Immunoglobulin (Ig) A, IgG, IgM, and IgE are found in human milk. The most abundant Ig is secretary IgA (sIgA), followed by secretary IgG. SIgA in human milk is not fully digested by gastric acid in preterm or term infants. It provides lower gastrointestinal immunity and prevents the binding of toxins, bacteria, and viruses to the intestinal mucosa by neutralizing them [13,14]. Although sIgA in human milk decreases over time, its production in breastfed infants increases over the first 6 months of life [15,16]. IgG is the main immunoglobulin in the blood. It is also present in human milk, albeit at low levels [17]. However, IgG levels tend to increase in human milk as breastfeeding continues. Several studies suggest the possibility of IgG in human milk as a protective role in decreasing infections in breastfed infants [12,17,18]. Human milk can transfer IgM to infants. Term infants can partially digest IgM, whereas preterm infants cannot [13]. Human milk also contains allergen-specific IgG and IgE that may help sensitize infants [19].

Lactoferrin is a rich whey protein in human milk that binds well with iron. Lactoferrin in human milk is known to prevent bacterial, fungal, and viral infections. As iron is essential for microorganism growth, the iron-binding affinity of lactoferrin may inhibit bacterial growth by competing with bacteria to bind iron. However, the reported protective effects of recombinant or bovine lactoferrin are conflicting [20,21].

Glutamine, which is abundant in human milk, has been considered a fuel for rapidly dividing enterocytes and immune cells. It is a conditionally essential amino acid in critically ill patients, especially preterm in infants [20,22]. A blinded randomized study reported that glutamine supplementation for 1 month can reduce the rate of sepsis in preterm infants by blunting HLA-DR⁺ and CD16⁺ T lymphocytes [23]. Glutamine has been theoretically considered to prevent necrotizing enterocolitis (NEC), but recent well-designed randomized controlled trials have not proven the benefit of glutamine supplementation for reducing NEC rates [8].

Human milk contains a considerable amount of long-chain polyunsaturated fatty acids (LCPUFAs). Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are essential LCPUFAs that can modulate diverse immune responses in the body via lipoxins, prostaglandins, and thromboxanes [24]. LCPUFA supplementation can significantly reduce bronchopulmonary dysplasia and NEC in preterm infants by downregulating platelet-activating factor (PAF) and PAF receptor, and by downregulating endotoxin translocation into the systemic circulation [20,24]. It can also help develop retinal and cognitive function in infants [25,26]. The mean levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) in human milk are about 0.5% and 0.3% of total fatty acids, respectively, but the ratio of AA/DHA in mature human milk is considerably variable depending on a mother’s diet and region [27]. Both DHA and AA are included in infant formula at amounts at least equal to DHA in human milk [27]. However, the recommended dosages or ratios remain unclear.

Human milk oligosaccharides (HMOs) is the third most abundant substance in human milk, followed by lactose and lipids. The profiles of HMOs are quite diverse, with more than 200 distinct structures that differ from those of other mammals [28-30]. The secretor and Lewis genes determine HMO diversity [12]. HMOs are not digested by gastric acid and act as prebiotics by stimulating the growth of intestinal microbiota [12]. The interaction of HMOs occurs mainly in Bifidobacterium infantis, Bifidobacterium bifidum, Bacteroides fragilis, and Bacteroides vulgatus [31,32]. Another commensal bacteria, Escherichia coli, benefits indirectly from HMOs using metabolites from B. fragilis when HMOs are degraded [33]. HMOs also provide antimicrobial activities by increasing the sensitivity of Group B Streptococcus, Staphylococcus aureus, or Acinetobacter baumannii to antibiotics [34,35]. They also provide antiviral activity against rotavirus, norovirus, influenza, and human immunodeficiency virus by interacting with immune cells and inhibiting viral invasion [36,37]. In addition, as an anti-pathogen, 2'-fucosyllactose (FL) of HMOs acts as a soluble decoy receptor for Campylobacter jejuni and reduces colonization [38,39]. Lacto-N-fucopentaose I and 2'-FL reduce enteropathogenic E. coli adhesion and reduce pathogenicity by binding to heat-labile enterotoxin type 1 [40]. HMOs improve gut barrier function by promoting epithelial cell maturation via short-chain fatty acids, microbiota metabolites of HMOs, and glycocalyx alterations [12,41]. They can also enhance the local and systemic immune system by inhibiting Toll-like receptors and interacting with dendritic cells, leading to T-cell proliferation [12,42-44]. HMOs as immunonutrients play a role in promoting healthy gut microbiota, protecting against bacterial and viral infections, improving gut barrier function, and optimizing immune function (Table 2). Therefore, HMOs play a potential role in preventing infection and allergic diseases (Table 3) [45].

