Human milk oligosaccharide secretion dynamics during breastfeeding and its antimicrobial role: A systematic review

Table 1 Breastfeeding secretion dynamics in the first two years of lactation

Stage of lactation	Time period	Milk volume	Key nutritional components	Key bioactive components	Primary functions	Additional notes
Colostrum	First 3-5 days postpartum	Low (10-40 mL per feed)	High in protein, low in fat and lactose	High IgA, lactoferrin, HMOs, leukocytes, growth factors (e.g., EGF)	Provides passive immunity, protects gut mucosa, supports gut microbiota development, aids intestinal repair	Proteins: Peak concentration, casein: whey ratio approximately 20: 80- HMOs: Highest (approximately 15-20 g/L)- Growth factors: Abundant EGF for gut development- Fats: Minimal but rich in essential fatty acids
Transitional milk	Days 5–14 postpartum	Increasing (approximately 500-750 mL/day)	Protein decreases, fat and lactose increase	Reduced IgA and leukocytes; lactoferrin and lysozyme persist	Supports growing caloric needs, continues immune protection, aids microbiota development	Proteins: Decline begins- Lactose: Increases to support energy needs- HMOs: Decline slightly but remain significant- fats: Gradual increase, influenced by maternal diet
Mature milk	Weeks 3 onward	High (700-900 mL/day)	Stable macronutrients: approximately 0.8%-1% protein, approximately 4%-5% fat, approximately 7% lactose	Balanced levels of HMOs, lactoferrin, lysozyme, and antibodies	Provides complete nutrition, supports immune and brain development, protects against infections	Proteins: Casein: Whey ratio shifts to approximately 50: 50- HMOs: Decline (approximately 5-10 g/L) but bioactive- Immune Factors: IgA, lysozyme, and lactoferrin persist- Fats: Higher variability based on maternal diet
Months 6-12	Late lactation phase	Gradual reduction (approximately 500-800 mL/day)	Similar macronutrient composition as mature milk	Persistent immune factors (IgA, lysozyme, HMOs, lactoferrin)	Complements solid foods, supports immune and microbiota maturation	Fats: Continue to vary, contributing to brain and energy needs- HMOs: Moderate levels (approximately 2-5 g/L)- Lactose: Steady for energy support- Immune Factors: Immune benefits adapted for growing infant
Months 12-24	Toddler phase	Further reduction (approximately 300-500 mL/day)	Macronutrients adapt to reduced dependence on milk	Sustained immune components (IgA, lysozyme, lactoferrin)	Provides immune protection, complements toddler diet, supports growth and immunity	Proteins: Further adapted to complement the solid diet- HMOs: Persist at low levels (< 2 g/L)- Immune factors: Support immune resilience- Fats: Important for energy and essential fatty acids

This table provides a detailed view of the changes in breastfeeding dynamics and their biological significance. EGF: Epidermal growth factor; HMOs: Human milk oligosaccharides; IgA: Immunoglobulin A.

Table 2 Maternal factors affecting human milk oligosaccharides composition

Factor	Key influence	Examples and mechanisms
Maternal genetics	Primary determinant of HMO diversity and abundance	Secretor Status: Active FUT2 gene leads to higher 2′-FL levels; Lewis blood type: Influences specific HMO fucosylation patterns
Dietary intake	Availability of substrates and cofactors for HMO synthesis	Carbohydrates: Essential for lactose core formation; PUFAs: Support sialylated HMO production; Micronutrients: Iron, zinc, and B vitamins enhance enzymatic activity
Maternal health status	Alters HMO composition and concentration	Obesity: Reduces fucosylated HMOs (e.g., 2′-FL); Diabetes: Lowers fucosylation and sialylation; Infections/Inflammation: Downregulates glycosylation enzymes
Stress levels	Impacts hormonal and metabolic pathways	Cortisol: Reduces sialylated HMO levels; Chronic Stress: Alters glycosylation balance and overall HMO diversity
Parity (number of pregnancies)	Enhanced mammary adaptation and metabolic efficiency in multiparous mothers	Higher HMO concentrations in multiparous mothers; Primiparous mothers may produce less diverse HMO profiles
Lactation stage	Dynamic changes in HMO levels during breastfeeding	Colostrum: High fucosylated and sialylated HMOs for immune protection; Mature Milk: Decline in total HMOs; increase in 3-FL for gut microbiota diversity
Environmental Conditions	Geographic, pollutant, and cultural influences on HMO secretion	Rural areas: Prolonged breastfeeding supports 3-FL and LNT secretion (e.g., Kenyan mothers); Pollutants: Disrupt metabolic pathways and reduce HMO diversity
Breastfeeding practices	Duration and exclusivity influence HMO exposure	Extended Breastfeeding: Enhances long-term microbial and immune benefits; Early Weaning: Reduces HMO exposure in critical periods

