.
      • Hornef M.W.
      • Torow N.
      Layered immunity' and the 'neonatal window of opportunity'—timed succession of non-redundant phases to establish mucosal host-microbial homeostasis after birth.
      ,
      • Xu C.
      • Zhu H.
      • Qiu P.
      Aging progression of human gut microbiota.
      Dysbiosis refers to alterations in the microbiota of a given niche associated with disease. In humans, the most common examples include Clostridium difficile colitis and antibiotic-associated diarrhea. In both cases, exposure to antibiotics alters the intestinal microbiota, creating an opportunity for pathobionts to flourish, which leads to inflammation that may be acute or chronic. The recent descriptions of associations between alterations in the intestinal microbiota and a wide variety of autoimmune and acute and chronic inflammatory diseases (Table II) have challenged assumptions about the origins and pathogenesis of many diseases that have been increasing over the past several decades.
      • Wu W.H.
      • Zegarra-Ruiz D.F.
      • Diehl G.E.
      Intestinal microbes in autoimmune and inflammatory disease.
      It seems likely that significant changes in hygiene, diet, exposure to antibiotics (both prescribed and in foods), and many life-saving medical advances (eg, cesarean delivery) have had the unexpected consequence of fundamentally altering the intestinal microbiota and that these changes now span several generations such that mothers delivering infants today are not colonized with and therefore unable to transfer many bacterial species that colonized our ancestors (Figure 2).
      • Rachid R.
      • Stephen-Victor E.
      • Chatila T.A.
      The microbial origins of food allergy.
      ,
      • Ronan V.
      • Yeasin R.
      • Claud E.C.
      Childhood development and the microbiome—the intestinal microbiota in maintenance of health and development of disease during childhood development.
      The result may be a loss of some or all of the foundational protective bacteria that evolved with humans over thousands of years.
      • Henrick B.M.
      • et al.
      Elevated fecal pH indicates a profound change in the breastfed infant gut microbiome due to reduction of Bifidobacterium over the past century.
      Mechanisms by which gut microbes influence the developing immune system include direct impacts on T helper type 1/T helper type 2/regulatory T cell (Treg) balance,
      • Walker W.A.
      • Iyengar R.S.
      Breast milk, microbiota, and intestinal immune homeostasis.
      intestinal inflammation and permeability,
      • He Y.
      • Lawlor N.T.
      • Newburg D.S.
      Human milk components modulate Toll-like receptor-mediated inflammation.
      and indirect effects of microbial metabolites, most notably butyrate,
      • Di Costanzo M.
      • De Paulis N.
      • Biasucci G.
      Butyrate: a link between early life nutrition and gut microbiome in the development of food allergy.
      indole-3-lactic acid,
      • Meng D.
      • et al.
      Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine.
      ,
      • Ehrlich A.M.
      • et al.
      Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells.
      and other bacterial tryptophan metabolites.
      • Scott S.A.
      • Fu J.
      • Chang P.V.
      Microbial tryptophan metabolites regulate gut barrier function via the aryl hydrocarbon receptor.
      Figure 2
      Figure 2Role of breastfeeding in establishing an intestinal microbiota rich in Bifidobacterium and Bacteroides species. Factors that can disrupt this process such as delivery by cesarean section, antibiotic therapy, and formula feeding are shown. Secretory immunoglobulin A (SIgA) in breast milk provides local, mucosal immunity in the intestine. Immunoglobulin G (IgG) antibodies in the breast milk may be absorbed to provide systemic immunity. Breast milk can also enhance memory lymphocytes to reside in the Peyer's patches, leading to long-standing immunity against enteral pathogens. FcRn = neonatal Fc receptor; hMO = human milk oligosaccharide. Copyright Satyan Lakshminrusimha.

      Human Milk Components That Shape the Infant Intestinal Microbiota

      Immunoglobulins

      Neonates are born immunologically immature and susceptible into an environment abundant in pathogens. This susceptibility is evident in the high rates of infant mortality from diarrheal and respiratory infections and has been attributed to immature innate and adaptive immunity, as well as a deficiency in diversity of the commensal microbiota.
      • Kamada N.
      • Chen G.Y.
      • Inohara N.
      • Nunez G.
      Control of pathogens and pathobionts by the gut microbiota.
      Transfer of maternal antibodies by milk provides protection against pathogens until immune system maturation, particularly against organisms that gain access via mucosal barriers (Figure 3).
      Figure 3
      Figure 3Components of breast milk with immunomodulatory function that are harmed by pasteurization (blue boxes) or unharmed by pasteurization (red boxes). SIgA = secretory immunoglobulin A. Copyright Satyan Lakshminrusimha.
      Breast milk antibodies are predominantly made up of secretory immunoglobulin A (SIgA) (>90%) but also consist of immunoglobulin M and immunoglobulin G (IgG). SIgA (and immunoglobulin M, to a lesser extent) work locally by protecting the neonatal mucosal surfaces through mechanisms such as toxin or adhesin neutralization, bacterial agglutination, and enchained growth,
      • Harris N.L.
      • et al.
      Mechanisms of neonatal mucosal antibody protection.
      • Zheng W.
      • et al.
      Microbiota-targeted maternal antibodies protect neonates from enteric infection.
      • Moor K.
      • et al.
      High-avidity IgA protects the intestine by enchaining growing bacteria.
      ultimately physically impeding bacterial interactions with the gut tissues (“immune exclusion”) and thus preventing inflammation (Figure 2). This ability for exclusion of luminal bacteria is not critically dependent on antibody specificity.
      • Moor K.
      • et al.
      High-avidity IgA protects the intestine by enchaining growing bacteria.
      ,
      • Rogier E.W.
      • et al.
      Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.
      Interactions between milk SIgA and a subclass of Tregs may be of particular importance. Tregs limit inflammation but delay pathogen clearance. Although they are important during initial colonization of the neonate, mechanisms regarding regulation of Tregs are unclear. In mice, Tregs are induced by the intestinal microbiota around day 14 of life (early weaning), but maternal milk SIgA influenced Treg numbers much earlier (the first 3 days of life), and this impact persisted over the lifetime of the mouse and even into subsequent generations. Milk SIgA seems to create a set-point for Tregs that is inherited from mother to pup in a nongenetic, nonepigenetic, nonmicrobe mechanism with long-term multigenerational consequences.
      • Ramanan D.
      • et al.
      An immunologic mode of multigenerational transmission governs a gut treg setpoint.
      ,
      • Zimmermann J.
      • Macpherson A.J.
      Breast milk modulates transgenerational immune inheritance.
      Antibody-specific SIgA is also present in breast milk and targets common intestinal and respiratory pathogens in the mother's environment.
      • Brandtzaeg P.
      Mucosal immunity.
      The mother's intestinal microbiota influences production of plasma cells in the Peyer's patches of the maternal small intestine; these plasma cells, programmed by maternal gut microbes, secrete SIgA into the milk.
      • Usami K.
      • et al.
      The gut microbiota induces Peyer's-patch-dependent secretion of maternal IgA into milk.
      SIgA, however, appears to have limited importance in supporting systemic immunity, except perhaps in preterm infants
      • Weaver L.T.
      • Wadd N.
      • Taylor C.E
      • Greenwell J.
      • Toms G.L
      The ontogeny of serum IgA in the newborn.
      in whom IgA absorption is believed to be secondary to greater passive intestinal permeability. In addition, neonatal IgA production is enhanced from maternally derived microbiota and can be transported to mucosal secretions, although evidence to support a protective role of this particular IgA against common enteric pathogens compared with SIgA found in breast milk is lacking.
      • Mu Q.
      • et al.
      Regulation of neonatal IgA production by the maternal microbiota.
      The impact of milk SIgA on the infant intestinal microbiota is supported by the following observations: individuals and animals deficient in IgA have an increased relative abundance of Enterobacteriaceae in their feces, and IgA appears to support the growth of obligate anaerobes such as Bacteroides in the gut.
      • Gopalakrishna K.P.
      • Hand T.W.
      Influence of maternal milk on the neonatal intestinal microbiome.
      Provision of maternal SIgA in breast milk not only altered the intestinal microbiota of the pups but resulted in alterations of intestinal epithelial cell gene expression that persisted into adulthood.
      • Rogier E.W.
      • et al.
      Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.
      Passive immunity is also transferred to the neonate by maternal antigen-specific IgG induced by the maternal microbiota or vaccination
      • Zheng W.
      • et al.
      Microbiota-targeted maternal antibodies protect neonates from enteric infection.
      (Figure 2). The neonatal Fc receptor expressed on intestinal epithelial cells mediates the neonatal uptake of maternal-derived IgG from ingested milk into the small intestine, which is the same receptor that aids transport of maternal IgG across the placenta. In the mouse, maternal pathogen-specific IgG in the neonatal gut lumen, but not IgA or immunoglobulin M, plays a critical role in neonatal protection.
      • Caballero-Flores G.
      • et al.
      Maternal immunization confers protection to the offspring against an attaching and effacing pathogen through delivery of IgG in breast milk.
      In this model, protective immunity against enteric infection was induced after oral infection but not parenteral immunization, correlating with the presence of pathogen-specific IgA and IgG in the intestinal lumen. Both oral and parenteral immunization of dams protected offspring against oral pathogen challenge, correlating with lower pathogen loads in feces, liver, and spleen of the pups as well as reduced inflammation and mortality. Maternal IgG opsonizes pathogens, increases phagocytosis by neutrophils, and reduces pathogen attachment to the intestinal epithelium during the neonatal period. Long-lasting immunologic memory relies on the generation of memory B cells in the germinal centers of intestinal Peyer's patches and lymphoid follicles, which differentiate into plasma cells that produce mucosal antibodies in the gut.
      • Pasetti M.F.
      • Simon J.K.
      • Sztein M.B.
      • Levine M.M.
      Immunology of gut mucosal vaccines.
      ,
      • Czerkinsky C.
      • Holmgren J.
      Vaccines against enteric infections for the developing world.
      Maternal breast milk–derived IgG also plays an important role in establishing intestinal immune homeostasis after microbial colonization and exposure to commensal antigens. Maternal antibody isotypes IgG2b and IgG3 in breast milk are important in promoting functional mutualism with the developing neonatal intestinal microbiota by limiting mucosal adaptive immune responses through dampening the development of T follicular helper cells and subsequent germinal center responses against intestinal microorganisms.
      • Koch M.A.
      • et al.
      Maternal IgG and IgA antibodies dampen mucosal T helper cell responses in early life.
      These antibody responses are T-cell independent and largely Toll-like receptor (TLR) dependent. This modulation of the interaction between the microbiota and intestinal mucosa appears to play an important role in the prevention of necrotizing enterocolitis (NEC).
      • Gopalakrishna K.P.
      • et al.
      Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants.
      Maternal natural antibodies (mNabs) are antibodies acquired without known exposure to the pathogen or through immunization. Using a murine model, Zheng et al
      • Zheng W.
      • et al.
      Microbiota-targeted maternal antibodies protect neonates from enteric infection.
      showed how a single commensal species can induce cross-reactive mNabs that protect against pathogens. These IgG mNabs can be assimilated directly from ingested milk into serum by a neonatal Fc receptor–dependent process. IgG binding to the microbial surface and entering the bloodstream can additionally drive immune-effector functions such as complement-dependent bacteriolysis and opsonization, protecting against invasive pathogenic infections.