The initial formation of neonatal intestinal microbiota depends on maternal microbiota via adherence to intestinal cells and suppression of inflammation by the interaction with dendritic cell and the induction of T regulatory cell production [46]. The maternal microbiota is affected by diverse conditions, such as the prenatal use of antibiotics, health status, body mass index, and gestational age [47]. The complex human milk microbiota changes over time and varies among mothers. However, they generally include Bifidobacterium and Lactobacillus spp, Streptococcus, Staphylococcus, Bacteroides, and Enterobacter [46,47]. The human milk microbiota can contribute to establishing healthy intestinal bacteria in neonates and infants. Lactobacillus in the human milk induces Th1 cytokines and natural killer cells. They can adhere to intestinal cells, leading to the increased colonization of beneficial bacteria and the inhibition of pathogenic adhesion in the gut [48,49]. Bifidobacterium may suppress interleukin-8 in the presence of pathogenic Salmonella [50] and Bacteroides may suppress inflammation in the laminar propria by increasing FOXP3 T cells using surface polysaccharide A [51]. Although human milk contains diverse beneficial microbiota, intestinal microbiota compositions differ between infants fed expressed human milk and those directly breastfed. The intestinal microbiota of expressed breastmilk-fed infants show higher amounts of potential pathogens such as Enterobacteriaceae but lower amounts of Bifidobacterium [47,52]. Human milk plays crucial roles in preventing infections and influencing long-term health like obesity or allergic diseases via diverse immune mechanisms [45]. However, further research is required regarding the combined effect of bioactive supplementation in formula.

Infants and children with iron deficiency can be susceptible to infections due to impaired T-cell function which is associated with decreased production of interleukin 2 [53]. Iron supplementation can reverse the T-cell dysfunction.

Zn, the second most abundant metal in the human body following Fe, is a cofactor involved in DNA synthesis and repair. It is fundamental in protein synthesis as well as cell growth and differentiation. Zn deficiency occurs in acrodermatitis enteropathica, chronic malabsorption such as short bowel syndrome, or long-term insufficient supplementation of total parenteral nutrition [3]. Inadequate Zn intake increases diarrhea and pneumonia, especially in children younger than 5 years of age, and Zn supplementation can reduce the duration of diarrhea and respiratory illnesses [54]. Zn acts as a signaling molecule in the immune system [55]. Zn deficiency induces lymphopenia caused by decreased B cell differentiation in the bone marrow and interferon-γ production [56,57].

Vitamins act as antioxidants by suppressing reactive oxygen radicals and activating antioxidant enzymes [58]. Antioxidants can play important roles in the immune response via phagocytic function, cytokine and immunoglobulin production, and cell- mediated responses [3]. Oxidative stress or free radicals are involved in the development of diverse illnesses including chronic inflammatory diseases, aging, and cancers. Vitamin E is a membrane-bound antioxidant that can act as a free radical scavenger that prevents damage from lipid peroxidation. When vitamin E deficiency is caused by chronic cholestasis or a chronic malabsorptive state, decreased activities of antioxidative enzymes and increased hepatic lipid peroxidation can occur. Vitamin C, a hydrophilic antioxidant that cannot be synthesized by the human body, acts as a cofactor in collagen synthesis by activating hydroxylase by reducing Fe³⁺ to Fe²⁺. It is involved in inhibition of the redox potential under oxygen radical reactions in the body [59]. Excess free radial generation and oxidant cellular damage have been postulated in the pathogenesis of NEC in preterm infants and inflammatory bowel disease in children and adults. Although further clinical studies are necessary, vitamins can be used as antioxidant and immunologic nutrients [60,61].