2′-FL: 2′-Fucosyllactose; 3′-FL: 3′-Fucosyllactose; HMOs: Human milk oligosaccharides; LNT: Lacto-N-Tetraose; PUFAs: Polyunsaturated fatty acids.

Table 3 Specific anti-microbial roles of some human milk oligosaccharides

HMO type	Key pathogens targeted	Mode of action
2'-FL	Campylobacter jejuni, EPEC, Norovirus, RSV, Rotavirus	Anti-adhesive activity, inhibition of pathogen attachment to epithelial cells, by acting as a decoy receptor
3-FL	EPEC, Pseudomonas aeruginosa, Salmonella enterica, and Norovirus (pathogens commonly encountered later in infancy)	The prebiotic effect promotes beneficial bacteria and blocks pathogen adhesion
DFL	Escherichia coli, Campylobacter jejuni, Salmonella enterica, Helicobacter pylori	Anti-adhesive activity preventing pathogen attachment to epithelial cells; supports immune modulation and gut barrier function
LNT	Entamoeba histolytica, GB. streptococcus, Vibrio cholera toxin	Neutralization of bacterial toxins and prevention of protozoal adhesion
3'-SL	EPEC, Helicobacter pylori, Pseudomonas aeruginosa, Vibrio cholera toxin, RSV, Rotavirus	Blocking bacterial adhesion and neutralizing bacterial toxins
6'-SL	EPEC, Helicobacter pylori, Pseudomonas aeruginosa, Influenza virus, Rotavirus	Inhibition of bacterial and viral adherence, especially in respiratory and gastrointestinal infections

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; EPEC: Enteropathogenic E. coli; GB. streptococcus: Group B streptococci; LNT: Lacto-N-Tetraose; RSV: Respiratory syncytial virus; HMO: Human milk oligosaccharide.