      Future Directions

      Further research is needed to understand the mechanisms underlying milk immunoglobulin production (eg, how do alterations in the maternal gut microbiota, maternal disease processes and maternal genetics/diet/medications influence milk SIgA levels?), the impact of milk SIgA on the milk microbiota, mechanisms by which milk SIgA shape the infant gut microbiota, and the importance of antibodies found in relatively low abundance (eg, specific high-affinity IgG antibodies, mNabs). Standard pasteurization denatures milk immunoglobulins; development of novel methods to ensure safety of human milk without damaging antibodies would improve the protective effects of donor milk. Immunization during pregnancy is currently limited to influenza, pertussis, and coronavirus disease 2019, with strong evidence that transfer of antibodies through milk is effective at preventing disease in the infant for influenza and pertussis. Novel vaccines against common causes of diarrhea and respiratory infections that could be safely given during pregnancy and lactation may be beneficial in decreasing morbidity and mortality in developing countries. Clinical trials of these vaccines should include analysis of milk immunoglobulins and the infant gut microbiota.

      Human Milk Oligosaccharides

      All mammals produce oligosaccharides of varying structure, diversity, and size, with humans exhibiting the highest degree of complexity.
      • Tao N.
      • et al.
      Evolutionary glycomics: characterization of milk oligosaccharides in primates.
      ,
      • Wrigglesworth D.J.
      • et al.
      High-throughput glycomic analyses reveal unique oligosaccharide profiles of canine and feline milk samples.
      More than 200 structures of human milk oligosaccharides (hMOs) have been characterized, with most women producing several dozen structures,
      • Wu L.D.
      • Ruhaak L.R.
      • Lebrilla C.B.
      Analysis of milk oligosaccharides by mass spectrometry.
      commonly divided into categories (fucosylated, sialylated, or nondecorated [meaning no fucose or sialic acid]). hMOs are diverse, as well as highly abundant, representing a greater volume in human milk than protein. Although there is variation between women and across geographic regions, hMO abundance is 9 to 22 g/L in colostrum, 8 to 19 g/L in transitional milk, 6 to 15 g/L at 1 month of lactation, and 4 to 6 g/L after 6 months
      • Thum C.
      • et al.
      Changes in HMO concentrations throughout lactation: influencing factors, health effects and opportunities.
      ; however, the human infant is unable to digest these oligosaccharides to utilize them as an energy source. The compelling question has been, why does a mother expend tremendous energy to produce a wide variety and large volume of hMOs at great cost to herself when these relatively small sugar molecules are not digestible by her infant? Analyses involving culture media in which hMOs are the only carbon source have shown that very few species of bacteria that inhabit the human gut are able to transport or digest hMOs (mostly limited to just a few species and subspecies of Bifidobacterium and Bacteroides).
      • Salli K.
      • et al.
      Selective utilization of the human milk oligosaccharides 2′-fucosyllactose, 3-fucosyllactose, and difucosyllactose by various probiotic and pathogenic bacteria.
      • Yu Z.T.
      • Chen C.
      • Newburg D.S.
      Utilization of major fucosylated and sialylated human milk oligosaccharides by isolated human gut microbes.
      • Sela D.A.
      • Mills D.A.
      Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides.
      Thus, the primary purpose of hMOs in mother's milk appears to be to provide a selective advantage to a few commensal bacteria to shape the intestinal microbiota of the infant.
      • Wang M.
      • et al.
      Fecal microbiota composition of breast-fed infants is correlated with human milk oligosaccharides consumed.
      This hypothesis is supported by the following direct observations: (1) increased total hMOs in milk are associated with increased abundance of Bifidobacterium species in the milk, with specific categories of hMOs influencing abundance of individual species
      • Aakko J.
      • et al.
      Human milk oligosaccharide categories define the microbiota composition in human colostrum.
      ; (2) abundance of categories of hMOs in milk are associated with alterations in the fecal microbiota of term and preterm infants
      • Wang M.
      • et al.
      Fecal microbiota composition of breast-fed infants is correlated with human milk oligosaccharides consumed.
      ,
      • De Leoz M.L.
      • et al.
      Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota: a proof-of-concept study.
      • Underwood M.A.
      • et al.
      Human milk oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota.
      • Underwood M.A.
      • et al.
      Digestion of human milk oligosaccharides by Bifidobacterium breve in the premature infant.
      ; (3) in retrospective human studies and animal models, individual hMO structures have a protective effect against NEC
      • Masi A.C.
      • et al.
      Human milk oligosaccharide DSLNT and gut microbiome in preterm infants predicts necrotising enterocolitis.
      ,
      • Li B.
      • et al.
      Human milk oligosaccharides protect against necrotizing enterocolitis by activating intestinal cell differentiation.
      ; and (4) hMO composition is a risk factor for stunting and malnutrition in infants, with causality shown in animal models between specific hMO structures, the intestinal microbiota, and growth.
      • Charbonneau M.R.
      • et al.
      Sialylated milk oligosaccharides promote microbiota-dependent growth in models of infant undernutrition.
      In addition to shaping the infant microbiota with resulting effects on the developing immune system, hMOs appear to have direct antimicrobial effects and to influence gut maturation and barrier function.
      • Li B.
      • et al.
      Human milk oligosaccharides protect against necrotizing enterocolitis by activating intestinal cell differentiation.
      ,
      • Moubareck C.A.
      Human milk microbiota and oligosaccharides: a glimpse into benefits, diversity, and correlations.
      A recent review summarized several pathways underlying the immunomodulatory effects of hMOs and their potential role in prevention of allergic diseases early in life.
      • Zuurveld M.
      • et al.
      Immunomodulation by human milk oligosaccharides: the potential role in prevention of allergic diseases.
      Three strategies for imitating the prebiotic effects of hMOs have been explored. Synthetic prebiotic glycans such as inulin, galacto-oligosaccharides, and fructo-oligosaccharides have been added to infant formula or administered directly to infants in hopes of altering the intestinal microbiota, improving feeding tolerance, and decreasing the risk of sepsis and NEC. A recent meta-analysis of 18 randomized controlled trials (1322 preterm infants) found a decrease in sepsis, mortality, and feeding intolerance (but not NEC) with prebiotic administration.
      • Chi C.
      • Buys N.
      • Li C.
      • Sun J.
      • Yin C.
      Effects of prebiotics on sepsis, necrotizing enterocolitis, mortality, feeding intolerance, time to full enteral feeding, length of hospital stay, and stool frequency in preterm infants: a meta-analysis.
      Second, a limited number of hMO structures (eg, 2′-fucosyllactose, lacto-N-neotetraose) have been added to term infant formula, with small studies showing lower levels of cytokines and fewer episodes of respiratory infection and alteration in the fecal microbiota,
      • Puccio G.
      • et al.
      Effects of infant formula with human milk oligosaccharides on growth and morbidity: a randomized multicenter trial.
      • Goehring K.C.
      • et al.
      Similar to those who are breastfed, infants fed a formula containing 2′-fucosyllactose have lower inflammatory cytokines in a randomized controlled trial.
      • Berger B.
      • et al.
      Linking human milk oligosaccharides, infant fecal community types, and later risk to require antibiotics.
      although a single larger study showed no benefit.
      • Leung T.F.
      • et al.
      A randomized controlled trial of different young child formulas on upper respiratory and gastrointestinal tract infections in Chinese toddlers.
      Finally, a mixture of bovine milk oligosaccharides added to infant formula in a randomized trial exhibited increased relative abundance of fecal Bifidobacterium, decreased relative abundance of fecal Clostridium, and increased fecal IgA compared with a control formula without added oligosaccharides.
      • Estorninos E.
      • et al.
      Term infant formula supplemented with milk-derived oligosaccharides shifts the gut microbiota closer to that of human milk-fed infants and improves intestinal immune defense: a randomized controlled trial.