Table 4 Studies of anti-bacterial effects of human milk oligosaccharides

Ref.	Type of article	Microbes investigated	Types of HMOs	Mechanisms of action	Key findings	Patient population
Morrow et al[76]	Observational study	Campylobacter jejuni, Calicivirus	2′-FL, LNDFH	Inhibits pathogen adhesion by mimicking host glycans; Modulates immune responses, reducing intestinal inflammation	Significant reduction in diarrhea incidence (P = 0.004 for Campylobacterjejuni, P = 0.012 for Calicivirus)	Breastfed human infants
Yu et al[77]	Experimental study	Campylobacter jejuni	2′-FL	Prevents bacterial adhesion to epithelial cells; Suppresses pro-inflammatory cytokines (IL-8, IL-1β)	Colonization reduced by 80% in mouse models; Lowered intestinal inflammation	Mouse model
Facinelli et al[78]	Experimental in vitro study	E. coli, Salmonella fyris	2'-FL, 6'-SL	2'-FL and 6'-SL significantly reduced the adhesion of Escherichia coli to Caco-2 cells at human milk concentrations but no effect on Salmonella fyris	Reducing the adhesion of harmful bacteria like E. coli	Bacterial adhesion to Caco-2 cells (a human intestinal cell line) cultures
Wang et al[79]	Experimental study	E. coli O157: H7	2′-FL	Acts as a soluble decoy receptor; Enhances mucin secretion (MUC2); Promotes microbiota balance	Colonization reduced by > 90% in mouse intestines; Lowered pro-inflammatory cytokines (IL-6, TNF-α)	Mouse model
Lin et al[99]	Experimental study	UPEC	Neutral and sialic acid-rich HMOs	Reduces bacterial internalization into epithelial cells. Suppresses UPEC-induced proinflammatory signaling (MAPK, NF-κB)	Reduced cytotoxicity and improved bladder epithelial integrity; Decreased cell death and detachment	In vitro bladder epithelial cells
Martín-Sosa et al[100]	Experimental study	ETEC, UPEC	Sialylated oligosaccharides	HMOs strongly inhibited hemagglutination mediated by ETEC strains expressing CFA/I and CFA/II. HMOs also inhibited hemagglutination by UPEC strains	The inhibitory capacity was significantly reduced when HMOs were desialylated, highlighting the importance of the sialylated fraction	In vitro
Coppa et al[102]	Experimental study	EPEC, Vibrio cholerae, Salmonella fyris	Acidic, neutral high-molecular-weight, and neutral low-molecular-weight HMOs	Blocks bacterial adhesion to Caco-2 cells by acting as decoy receptors	Acidic HMOs inhibit all three pathogens. Neutral high-molecular-weight HMOs inhibit E. coli and V. cholerae. Neutral low-molecular-weight HMOs inhibit E. coli and S. fyris	In vitro with Caco-2 cells
Newburg et al[103]	Experimental study	E. coli (heat-stable enterotoxin, (ST)	Fucosylated HMOs	Oligosaccharide fraction provided significant protection against the diarrheagenic effects of E. coli heat-ST), while the lactose fraction did not	Among HMOs, the neutral fraction exhibited protective activity, whereas the acidic fraction did not (22% vs 57% mortality, respectively	Suckling mouse model
Noguera-Obenza et al[101]	Observational study	EHEC, EPEC	Not specified; focus on secretory IgA (sIgA) in human milk	Neutralizes bacterial adhesion by targeting virulence antigens (e.g., EspA, EspB, intimin)	sIgA in human milk reduces adhesion of EHEC/EPEC to gut epithelium. - Antibodies are regionally variable and linked to pathogen exposure history	Milk samples from breastfeeding mothers (United States and Mexico)
He et al[109]	Experimental study	AIEC	Exosome-encapsulated HMOs (e.g., 2’-FL, Sialyl lactose)	Modulates immunity by reducing LPS-induced inflammation. Promotes anti-inflammatory macrophage phenotype	Reduced intestinal inflammation and bacterial load in AIEC-infected mice	In vivo murine model
Manthey et al[80]	Experimental study	EPEC	Various HMOs	Blocks bacterial attachment by acting as decoy receptors	Reduced EPEC attachment in vitro; Decreased intestinal colonization in mouse models	In vitro epithelial cells and suckling mice models
Chambers et al[81]	Experimental study	GB. streptococcus	Pooled and single-entity HMOs	Increases cell permeability; Bioorthogonal probes retain activity and identify cellular targets	Up to 90% growth inhibition by pooled HMOs; DFL, LNT-II, and LSTa exhibit significant antibacterial activity (35%-60%)	In vitro GB. streptococcus cultures
Chambers et al[117]	Experimental study	GB. streptococcus	Various HMOs	Increases membrane permeability. Perturbs metabolic pathways, including glycerophospholipid and linoleic acid metabolism	Potentiated trimethoprim activity, reducing MIC by up to 512-fold; Synergistic effects observed in combination therapy	In vitro studies with clinical GB. streptococcus isolates
Ackerman et al[82]	Experimental study	GB. streptococcus	Pooled donor HMOs	Disrupts biofilm formation; Alters bacterial cell arrangement	Donor-specific effects: HMOs from some donors inhibited biofilm production and bacterial growth by 40%; Biofilm structure changes were observed using electron microscopy	In vitro GB. streptococcus cultures from human donors
Lin et al[83]	Experimental study	GB. streptococcus	Neutral (non-sialylated) HMOs	Direct bacteriostatic effect; Neutral HMOs inhibit growth by impairing glycosyltransferase functions; Synergistic effects with antibiotics	Neutral HMOs reduce GB. streptococcus growth by up to 98%; Identified glycosyltransferase (gbs0738) as a target for HMO-mediated inhibition; Synergy observed with vancomycin and ciprofloxacin, significantly lowering the required antibiotic dose	In vitro GB. streptococcus cultures
Moore et al[84]	Experimental study	GB. streptococcus	Purified HMOs	Inhibits biofilm formation in colonizing and invasive strains; Exhibits strain-specific effects based on capsular serotype and sequence type	50% biofilm inhibition for colonizing strains, 45% for invasive strains; Significant inhibition across multiple capsular types (CpsIb, CpsII, etc.); Effective biofilm dismantling of mature biofilms	30 diverse clinical and laboratory strains of GB. streptococcus (in vitro analysis)
Andreas et al[118]	Clinical and experimental study	GB. streptococcus	Specific fucosylated HMOs	Reduces GB. streptococcus colonization in mothers and infants; Acts bacteriostatically, impairing glycosyltransferase-dependent cell proliferation	LNDFHI and related HMOs reduce GB. Streptococcus growth by 50%; Lewis-positive mothers produce HMOs that reduce maternal and infant colonization; Lewis-negative mothers could benefit from synthetic HMO supplementation	Gambian mother-infant pairs and in vitro analysis
Mysore et al[85]	Preclinical study	Helicobacter pylori	38-SL, a natural HMO analog	Inhibits bacterial adhesion to gastric epithelial cells; Acts as a decoy receptor preventing colonization	2 out of 6 animals achieved permanent H. pylori eradication; Safe and showed efficacy in rhesus monkey model; Transient decrease in bacterial load observed in other regimens	H. pylori-positive rhesus monkeys
Parente et al[86]	Double-blind, placebo-controlled clinical trial	Helicobacter pylori	3’SL	3’SL fail to eradicate or suppress H. pylori infection		Adults with dyspepsia and confirmed H. pylori infection
Jantscher-Krenn et al[116]	Experimental study	Entamoeba histolytica	Pooled HMOs	Reduces trophozoite attachment to intestinal epithelium	LNT has significant protection (up to 80%) while fucosylated HMOs are ineffective. GOS completely block E. histolytica attachment and cytotoxicity at 8 mg/mL	In vitro cultures of E. histolytica and human intestinal epithelial HT-29 cells
Nguyen et al[105]	Experimental study	Clostridium difficile, specifically focusing on its toxin A (TcdA) and the carbohydrate binding site (TcdA-f2)	LNFPV & LNH	A mixture of HMOs binds to the carbohydrate binding site of Clostridium difficile toxin A (TcdA-f2) at a concentration of 20 g/mL	LNFPV and LNH demonstrated strong binding with TcdA-f2, exhibiting docking energies of -9.48 kcal/mol and -12.81 kcal/mol, respectively