      Future Directions

      Further research is needed to understand differences in the impact of specific hMO structures (eg, are some hMOs more important in shaping the microbiota or influencing gut maturation or inflammation than others?), the impact of hMOs that are absorbed from the gut (eg, do hMOs in the bloodstream and urinary tract play an immunologic or antibacterial role?), and the importance of genetic differences in hMO production in preventing intestinal dysbiosis (eg, how do common mutations in fucosyltransferases affect the milk microbiota, the intestinal microbiota, and infant susceptibility to disease?; is supplementation of specific hMOs to infants of mothers who are unable to secrete those hMOs protective?). As a wider array of milk oligosaccharides (either synthetic or from animal sources) becomes available, studies of combinations of these glycans for the prevention of dysbiosis-associated diseases have high potential.

      Lactoferrin

      Lactoferrin (LF), the second most abundant protein in breast milk,
      • Ochoa T.J.
      • Pezo A.
      • Cruz K.
      • Chea-Woo E.
      • Cleary T.G.
      Clinical studies of lactoferrin in children.
      is a multifunctional bioactive glycoprotein. It is known to have antimicrobial, anti-inflammatory, immunomodulatory, and growth-promoting properties that may prevent bacterial translocation in the intestines of premature neonates.
      • Sherman M.P.
      New concepts of microbial translocation in the neonatal intestine: mechanisms and prevention.
      Higher concentrations of LF are found in colostrum and in milk from mothers who deliver preterm.
      • Embleton N.D.
      • Berrington J.E.
      Clinical trials of lactoferrin in the newborn: effects on infection and the gut microbiome.
      LF exerts its antimicrobial and antifungal effects through a variety of mechanisms. It is bacteriostatic by binding to iron and making it inaccessible to pathogens in the intestinal lumen that require iron to proliferate. It also binds to lipopolysaccharide (LPS) and lipoteichoic acid on the cell surface, disrupting the bacterial cell membrane and decreasing biofilm formation.
      • Appelmelk B.J.
      • et al.
      Lactoferrin is a lipid A-binding protein.
      • Brandenburg K.
      • Jurgens G.
      • Muller M.
      • Fukuoka S.
      • Koch M.H.
      Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.
      • Lingappan K.
      • Arunachalam A.
      • Pammi M.
      Lactoferrin and the newborn: current perspectives.
      Several studies have shown potent bactericidal activity against pathogens as a result of formation of lactoferricin, a peptide released during digestion of LF.
      • Tomita M.
      • et al.
      Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin.
      ,
      • Lonnerdal B.
      Bioactive proteins in breast milk.
      Lactoferricin degrades the protein structures of bacteria and yeast needed to attach to intestinal cells and invade,
      • Ochoa T.J.
      • Cleary T.G.
      Effect of lactoferrin on enteric pathogens.
      • Edde L.
      • et al.
      Lactoferrin protects neonatal rats from gut-related systemic infection.
      • Lonnerdal B.
      Nutritional and physiologic significance of human milk proteins.
      • Embleton N.D.
      • Berrington J.E.
      • McGuire W.
      • Stewart C.J.
      • Cummings S.P.
      Lactoferrin: antimicrobial activity and therapeutic potential.
      • Gomez H.F.
      • Ochoa T.J.
      • Carlin L.G.
      • Cleary T.G.
      Human lactoferrin impairs virulence of Shigella flexneri.
      • Qiu J.
      • et al.
      Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.
      and it inhibits endocytosis of pathogens into host cells.
      • Suzuki Y.A.
      • Wong H.
      • Ashida K.Y.
      • Schryvers A.B.
      • Lonnerdal B.
      The N1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus.
      Several translational investigators of LF prophylaxis have reported improved bacterial clearance and survival in mice and piglets receiving LF prophylaxis.
      • Edde L.
      • et al.
      Lactoferrin protects neonatal rats from gut-related systemic infection.
      ,
      • Zagulski T.
      • Lipinski P.
      • Zagulska A.
      • Broniek S.
      • Jarzabek Z.
      Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo.
      • Venkatesh M.P.
      • Pham D.
      • Kong L.
      • Weisman L.E.
      Prophylaxis with lactoferrin, a novel antimicrobial agent, in a neonatal rat model of coinfection.
      • Artym J.
      • Zimecki M.
      • Kruzel M.L.
      Enhanced clearance of Escherichia coli and Staphylococcus aureus in mice treated with cyclophosphamide and lactoferrin.
      • Lee W.J.
      • Farmer J.L.
      • Hilty M.
      • Kim Y.B.
      The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets.
      LF has immunomodulatory effects, including regulation of T helper type 1/T helper type 2 balance,
      • Fischer R.
      • Debbabi H.
      • Dubarry M.
      • Boyaka P.
      • Tome D.
      Regulation of physiological and pathological Th1 and Th2 responses by lactoferrin.
      transcriptional regulation of immune and cell-signaling proteins in enterocytes,
      • Lonnerdal B.
      Bioactive proteins in breast milk.
      binding to LPS (resulting in reduction in upregulation of inflammatory cytokines), and decreasing the production of tumor necrosis factor and other pro-inflammatory cytokines.
      • Berlutti F.
      • et al.
      Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.
      ,
      • Otsuki K.
      • et al.
      Recombinant human lactoferrin has preventive effects on lipopolysaccharide-induced preterm delivery and production of inflammatory cytokines in mice.
      By sequestering free iron at inflammatory sites, LF prevents catalysis and production of reactive oxygen species.
      • Guillen C.
      • et al.
      Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice.
      LF also serves as a prebiotic shaping the intestinal microbiota of the infant.
      • Ochoa T.J.
      • Pezo A.
      • Cruz K.
      • Chea-Woo E.
      • Cleary T.G.
      Clinical studies of lactoferrin in children.
      LF promotes growth of beneficial bacteria, specifically Lactobacillus and Bifidobacterium, which, in turn, can limit the growth of pathogens by decreasing intestinal pH.
      • Lonnerdal B.
      Nutritional and physiologic significance of human milk proteins.
      ,
      • Petschow B.W.
      • Talbott R.D.
      • Batema R.P.
      Ability of lactoferrin to promote the growth of Bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level.
      • Tian H.
      • Maddox I.S.
      • Ferguson L.R.
      • Shu Q.
      Influence of bovine lactoferrin on selected probiotic bacteria and intestinal pathogens.
      • Mastromarino P.
      • et al.
      Correlation between lactoferrin and beneficial microbiota in breast milk and infant's feces.
      In animal experiments and in vitro, LF reportedly increases cell proliferation and affects crypt cell development in the small intestine.
      • Nichols B.L.
      • McKee K.S.
      • Henry J.F.
      • Putman M.
      Human lactoferrin stimulates thymidine incorporation into DNA of rat crypt cells.
      • Lonnerdal B.
      • Jiang R.
      • Du X.
      Bovine lactoferrin can be taken up by the human intestinal lactoferrin receptor and exert bioactivities.
      • Buccigrossi V.
      • et al.
      Lactoferrin induces concentration-dependent functional modulation of intestinal proliferation and differentiation.
      Its mitogenic effect has been proposed to aid in the rapid development of the intestinal mucosa of newborns.
      • Berseth C.L.
      • Lichtenberger L.M.
      • Morriss Jr, F.H.
      Comparison of the gastrointestinal growth-promoting effects of rat colostrum and mature milk in newborn rats in vivo.
      ,
      • Heird W.C.
      • Schwarz S.M.
      • Hansen I.H.
      Colostrum-induced enteric mucosal growth in beagle puppies.
      These biological activities have been shown to be promoted by both bovine and human LF.
      • Liao Y.
      • Jiang R.
      • Lonnerdal B.
      Biochemical and molecular impacts of lactoferrin on small intestinal growth and development during early life.
      ,
      • Hu W.
      • et al.
      Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets.
      Clinical trials of LF in neonates have been mixed. Manzoni et al
      • Manzoni P.
      • et al.
      Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low-birth-weight neonates: a randomized trial.
      found a significant reduction of late-onset sepsis, NEC, and death in preterm infants receiving prophylactic bovine LF compared with placebo, with the most pronounced impact seen in preterm infants with birth weight <1000 g. In a secondary analysis, the group found a significant decrease in invasive fungal infections.
      • Manzoni P.
      • et al.
      Bovine lactoferrin prevents invasive fungal infections in very low birth weight infants: a randomized controlled trial.
      Similar trends were obtained in small studies by other investigators.
      • Ochoa T.J.
      • et al.
      Randomized controlled trial of lactoferrin for prevention of sepsis in peruvian neonates less than 2500 g.
      • Akin I.M.
      • et al.
      Oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on T-regulatory cells.
      • Sherman M.P.
      • et al.
      Randomized controlled trial of talactoferrin oral solution in preterm infants.
      A recent, large, multicenter randomized placebo-controlled trial, the ELFIN (Enteral Lactoferrin in Neonates) trial
      • group T.E.t.i.
      Enteral lactoferrin supplementation for very preterm infants: a randomised placebo-controlled trial.
      in the United Kingdom, however, failed to show any significant reductions in either sepsis or NEC. LIFT (Lactoferrin Infant Feeding Trial)
      • Tarnow-Mordi W.O.
      • et al.
      The effect of lactoferrin supplementation on death or major morbidity in very low birthweight infants (LIFT): a multicentre, double-blind, randomised controlled trial.
      in Australia and New Zealand also showed no difference in mortality or major morbidity (including NEC); however, the group performed a Cochrane meta-analysis of >5000 infants, which found that although there was no improvement in death or major morbidity, LF did reduce late-onset sepsis.
      • Pammi M.
      • Preidis G.A.
      • Tarnow-Mordi W.O.
      Evidence from systematic reviews of randomized trials on enteral lactoferrin supplementation in preterm neonates.
      A recent nested study of a subset of the infants in the ELFIN trial showed that LF altered the fecal microbiota compared with matched control infants (with decreases in fecal Staphylococcus and other potential pathogens); however, the changes were not as large as those associated with other clinical variables. In addition, LF administration did not appear to alter the infant metabolome.
      • Embleton N.
      • et al.
      Lactoferrin impact on gut microbiota in preterm infants with late-onset sepsis or necrotising enterocolitis: the MAGPIE mechanisms of action study.