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; 38-SL: 38-sialyllactose; AIEC: Adherent-Invasive E. coli; CFAs: Colonization factor antigens; CD4: Cluster of differentiation 4+; CD8: cluster of differentiation 8+; DFL: Difucosyllactose; Escherichia coli: E. coli, EHEC: Enterohemorrhagic E. coli; EPEC: Enteropathogenic E. coli; ETEC: Enterotoxigenic Escherichia coli; GB. streptococcus: Group B streptococci; HBGAs: Histo-blood group antigens; HIEs: Human intestinal enteroids; HMOs: Human milk oligosaccharides; IgA: Immunoglobulin A; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; IL-8: Interleukin-8; LNFPV: Lacto-N-fucopentaose; LNDFH: Lacto-N-Difucohexaose; LNDFHI: lacto-N-difucohexaose I; LNH: Lacto-N-neohexaose; LNT: Lacto-N-Tetraose; LNT-II: lacto-N-triose II; LPS: Lipopolysaccharides; LSTa: LS-tetrasaccharide a; MIC: Minimum inhibitory concentration; NF-κB: Nuclear factor kappa B; PBMCs: Peripheral blood mononuclear cells; RSV: Respiratory syncytial virus; RV: Rota virus; SA: Sialic acid; TcdA-f2: Clostridium difficile toxin A; Th1: T helper 1; Th2: T helper 2; TNF-α: Tumor necrosis factor; UPEC: Uropathogenic E. coli.