      Future Directions

      Further research is needed to understand the impact of digestion (partial digestion releases the highly active peptide lactoferricin, and yet further digestion reduces bioactivity; is the limited capacity for proteolysis in the preterm infant advantageous in this regard?) and absorption of LF (does absorption of intact LF or lactoferricin result in significant tissue concentration in infection? is a leakier intestinal mucosa, as seen in the preterm infant, advantageous?), the antiviral effects of LF, the impact of LF on the milk microbiota, and the synergistic effects of LF with other bioactive milk molecules. More detailed study of the impact of glycosylation on the antimicrobial and immunostimulatory effects of LF is needed. Further clinical trials of LF in preterm infants (beyond those in progress) may be difficult to justify; however, it is possible that LF in combination with other human milk bioactive molecules could alter the intestinal microbiota and help decrease the risk of NEC and late-onset sepsis in preterm infants and dysbiosis-associated disease in term infants.

      Lysozyme

      Lysozyme is a ubiquitous antibacterial enzyme abundant in the granules of macrophages, Paneth cells, and neutrophils and in many secretions, including mucus, amniotic fluid, and breast milk.
      • Lonnerdal B.
      Nutritional and physiologic significance of human milk proteins.
      Concentrations decrease from colostrum to transitional milk, then slowly rise but do not surpass colostrum levels until >2 months’ postpartum.
      • Montagne P.
      • Cuilliere M.L.
      • Mole C.
      • Bene M.C.
      • Faure G.
      Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation.
      Lysozymes enact their antimicrobial properties through the degradation of the outer wall of gram-positive bacteria, hydrolyzing β-1,4 bonds between N-acetylmuramic acid and N-acetyl-d-glucosamine in the peptidoglycan layer.
      • Chipman D.M.
      • Sharon N.
      Mechanism of lysozyme action.
      ,
      • Phillips D.C.
      The three-dimensional structure of an enzyme molecule.
      They also have bactericidal activity against gram-negative bacteria in vitro, acting synergistically with LF, which binds to the LPS in the outer bacterial membrane, removing it and allowing lysozyme to access and degrade the internal proteoglycan matrix of the membrane, thereby killing the bacteria.
      • Ellison 3rd, R.T.
      • Giehl T.J.
      Killing of gram-negative bacteria by lactoferrin and lysozyme.
      These mechanisms are also important in preventing bacterial binding to intestinal epithelial cell receptors and therefore bacterial translocation.
      Most strains of infant-type human-residential bifidobacteria (HRB) can grow and survive in human milk, although the capacity to do so appears to be strain dependent and breast milk dependent.
      • Minami J.
      • Odamaki T.
      • Hashikura N.
      • Abe F.
      • Xiao J.Z.
      Lysozyme in breast milk is a selection factor for bifidobacterial colonisation in the infant intestine.
      Infant-type HRB are tolerant of high lysozyme concentrations, correlating well with growth in breast milk; however, adult-type HRB and non-HRB were generally susceptible to lysozymes and therefore unable to grow in breast milk, suggesting that lysozyme in human milk could selectively exclude colonization of non-HRB in the intestines of breastfed infants. The mechanism behind this differential tolerance is still unknown.
      Pigs consuming transgenic goat milk containing human lysozyme (HLZ) had reduced clinical severity and improved recovery from enterotoxigenic Escherichia coli infection,
      • Garas L.C.
      • et al.
      Young pigs consuming lysozyme transgenic goat milk are protected from clinical symptoms of enterotoxigenic Escherichia coli infection.
      ,
      • Cooper C.A.
      • Garas Klobas L.C.
      • Maga E.A.
      • Murray J.D.
      Consuming transgenic goats' milk containing the antimicrobial protein lysozyme helps resolve diarrhea in young pigs.
      longer intestinal villi, thinner lamina propria, a rise in intraepithelial lymphocytes and Goblet cells,
      • Garas L.C.
      • et al.
      Young pigs consuming lysozyme transgenic goat milk are protected from clinical symptoms of enterotoxigenic Escherichia coli infection.
      ,
      • Cooper C.A.
      • Brundige D.R.
      • Reh W.A.
      • Maga E.A.
      • Murray J.D.
      Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology.
      and increased numbers of intestinal Lactobacillaceae and Bifidobacteriacea and decreased numbers of E coli and other coliforms.
      • Garas L.C.
      • et al.
      Young pigs consuming lysozyme transgenic goat milk are protected from clinical symptoms of enterotoxigenic Escherichia coli infection.
      ,
      • Maga E.A.
      • Cullor J.S.
      • Smith W.
      • Anderson G.B.
      • Murray J.D.
      Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.
      ,
      • Maga E.A.
      • et al.
      Consumption of lysozyme-rich milk can alter microbial fecal populations.
      Mechanistic studies show increased expression of TLR4 and interleukin-10 in pigs fed HLZ goat milk
      • Garas L.C.
      • et al.
      Young pigs consuming lysozyme transgenic goat milk are protected from clinical symptoms of enterotoxigenic Escherichia coli infection.
      ; TLR4 binds LPS from gram-negative bacteria and stimulates the innate immune system, whereas interleukin-10 is an anti-inflammatory cytokine produced by monocytes and regulatory T cells that inhibits LPS-induced stimulation of inflammatory cytokines. Oral administration of hen egg white lysozyme in a porcine model of chemically induced colitis led to decreased expression of the pro-inflammatory cytokines tumor necrosis factor-α and interleukin-8, as well as increased expression of anti-inflammatory transforming growth factor-β1.
      • Lee M.
      • et al.
      Hen egg lysozyme attenuates inflammation and modulates local gene expression in a porcine model of dextran sodium sulfate (DSS)-induced colitis.
      Cooper et al
      • Cooper C.A.
      • Brundige D.R.
      • Reh W.A.
      • Maga E.A.
      • Murray J.D.
      Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology.
      observed similar findings on transforming growth factor-β1 expression in the ileum of pigs fed goat's milk containing HLZ. Interestingly, transgenic HLZ goat milk also has reduced spoilage compared with milk from nontransgenic control goats, which could be of benefit to regions with poor access to refrigeration.
      • Maga E.A.
      • Cullor J.S.
      • Smith W.
      • Anderson G.B.
      • Murray J.D.
      Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.
      Both HLZ and human LF have been expressed in rice, a common food for infants. A randomized trial of oral rehydration solution containing both human rice proteins administered to infants with acute diarrhea and dehydration showed shorter duration of diarrhea compared with standard oral rehydration solutions.
      • Zavaleta N.
      • et al.
      Efficacy of rice-based oral rehydration solution containing recombinant human lactoferrin and lysozyme in Peruvian children with acute diarrhea.
      As noted earlier, lysozymes are also produced by macrophages, neutrophils, and Paneth cells (secretory cells at the base of the small intestinal crypts that protect stem cells and shape the intestinal microbiota). Paneth cell immaturity in the premature infant may play an important role in the pathogenesis of NEC. Infants with NEC have fewer Paneth cells than matched controls, and a Paneth cell ablation mouse model generates a NEC-like phenotype.
      • Coutinho H.B.
      • et al.
      Absence of lysozyme (muramidase) in the intestinal Paneth cells of newborn infants with necrotising enterocolitis.
      ,
      • Lueschow S.R.
      • McElroy S.J.
      The paneth cell: the curator and defender of the immature small intestine.
      It remains uncertain which of the many antimicrobial proteins and peptides secreted by Paneth cells are most important in shaping the microbiota of the small intestine; however, given that Paneth cells are not fully functional until at or following term gestation, it is likely that this important function (influencing the composition and function of the intestinal microbiota) is performed sequentially by swallowed amniotic fluid, human milk, and then Paneth cells, all containing lysozyme. No clinical trials, however, have yet to assess the benefits of lysozyme supplementation in the neonatal population.