Table 5 Studies of the role of human milk oligosaccharides against viral infections

Ref.	Virus investigated	Type of HMOs	Mechanisms of action	Key findings	Patient population
Patil et al[112]	Human Norovirus (HuNoV)	2'-FL	Acts as decoy receptors, blocking binding to HBGAs	Significant inhibition of Norovirus GII.4 Sydney [P16] replication in adult and pediatric HIEs. Less effective in infant HIEs due to low HBGA expression	Adults and pediatric intestinal organoids
Koromyslova et al[87]	GI.1 and GII.17 Noroviruses	2'-FL	Inhibits binding to HBGAs by mimicking natural attachment sites	Broad inhibition of GI.1 and GII.17 norovirus binding across genogroups. Demonstrated dose-dependent inhibition with 2'-FL in vitro	Not specified (laboratory models)
Hanisch et al[88]	Norovirus GII.4 (Sydney 2012) and GII.10 (Vietnam 026)	High-molecular mass HMOs rich in fucose	Multivalent fucose presentation enhances steric and valency effects for strong virus binding inhibition	High-mass HMOs with terminal blood group H1 or Lewis-b antigens showed potent binding to GII.4; distinct preferences observed for GII.10	Not specified (laboratory models)
Weichert et al[89]	Norovirus (not strain-specific)	2'-FL and 3-FL	Structurally mimic HBGAs and block norovirus binding to surrogate HBGA samples	2'-FL and 3-FL bind to HBGA pockets on the capsid, acting as naturally occurring decoys to inhibit norovirus attachment	Not specified (laboratory models)
Derya et al[90]	Norovirus GII.17 and GII.4	LNFP I and simpler fucosylated HMOs	Structurally similar to HBGAs; block binding to natural receptors	Simple fucosylated HMOs were more effective than complex ones (e.g., LNFP I) in inhibiting GII.17 and GII.4 binding to human gastric mucins	Not specified (laboratory models)
Hester et al[91]	RV OSU and Wa strains	SA-containing HMOs (e.g., 3'-SL, 6'-SL), neutral HMOs (e.g., LNnT)	Blocks viral binding and decreases infectivity through decoy receptor mechanisms.	SA-containing HMOs inhibited RV OSU infectivity in vitro. Both neutral and SA-containing HMOs reduced viral replication during acute infection in situ	In vitro and 21-day-old piglets
Comstock et al[92]	Rotavirus OSU strain	2'-FL, LNnT, 6'-SL, 3'-SL, and free SA	Modulates systemic and gastrointestinal immune cells to alter infection susceptibility	HMO-fed pigs showed increased NK and memory T cells and reduced immune cell populations linked to infection compared to formula-fed pigs	Colostrum-deprived neonatal piglets
Li et al[93]	Rotavirus OSU strain	4 g/L HMOs (2'-FL, LNnT, 6'-SL, 3'-SL)	Modulates immune response and alters colonic microbiota to reduce diarrhea duration	HMO-fed piglets had shorter diarrhea duration, enhanced anti-inflammatory and Th1 cytokine expression, altered colonic microbiota, and increased pH	Formula-fed newborn piglets
Laucirica et al[94]	Human Rotavirus G1P[8] and G2P[4]	2'-FL, 3'-SL, 6'-SL, galacto-oligosaccharides	Acts as soluble decoy receptors, directly affecting the virus to block binding	All oligosaccharides reduced infectivity in vitro. Maximum reduction for G1P[8] (62%) with 2'-FL and for G2P[4] (73%) with 3'-SL + 6'-SL mixture	African green monkey kidney epithelial cells (MA104)
Gozalbo-Rovira et al[113]	Rotavirus P[8] genotype	LNB, precursor of H1 antigen	LNB binds to the VP8* domain of the P[8] VP4 spike protein, inhibiting viral attachment	LNB binds with reduced affinity but induces conformational changes that inhibit rotavirus infection. Differences in ligand affinity explain variability in susceptibility among secretor and non-secretor phenotypes	Not specified (laboratory models)
Xiao et al[95]	Influenza virus	2'-FL	Enhances both innate and adaptive immune responses to vaccination	2'-FL improved vaccine-specific humoral and cellular immune responses, including CD4+ and CD8+ T-cell proliferation, dendritic cell maturation, and antigen presentation	6-week-old female C57Bl/6JOlaHsd mice
Mahaboob Ali et al[96]	Influenza virus, respiratory syncytial virus, human metapneumovirus, SARS-CoV-2	Multiple HMOs (e.g., 2'-FL, LNnT)	Mimic host cell receptors to inhibit viral entry by binding to viral surface proteins	In silico studies identified HMOs with high binding affinities to viral proteins, suggesting their potential as viral entry inhibitors and antiviral agents	Computational in silico modeling
Pandey et al[97]	Avian Influenza (H9N2 and other subtypes)	3'-SL, 6'-SL	Bind hemagglutinin and block viral attachment to host cells	3'-SL showed broad-spectrum activity against avian influenza in vitro. In vivo, 3'-SL eliminated H9N2 in chickens and improved clinical symptoms	Pathogen-free chickens
Duska-McEwen et al[98]	RSV, Influenza	2'-FL, 6'-SL, 3'-SL, LNnT	Enhance innate immunity by reducing viral load, cytokines, and inflammation	2'-FL reduced RSV viral load and cytokines. LNnT and 6'-SL decreased Influenza viral load. 6'-SL reduced IP-10 and TNF-α in RSV-infected PBMCs	In vitro respiratory epithelial cells and PBMCs
Schijf et al[114]	RSV	scGOS, lcFOS, pAOS	Modulates Th1/Th2 immune responses and enhances RSV-specific CD4+ and CD8+ T cells	Increased RSV clearance, enhanced Th1 response, reduced Th2 cytokines, and lower airway eosinophilia in RSV-infected mice	RSV-infected C57BL/6 mice
Guo et al[115]	Influenza virus	3'-SL	Synergistically reduces viral load and inflammation when combined with OPN	3'-SL and OPN reduced viral load by 75%, suppressed cytokine levels (TNF-α, IL-6), and exhibited anti-inflammatory effects	Human laryngeal carcinoma cell line (HEP-2)