      Future Directions

      Further research to understand the selective effects of lysozyme on bifidobacteria and other commensal gut microbes is needed (how do select commensal bacteria resist killing by lysozyme, and why has this apparent tolerance factor not been acquired by pathogens?). Further study of the impact of combined milk bioactives (eg, lysozyme plus LF) on the microbiota and intestinal inflammation and permeability would be of value in preparation for clinical trials. The milk of transgenic livestock delivering HLZ has the potential to decrease dysbiosis-associated diseases, including diarrheal diseases in developing countries; given the substantial mortality of diarrhea in infants, clinical trials of this approach are certainly justified.

      Milk Fat Globule Membrane

      Milk is essentially an emulsification of milk fat globules (MFGs) in an aqueous solution. MFGs are bags of fat of varying sizes (range of 0.2 to 15 μm in human milk). They consist of a membrane surrounding a core of triacylglycerols that provide about one half of human milk's caloric content.
      • Koletzko B.
      Human milk lipids.
      The membrane is a tri-layer with an inner polar lipid monolayer derived from the endoplasmic reticulum, a central protein-rich layer, and an outer polar lipid bilayer derived from the apical membrane of the mammary epithelial cell. The MFG membrane maintains the stability of the MFG in the lipid-in-water emulsion and protects the triacylglycerols from enzymatic digestion and coalescence. This MFG membrane is biologically active with a variety of phospholipids, sphingolipids, glycolipids, proteins, glycoproteins, and cholesterols. In addition to nutritional, metabolic, and synthetic properties (eg, the MFG membrane contains the majority of choline-containing phospholipids in milk), the MFG membrane also has antimicrobial, anti-inflammatory, and anticancer properties.
      MFG membranes have predominantly been studied in bovine milk; however, a recent summary of metabolomic studies showed both similarities and differences in MFG membrane proteins and glycoproteins across stages of lactation and among 5 species of mammals (bovine, goat, yak, buffalo, and human).
      • Manoni M.
      • Di Lorenzo C.
      • Ottoboni M.
      • Tretola M.
      • Pinotti L.
      Comparative proteomics of milk fat globule membrane (MFGM) proteome across species and lactation stages and the potentials of MFGM fractions in infant formula preparation.
      To date, 191 proteins have been identified in human MFG membrane and 120 in bovine MFG membrane.
      • Brink L.R.
      • Lonnerdal B.
      Milk fat globule membrane: the role of its various components in infant health and development.
      Table III summarizes some of the most abundant MFG membrane components,
      • Manoni M.
      • Di Lorenzo C.
      • Ottoboni M.
      • Tretola M.
      • Pinotti L.
      Comparative proteomics of milk fat globule membrane (MFGM) proteome across species and lactation stages and the potentials of MFGM fractions in infant formula preparation.
      ,
      • Silva R.C.D.
      • Colleran H.L.
      • Ibrahim S.A.
      Milk fat globule membrane in infant nutrition: a dairy industry perspective.
      ,
      • Thum C.
      • Roy N.C.
      • Everett D.W.
      • McNabb W.C.
      Variation in milk fat globule size and composition: a source of bioactives for human health.
      with the components in bold type indicating some degree of impact on the intestinal microbiota in piglets and germ-free mice.
      • Brink L.R.
      • Lonnerdal B.
      Milk fat globule membrane: the role of its various components in infant health and development.
      Pasteurization does not appear to alter the size distribution of MFGs; however, many of the MFG proteins are altered with significant differences between goat, bovine, and human samples.
      • Ma Y.
      • Zhang L.
      • Wu Y.
      • Zhou P.
      Changes in milk fat globule membrane proteome after pasteurization in human, bovine and caprine species.
      A recent review of 6 double-blind, randomized controlled trials of infant formula with added MFG membrane found a positive effect on cognitive development in 2 of the studies and a protective effect against infection in 3 of the studies.
      • Hernell O.
      • Domellof M.
      • Grip T.
      • Lonnerdal B.
      • Timby N.
      Physiological effects of feeding infants and young children formula supplemented with milk fat globule membranes.
      A subsequent randomized controlled trial reported alterations in the infant fecal microbiome and metabolome.
      • He X.
      • et al.
      Fecal microbiome and metabolome of infants fed bovine MFGM supplemented formula or standard formula with breast-fed infants as reference: a randomized controlled trial.
      Table IIIMilk fat globule membrane components. Listed are some of the most abundant components; those in bold are either antimicrobial or have been shown to influence the infant intestinal microbiota.
      Butyrophilin
      Glycerophospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and phosphatidylserine)
      Xanthine dehydrogenase/oxidoreductase
       Adipophilin
       Sphingolipids (sphingomyelin)
       Glycosphingolipids (gangliosides, cerebrosides)
      Lactadherin (PAS VI/VII, MFG-EGF factor 8)
      Mucins (MUC1, MUC4, MUC15)
      Cholesterol
      Fatty acid synthase
      Ras-related protein Rab-18
      Glycomacropeptide (a peptide from digestion of kappa-casein)
      Lactoferrin/lactotransferrin
      Isracidin (a peptide from digestion of alpha-S1-casein)
      BSSL
      GlyCAM1/lactophorin/PP3
      TLR2
      CD14
      Lactoperoxidase
      BSSL = bile salt–stimulated lipase; GlyCAM-1 = glycosylation-dependent cell adhesion molecule 1; MFG = milk fat globule; PP3 = proteose peptone component 3; TLR = Toll-like receptor.

      Future Directions

      Because there is only one study of the impact of the MFG membrane on the infant fecal microbiota, further research is needed on this important question, underscoring the value of including fecal microbiota analysis in clinical trials. The hypothesis that the MFG membrane could be useful as a “protecting encapsulating matrix for lactic acid bacteria” has been proposed and is deserving of investigation.
      • Guerin J.
      • Burgain J.
      • Gomand F.
      • Scher J.
      • Gaiani C.
      Milk fat globule membrane glycoproteins: valuable ingredients for lactic acid bacteria encapsulation?.
      The ready availability of commercial bovine and caprine MFG membrane and preliminary data from clinical trials of infant formulas with added MFG membrane suggest that further clinical trials in term and preterm infants are warranted.