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; CD4: Cluster of differentiation 4+; CD8: cluster of differentiation 8+; HBGAs: Histo-blood group antigens; HIEs: Human intestinal enteroids; HMOs: Human milk oligosaccharides; LNnT: Lacto-N-neotetraose; NK: Natural Killer; OPN: Osteopontin; PBMCs: Peripheral blood mononuclear cells; RSV: Respiratory syncytial virus; RV: Rota virus; SA: Sialic acid; Th1: T helper 1; Th2: T helper 2; LNDFHI: Lacto-N-difucohexaose I; NLB: Lacto-N-biose; scGOS: Short-chain galactooligosaccharides; lcFOS: Long-chain fructooligosaccharides; pAOS: Pectin-derived acidic oligosaccharides.

Table 6 Studies on human milk oligosaccharides levels, geographic variations, and antimicrobial activity

Ref.	Population/region	Key findings on HMO levels	Key antimicrobial activity	Geographic influence
Mao et al[144]	Urban Chinese mothers	High 2′-FL and sialylated HMOs (6′-SL) in early lactation; Gradual decline of 2′-FL and HMOs in mature milk	2′-FL inhibits Campylobacter jejuni and EPEC; 6′-SL protects against Rotavirus and Influenza	Secretor status influences early lactation HMO profile, optimizing pathogen defense in neonates
Liu et al[146]	Chinese mothers	3-FL increases significantly during later lactation; Sialylated HMOs decline after the first month	3-FL supports microbial diversity and inhibits Salmonella enterica and Pseudomonas aeruginosa	Extended breastfeeding in rural areas maintains high HMO levels, supporting long-term pathogen defense
Austin et al[129]	Chinese urban settings	Early high levels of fucosylated HMOs (2′-FL); Non-fucosylated LNT remains stable across lactation stages.	LNT neutralizes Vibrio cholera toxin; 2′-FL is critical for early pathogen defense	Urban mothers exhibit shorter lactation duration, potentially limiting long-term HMO-mediated benefits
Siziba et al[145]	German infants	High variability in HMO profiles across lactation; Secretor mothers show enriched 2′-FL levels	Sialylated HMOs reduce inflammatory responses and protect against respiratory pathogens (RSV, Influenza)	Differences in breastfeeding practices and complementary feeding influence microbiota outcomes
Derrien et al[65]	Rural Kenyan infants	High prevalence of 3-FL and LNT in late lactation; Enriched secretor group III (Se+, Le-)	3-FL supports Bifidobacterium infantis, lowering pH and reducing enteropathogen colonization; LNT neutralizes toxins	Rural diet and prolonged breastfeeding enhance microbiota dominated by B. infants
Kortesniemi et al[147]	Chinese mothers across lactation stages	Rapid decline in total HMOs from colostrum to mature milk; Sialylated HMOs persist in significant amounts	Sialylated HMOs prevent Helicobacter pylori adhesion and inhibit rotavirus infections	Secretor mothers exhibit early robust protection through 2′-FL, but benefits decline as lactation progresses
Asher et al[142]	Israeli mothers (Tel Aviv)	2′-FL dominant in secretors; LNT and 3-FL dominate in non-secretors; Seasonal and sex-dependent variations in HMO levels	2′-FL protects against infections via TLR4 inhibition; High DSLNT in non-secretors' colostrum protects against NEC	Urban breastfeeding with lower 2′-FL levels compared to global averages; shorter lactation may limit long-term benefits

The table summarizes findings from seven studies investigating human milk oligosaccharide (HMO) levels, geographic variations, and antimicrobial activity. Each study highlights the unique aspects of HMO concentrations across lactation stages, the role of specific HMOs in pathogen defense, and the influence of geographic and environmental factors. From this table, we found that across all regions, 2′-FL peaks in early lactation provide strong pathogen defense in neonates. 3-FL increases in later lactation, maintaining microbiota and pathogen defense during weaning. Rural settings like Kenya show sustained HMO levels due to prolonged breastfeeding, enriching microbiota that is beneficial for pathogen resistance. Urban populations like Israel and China experience sharper declines in HMOs due to shorter breastfeeding periods, potentially affecting long-term antimicrobial activity. Fucosylated HMOs (e.g., 2′-FL) are critical for early pathogen protection. Sialylated HMOs (e.g., 6′-SL) offer dual gastrointestinal and respiratory protection. Non-fucosylated HMOs (e.g., LNT and DSLNT) neutralize toxins and promote gut health throughout lactation. 2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 6'-SL: 6'-Sialyllactose; DSLNT: Disialyllacto-N-tetraose; EPEC: Enteropathogenic E. coli; HMOs: Human milk oligosaccharides; LNT: Lacto-N-tetraose; NEC: Necrotizing enterocolitis; RSV: Respiratory syncytial virus; TLR4: Toll-like receptor 4.