      Bile Salt–Stimulated Lipase

      At birth, the enzymatic capacity of the gut of the term infant is immature. Production of gastric pepsin, pancreatic triglyceride lipase, bile salts, and phospholipase A2 are all low in the first weeks and months of life, a time of rapid brain growth and high metabolic demand. Digestion of human MFGs begins in the stomach with gastric lipase, which is similar in abundance and activity to that of adults, and continues in the proximal small intestine through the actions of pancreatic lipase-related protein 2 and bile salt–stimulated lipase (BSSL). The latter is abundant in human milk and becomes functional in the small intestine after the partial digestion of MFGs in the stomach.
      • He X.
      • McClorry S.
      • Hernell O.
      • Lonnerdal B.
      • Slupsky C.M.
      Digestion of human milk fat in healthy infants.
      Three points of clinical interest are noteworthy: first, in addition to its digestive function, BSSL provides some protection against viral infection
      • Naarding M.A.
      • et al.
      Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.
      ,
      • Ruvoen-Clouet N.
      • et al.
      Bile-salt-stimulated lipase and mucins from milk of 'secretor' mothers inhibit the binding of Norwalk virus capsids to their carbohydrate ligands.
      ; second, BSSL is denatured by standard pasteurization techniques; and third, post-pyloric feeding limits the exposure of MFGs to gastric lipase and therefore limits the functional activity of BSSL. As a result, both of these common interventions (post-pyloric feeding and pasteurization of human milk) may lead to limited lipolysis and fat malabsorption.
      BSSL is highly glycosylated with differences in glycosylation over time of lactation,
      • Landberg E.
      • et al.
      Changes in glycosylation of human bile-salt-stimulated lipase during lactation.
      with common polymorphisms, and between native and recombinant forms. Variation in glycosylation patterns does not seem to affect digestive function,
      • Blackberg L.
      • et al.
      Recombinant human-milk bile-salt-stimulated lipase. Functional properties are retained in the absence of glycosylation and the unique proline-rich repeats.
      although it may affect susceptibility to some viruses.
      • Steba G.S.
      • et al.
      Bile-salt stimulated lipase polymorphisms do not associate with HCV susceptibility.
      ,
      • Stax M.J.
      • et al.
      HIV-1 disease progression is associated with bile-salt stimulated lipase (BSSL) gene polymorphism.
      A pilot study of recombinant human BSSL in preterm infants showed improved weight gain and increased absorption of docosahexaenoic acid and arachidonic acid but no effect on total fat absorption.
      • Casper C.
      • et al.
      rhBSSL improves growth and LCPUFA absorption in preterm infants fed formula or pasteurized breast milk.
      A Phase III randomized trial of the same product in 415 preterm infants found no improvement in growth velocity, except in a predefined subgroup of small-for-gestational-age infants. This study reported increased adverse events in the BSSL group.
      • Casper C.
      • et al.
      Recombinant bile salt-stimulated lipase in preterm infant feeding: a randomized phase 3 study.

      Future Directions

      The study of the intestinal virome and its impact on the microbiota, immune development, and risk of dysbiosis-associated diseases is in its infancy. Evidence to date suggests that viral colonization of the infant gut follows bacterial colonization and is influenced by human milk.
      • Bushman F.
      • Liang G.
      Assembly of the virome in newborn human infants.
      ,
      • Comitini F.
      • Fanos V.
      The dark matter of microbiome: the mother-infant pair virome.
      Further research into the impact of various forms of BSSL on the intestinal microbiota and virome is needed. The description of expression of recombinant human BSSL in transgenic cows raises the possibility of large-scale production
      • Wang Y.
      • et al.
      Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.
      ; however, to date, clinical evidence of benefit is limited.

      Human Milk Microbes

      The milk microbiota and its role in seeding the infant gut have been extensively reviewed.
      • Jost T.
      • Lacroix C.
      • Braegger C.
      • Chassard C.
      Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment and gut health.
      • Oikonomou G.
      • et al.
      Milk microbiota: what are we exactly talking about?.
      • Boudry G.
      • et al.
      The relationship between breast milk components and the infant gut microbiota.
      Unpasteurized human milk contains both microbes from the mother's skin (predominantly Staphylococcus, Streptococcus, Propionibacterium, and Corynebacterium) and the infant's mouth (Streptococcus and Rothia). In addition, limited studies suggest the possibility of translocation of intestinal bacteria from the maternal gut into the milk via the lymphatics.
      • Jost T.
      • Lacroix C.
      • Braegger C.
      • Chassard C.
      Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment and gut health.
      ,
      • Jost T.
      • Lacroix C.
      • Braegger C.P.
      • Rochat F.
      • Chassard C.
      Vertical mother-neonate transfer of maternal gut bacteria via breastfeeding.
      ,
      • Kordy K.
      • et al.
      Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve.
      Factors influencing the milk microbiota include mode of breastfeeding (pumped vs direct), antibiotic exposure, probiotic ingestion, delivery mode, parity, maternal body mass index, mastitis, and preterm delivery. Several studies have shown strong community-level associations between the milk microbiota and the infant fecal microbiota.
      • Biagi E.
      • et al.
      Microbial community dynamics in mother's milk and infant's mouth and gut in moderately preterm infants.
      • Lackey K.A.
      • et al.
      What's normal? Microbiomes in human milk and infant feces are related to each other but vary geographically: the INSPIRE study.
      • Williams J.E.
      • et al.
      Strong multivariate relations exist among milk, oral, and fecal microbiomes in mother-infant dyads during the first six months postpartum.
      The earliest studies of the fecal microbiota of the breastfed infant were performed before the antibiotic era and found a predominance of bifidobacteria (the most effective consumers of hMOs).
      • Henrick B.M.
      • et al.
      Elevated fecal pH indicates a profound change in the breastfed infant gut microbiome due to reduction of Bifidobacterium over the past century.
      Studies have shown that a single subspecies, Bifidobacterium longum subspecies infantis, encodes in its genome all of the transferases and glycosidases necessary to transport all HMO structures into its cytoplasm and completely digest these hMOs into monosaccharides.
      • Underwood M.A.
      • German J.B.
      • Lebrilla C.B.
      • Mills D.A.
      Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut.
      Provision of a probiotic B infantis strain to breastfed infants for just 3 weeks beginning at day of life 7 led to prolonged colonization with this strain
      • Frese S.A.
      • et al.
      Persistence of supplemented Bifidobacterium longum subsp. infantis EVC001 in breastfed infants.
      ,
      • O'Brien C.E.
      • et al.
      Early probiotic supplementation with B. infantis in breastfed infants leads to persistent colonization at 1 year.
      and decreased intestinal inflammation
      • Henrick B.M.
      • et al.
      Colonization by B. infantis EVC001 modulates enteric inflammation in exclusively breastfed infants.
      and immune dysregulation,
      • Henrick B.M.
      • et al.
      Bifidobacteria-mediated immune system imprinting early in life.
      showing the synergy between hMOs and this bacterial subspecies; we can find no other examples of sustained alteration of the microbiota by a brief period of probiotic administration. In the pre-antibiotic era, how B infantis arrived in the infant gut is uncertain, but it seems likely that the origin was the mother's intestinal microbiota, where this microbe would have been a minor component, with passage to the infant either through the fecal–oral route or the milk microbiota, or both. As noted earlier, several components of human milk shape the milk microbiota, including hMOs and lysozyme.
      The human milk virome has recently been characterized. The major component consists of bacteriophages with differences in composition influenced by preterm birth, delivery mode, birth weight, and stage of lactation.
      • Dinleyici M.
      • et al.
      Human milk virome analysis: changing pattern regarding mode of delivery, birth weight, and lactational stage.
      The impact of the milk virome on the infant fecal virome, metabolome, and bacterial composition remains unknown.