Table 7 Comparison of synthetic human milk oligosaccharides by method

Feature	Microbial fermentation	Chemical synthesis	Enzymatic synthesis
Cost	Low to moderate	High	Moderate
Scalability	High	Low	Moderate
Structural diversity	Limited (simpler HMOs)	High (complex and rare HMOs)	High (with optimized enzyme cascades)
Environmental impact	Moderate	High	Low
Ease of production	Straightforward for common HMOs	Complex and labor-intensive	Depends on enzyme availability

HMOs: Human milk oligosaccharides.

Table 8 Natural vs synthetic human milk oligosaccharides: Key comparisons

Feature	Natural HMOs	Synthetic HMOs
Source	Human breast milk	Biotechnological production
Structural diversity	Over 200 distinct HMOs	Limited to a few key HMOs (approximately 15 synthetic HMOs)
Customizability	Adaptive to maternal and infant factors	Fixed composition in formula.
Microbiota interactions	Broad spectrum of microbial benefits	Focused on Bifidobacteria support
Pathogen defense	Multifaceted, including sialylated HMOs	Limited to fucosylated HMOs
Immune modulation	Complex systemic and local effects	Under investigation
Accessibility	Breastfed infants only	Available in formula-fed populations

2′-FL: 2'-Fucosyllactose; HMOs: Human milk oligosaccharides.

Table 9 Extended health implications of human milk oligosaccharides antimicrobial effects

Health domain	Extended benefits	Mechanisms involved	Key HMOs
Immune development	Reduced risk of allergies (e.g., eczema, asthma) and autoimmune diseases (e.g., type 1 diabetes, IBD)	Promotes immune tolerance through Tregs; Modulates cytokine balance; Enhances GALT and sIgA production	2′-FL, 3-FL, sialylated HMOs
Gut microbiota programming	Establishes a balanced microbiota; Prevents dysbiosis and infections; Promotes long-term gut health	Selective enrichment of Bifidobacterium species; SCFA production supports gut barrier integrity and pathogen inhibition	2′-FL, 3-FL, LNT
Metabolic health	Reduced risk of obesity and type 2 diabetes; Improved lipid profiles and glucose metabolism; Prevention of metabolic disorders.	SCFA-mediated regulation of energy balance; Early microbiota influence on metabolic pathways	Fucosylated and non-fucosylated HMOs
Cognitive development	Enhanced neurodevelopment and cognitive function; Reduced risk of neurodevelopmental disorders (e.g., ASD, ADHD)	Precursors for gangliosides (myelination and synaptic plasticity); Microbiota-driven production of neuroactive compounds (e.g., serotonin)	Sialylated HMOs (3′-SL, 6′-SL)
Infection resistance	Long-term protection against infections; Reduced reliance on antibiotics, mitigating antibiotic resistance	Early microbial programming enhances innate and adaptive immunity; Pathogen exclusion through SCFA production and microbiota stability	2′-FL, 3-FL, sialylated HMOs
Chronic inflammatory diseases	Lower risk of IBD and systemic inflammation; Reduced incidence of cardiovascular diseases	Strengthens gut barrier integrity; Modulates systemic and intestinal inflammation through cytokine regulation	Fucosylated and sialylated HMOs
Non-communicable diseases	Prevention of type 2 diabetes and colorectal cancer; Improved long-term health outcomes	Anti-inflammatory and anti-carcinogenic effects of SCFAs; Early metabolic programming by microbiota	2′-FL, SCFA-producing HMOs
Neonatal-infant transition	Sustained protection during early dietary diversification; Optimized health outcomes with prolonged breastfeeding practices	Continuous microbiota support during complementary feeding; Long-term resilience against pathogenic exposures	3-FL, LNT

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 6'-SL: 6'-Sialyllactose; ADHD: Attention deficit hyperactivity disorder; ASD: Autism spectrum disorder; GALT: Gut-associated lymphoid tissue; HMOs: Human milk oligosaccharides; IBD: Inflammatory bowel disease; LNT: Lacto-N-tetraose; SCFA: Short chain fatty acids.