      Future Directions

      One of the challenges of determining the functional impact of the milk microbiota on the infant intestinal microbiota is that gastric acid and bile acids inhibit growth of many bacteria, limiting passage of these microbes beyond the stomach and proximal small bowel. The impact of these microbes on the developing immune system is likely limited as they tend not to reach the distal small bowel with its rich collection of immune cells. Research emphasis on milk microbes that are resistant to gastric acid and bile acids (eg, Bifidobacterium and Lactobacillus species) is needed, as is detailed exploration of the impact of the milk virome on the developing microbiota and immune system. Further research into the impact of the maternal exposome on the relevant components of the milk microbiota is needed. Although improved methods of pasteurization may help limit damage to milk proteins, peptides, and glycoproteins, these are not likely to spare the milk microbiota. Consequently, research into the impact of adding probiotic microbes to pasteurized donor milk is needed.
      In addition to the human milk components discussed here, limited evidence for other components that influence the infant gut microbiota is summarized in Table IV.
      Table IVAdditional human milk components that may alter the intestinal microbiota.
      Alpha lactalbumin
      • Bruck W.M.
      • et al.
      Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants.
      • Bruck W.M.
      • Graverholt G.
      • Gibson G.R.
      Use of batch culture and a two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed populations of human gut bacteria.
      • Nielsen C.H.
      • et al.
      Alpha-lactalbumin enriched whey protein concentrate to improve gut, immunity and brain development in preterm pigs.
      N-glycans cleaved from glycosylated milk proteins
      • Garrido D.
      • et al.
      Endo-beta-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins.
      • Karav S.
      • et al.
      Oligosaccharides released from milk glycoproteins are selective growth substrates for infant-associated Bifidobacteria.
      • Kirmiz N.
      • Robinson R.C.
      • Shah I.M.
      • Barile D.
      • Mills D.A.
      Milk glycans and their interaction with the infant-gut microbiota.
      • Mu C.
      • et al.
      New insights into porcine milk N-glycome and the potential relation with offspring gut microbiome.
      Leptin, insulin, adiponectin
      • Gotteland M.
      • Magne F.
      Alterations in human milk leptin and insulin are associated with early changes in the infant intestinal microbiome.
      ,
      • Grases-Pinto B.
      • et al.
      Influence of leptin and adiponectin supplementation on intraepithelial lymphocyte and microbiota composition in suckling rats.
      Medium-chain fatty acids, monoacylglycerols
      • Nejrup R.G.
      • et al.
      Lipid hydrolysis products affect the composition of infant gut microbial communities in vitro.
      Sphingosine, sphingomyelin
      • Nejrup R.G.
      • et al.
      Lipid hydrolysis products affect the composition of infant gut microbial communities in vitro.
      ,
      • Norris G.H.
      • Milard M.
      • Michalski M.C.
      • Blesso C.N.
      Protective properties of milk sphingomyelin against dysfunctional lipid metabolism, gut dysbiosis, and inflammation.
      sn-2 Fatty acids
      • Jiang T.
      • et al.
      Association between sn-2 fatty acid profiles of breast milk and development of the infant intestinal microbiome.
      Gangliosides
      • Rueda R.
      • Sabatel J.L.
      • Maldonado J.
      • Molina-Font J.A.
      • Gil A.
      Addition of gangliosides to an adapted milk formula modifies levels of fecal Escherichia coli in preterm newborn infants.
      Lactose
      • Stein-Thoeringer C.K.
      • et al.
      Lactose drives Enterococcus expansion to promote graft-versus-host disease.
      ,
      • Pieper R.
      • Vahjen W.
      • Zentek J.
      Intestinal lactose and mineral concentration affect the microbial ecophysiology along the gastrointestinal tract of formula-fed neonatal piglets.
      ,
      • Jakobsen L.M.A.
      • Sundekilde U.K.
      • Andersen H.J.
      • Nielsen D.S.
      • Bertram H.C.
      Lactose and bovine milk oligosaccharides synergistically stimulate B. longum subsp. longum growth in a simplified model of the infant gut microbiome.

      Lacto-Engineering Within the Mammary Gland (in mamma) to Alter the Infant Microbiota

      Many women struggle to produce a sufficient quantity of milk. In addition, the content of macronutrients and bioactive molecules in the milk varies from woman to woman, from foremilk to hindmilk, and over the time of lactation. Increasing the quantity and quality of human milk production has great potential to improve not just infant growth and nutrition but also the intestinal microbiota and consequently the risk of dysbiosis-associated diseases. The term exposome refers to all environmental exposures that influence health. Maternal diseases (eg, obesity, diabetes), diet, phytochemicals, and galactogogues all influence milk production, growth and overgrowth of the infant, and the infant microbiota.
      • Strain J.
      • Spaans F.
      • Serhan M.
      • Davidge S.T.
      • Connor K.L.
      Programming of weight and obesity across the lifecourse by the maternal metabolic exposome: a systematic review.
      • Williams J.E.
      • et al.
      Human milk microbial community structure is relatively stable and related to variations in macronutrient and micronutrient intakes in healthy lactating women.
      • Quin C.
      • et al.
      Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers.
      • Seferovic M.D.
      • et al.
      Maternal diet alters human milk oligosaccharide composition with implications for the milk metagenome.
      • Kumar H.
      • et al.
      Distinct patterns in human milk microbiota and fatty acid profiles across specific geographic locations.
      The lack of research and understanding of optimization of the nutritional status of the mother–infant dyad have recently been reviewed and represent a tremendous opportunity to improve long-term health of both mother and infant.
      • Ford E.L.
      • Underwood M.A.
      • German J.B.
      Helping mom help baby: nutrition-based support for the mother-infant dyad during lactation.
      Nutritional guidance for lactating mothers is focused on consumption of a variety of sources of macronutrients
      CDC
      Diet Considerations for Breastfeeding Mothers.

      Nutritional Needs While Breastfeeding. Choose MyPlate. https://www.myplate.gov/moms-breastfeeding-nutritional-needs (2020).

      Making Healthy Choices in Each Food Group. Choose MyPlate. https://www.choosemyplate.gov/moms-makinghealthy-food-choices (2015).

      Philadelphia TCH of Diet for Breastfeeding Mothers. https://www.chop.edu/pages/diet-breastfeeding-mothers (2014).

      WHO. Healthy Eating during Pregnancy and Breastfeeding: Booklet for mothers. In: Regional Office for Europe: Nutrition and Food Security. (2001). p. 26.

      but provides only limited guidance for mothers and infants with unique health needs or genetics. None of these guidelines include analysis of milk quantity, macronutrients, bioactive components, or the composition of the infant microbiome or metabolome. Although personalized medicine is making important in-roads in many aspects of medicine, the opportunities for progress in care of the mother–infant dyad are significant but largely ignored by industry and funding agencies. For instance, the current understanding of the safety and efficacy of galactogogues is limited; pharmaceuticals (eg, domperidone, metoclopramide) have limited efficacy and significant potential side effects, and botanicals (eg, fenugreek) have only minimal evidence regarding mechanisms of action, dosing, safety, and efficacy.
      • Wagner C.L.
      • et al.
      The Safety of Mother's Milk(R) Tea: results of a randomized double-blind, controlled study in fully breastfeeding mothers and their infants.
      • Sewell A.C.
      • Mosandl A.
      • Bohles H.
      False diagnosis of maple syrup urine disease owing to ingestion of herbal tea.
      • Reeder C
      • O'connor-Von SK
      The effect of fenugreek on milk production and prolactin levels in mothers of preterm infants.
      Oxytocin receptor agonists and nicotinamide derivatives offer promising alternatives for increasing milk production and quality, with early clinical trials in progress.

      Trial Exploring the Efficacy and Safety of FE 202767–Full Text View–ClinicalTrials.gov NCT02545127. https://clinicaltrials.gov/ct2/show/ (2021).

      ,
      • Ear P.H.
      • et al.
      Maternal nicotinamide riboside enhances postpartum weight loss, juvenile offspring development, and neurogenesis of adult offspring.
      Clinical trials of interventions to alter the milk and/or the infant microbiota were recently summarized in a systematic review.
      • Zaidi A.Z.
      • Moore S.E.
      • Okala S.G.
      Impact of maternal nutritional supplementation during pregnancy and lactation on the infant gut or breastmilk microbiota: a systematic review.
      Maternal consumption of probiotics altered the infant fecal microbiota and the milk microbiota in several studies, and maternal consumption of lipid-based nutritional supplements increased bacterial diversity in the infant feces; maternal consumption of vitamin D or prebiotic supplements did not significantly alter the infant microbiota. A study of maternal fish oil consumption, not included in the previously noted meta-analysis, showed increased Bifidobacterium and Lactobacillus species in the infant feces.
      • Quin C.
      • et al.
      Fish oil supplementation reduces maternal defensive inflammation and predicts a gut bacteriome with reduced immune priming capacity in infants.
      Attempts to decrease risk of preterm delivery or increase infant birth weight by altering the maternal vaginal or intestinal microbiota with probiotics have been disappointing to date.
      • Perez-Castillo I.M.
      • et al.
      Reporting of perinatal outcomes in probiotic randomized controlled trials. A systematic review and meta-analysis.
      In a single small study, maternal consumption of probiotic dietary supplements after birth did not decrease the risk of NEC or mortality in preterm infants
      • Benor S.
      • et al.
      Probiotic supplementation in mothers of very low birth weight infants.
      ,
      • Grev J.
      • Berg M.
      • Soll R.
      Maternal probiotic supplementation for prevention of morbidity and mortality in preterm infants.
      ; however, a meta-analysis of maternal consumption of probiotics during pregnancy and lactation exhibited a significant reduction in atopic dermatitis but not other allergic diseases,
      • Sun S.
      • Chang G.
      • Zhang L.
      The prevention effect of probiotics against eczema in children: an update systematic review and meta-analysis.
      presumably by altering the maternal intestinal and milk microbiota.
      • Abrahamsson T.R.
      • Sinkiewicz G.
      • Jakobsson T.
      • Fredrikson M.
      • Bjorksten B.
      Probiotic lactobacilli in breast milk and infant stool in relation to oral intake during the first year of life.
      A full review of traditional botanicals and phytochemicals that influence lactation and the infant gut microbiota is beyond the scope of this article and would be a valuable addition to the literature.
      • Sibeko L.
      • Johns T.
      • Cordeiro L.S.
      Traditional plant use during lactation and postpartum recovery: infant development and maternal health roles.

      Lacto-Engineering Expressed Human Milk to Alter the Infant Microbiota

      Currently, approaches to fortify expressed human milk (either from the infant's own mother or from a donor) are limited to the addition of fortifiers derived from bovine or donor human milk. To date, attempts to produce components of human milk on a large scale have focused on extracting similar or equivalent components from bovine milk,
      • Robinson R.C.
      • Poulsen N.A.
      • Barile D.
      Multiplexed bovine milk oligosaccharide analysis with aminoxy tandem mass tags.
      developing transgenic plants and animals,
      • Yemets A.I.
      • Tanasienko I.V.
      • Krasylenko Y.A.
      • Blume Y.B.
      Plant-based biopharming of recombinant human lactoferrin.
      ,
      • Cooper C.A.
      • Maga E.A.
      • Murray J.D.
      Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.
      or producing bioactive molecules through fungal or bacterial technology or through chemical synthesis.
      • Sprenger G.A.
      • Baumgartner F.
      • Albermann C.
      Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations.
      These approaches have fostered the fortification of infant formula with milk oligosaccharides and MFG membrane for term infants, but the addition of milk bioactives to expressed human milk currently remains in the realm of research. This is due in part to the lack of capacity to measure the content of promising milk bioactive molecules in expressed milk or any functional aspects of the infant microbiota in the clinical setting. A milk analyzer that could measure hMOs, immunoglobulins, and LF and a bedside test to detect fecal dysbiosis would be major steps forward in the early identification of mother–infant dyads at particularly high risk for dysbiosis-related disease.
      Two additional challenges to altering human milk (or formula) to improve the infant microbiota are limited capacity to synthesize more complex structures (eg, SIgA and larger hMOs) and limited posttranslational modification (eg, glycosylation of bioactive proteins and peptides). In January 2020, 108Labs produced the first cell-cultured human milk using primary human mammary cells in three-dimensional hollow fiber bioreactors, with others such as Biomilk, Biomilq, and TurtleTree Labs since announcing their own cell-cultured milks. Cell-cultured human milk bioreactors produce a comprehensive range of human milk proteins, lipids, and hMOs simultaneously. Biosynthesis of human proteins in lactating primary human mammary cells may yield therapeutic proteins with more normal posttranscriptional modifications and higher quality glycans, with a higher possibility of N-linked, fucosylated, and large O-linked oligosaccharides shown to positively affect in vivo bioactivity and stability.
      • Sola R.J.
      • Griebenow K.
      Effects of glycosylation on the stability of protein pharmaceuticals.
      More than 70% of human milk proteins are highly glycosylated, including several discussed herein (SIgA, LF, lysozyme, and BSSL).
      • Goonatilleke E.
      • et al.
      Human milk proteins and their glycosylation exhibit quantitative dynamic variations during lactation.
      The failure of recombinant milk proteins in previous human trials may be partially attributable to abnormal posttranscriptional modifications and lesser quality glycan patterns typical of recombinant proteins compared with native proteins.
      • Landberg E.
      • et al.
      Glycosylation of bile-salt-stimulated lipase from human milk: comparison of native and recombinant forms.
      Biosynthesis of the first ex vivo human SIgA dimers using primary human cells by establishing a novel long-lived plasma cell niche
      • Lightman S.M.
      • Utley A.
      • Lee K.P.
      Survival of long-lived plasma cells (LLPC): piecing together the puzzle.
      function bioreactor represents a significant step forward in production of a primary complex human milk bioactive component
      • de Sousa-Pereira P.
      • Woof J.M.
      IgA: structure, function, and developability.
      (Figure 4). Cell-cultured human SIgA administered with or without other therapeutic milk proteins and hMO derived from ex vivo lactating human mammary cells could be added to expressed mother's milk, pasteurized donor human milk, or infant formula to treat or prevent intestinal dysbiosis in preterm infants and/or term infants at high risk for dysbiosis-associated disease.
      Figure 4
      Figure 4Electron microscopy of 108Labs' first batch of native human secretory immunoglobulin A dimers biosynthesized on January 20, 2020, in 108Labs' secretory immunoglobulin A bioreactor, isolated from supernatant by affinity resin and diluted to 0.005 μg/mL for single protein imaging.
      Although the American Academy of Pediatrics recommends pasteurized donor human milk for preterm infants whose mothers are unable to produce a sufficient supply of their own milk, and the US Food and Drug Administration recommends against obtaining donor milk from sources that do not screen donors or ensure the safety of donor milk, there is currently very little oversight of donor human milk at the state or federal level.
      • Cohen M.
      Should human milk be regulated?.
      It is likely that cell-cultured human milk–derived therapeutic proteins will be regulated and marketed exclusively as biologic drugs. Oversight of this process through the Investigational New Drug process by the US Food and Drug Administration requires a significant investment of time and resources but is essential to ensuring safety and efficacy.

      Conclusions

      Human milk is a complex tissue containing macronutrients and micronutrients, bioactive cells and molecules, and potentially beneficial microbes that nourish and protect the fragile infant during a period of rapid growth and high vulnerability to infectious diseases. Immunoglobulins, hMOs, LF, lysozyme, and human MFG membranes not only protect the infant from infections but help shape the infant intestinal microbiota, potentially protecting the infant against a long list of intestinal dysbiosis-associated diseases. Novel methods of pasteurization that destroy pathogens but leave bioactive molecules intact would improve the protective benefits of donor milk. Methods of boosting specific bioactive molecules in milk (eg, maternal vaccination during pregnancy or immediately after preterm birth, dietary supplements, and novel galactogogues) are promising. Bioengineering some or several of the bioactive molecules described herein as additives to mother's milk (eg, α1,2 fucosylated hMOs for mothers with a common mutation that precludes production of these hMOs) or to donor milk or infant formulas may be helpful in bridging the gaps between human milk–fed and formula-fed infants in partially restoring our ancestral infant microbiota and in reducing risk for dysbiosis-associated disease.

      Acknowledgments

      The National Institutes of Health have provided grant support to Dr. Lakshminrusimha (R01 HD072929) and Dr. Underwood (R21 HD096247).
      Dr. Zeinali wrote the initial draft of several sections and edited the entire manuscript. Dr. Giuliano wrote the sections on three-dimensional mammary cell culture, biosynthesis of immunoglobulins, and other bioactive human milk molecules. Dr. Lakshminrusimha created Figures 1 to 3 and edited the manuscript. Dr. Underwood wrote the initial draft of several sections and edited the entire manuscript. All authors approved the final version as submitted.

      Disclosures

      Dr. Giuliano is a co-founder and the CEO of 108Labs. Dr. Underwood has received an honorarium from Prolacta for speaking at conferences on probiotics. Drs Zeinali and Lakshminrusimha have indicated that they have no conflicts of interest regarding the content of this article.

      Supplementary materials

      A co-culture of human primary mammary epithelial and B cells were grown and serial passaged for 30 days using mammary growth medium. The cells were then seeded into a hollow fiber bioreactor and cultured with growth medium for 10 more days until glucose consumption stabilization was observed. Lactation medium was then used to stimulate milk production and LLPC niche function, corresponding with an observed increase in the production of secretory IgA as measured by SIgA ELISA. Supernatant was harvested from the bioreactor 25 days after seeding. Secretory antibodies were isolated from supernatant using an affinity resin, and directly imaged with electron microscopy at 0.005 ug/mL.

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