Abstract
Mother's own human milk is the best nutrition for infants, especially preterm very-low-birth-weight (VLBW) (≤1,500 g) infants, because of its immune-modulatory constituents that strengthen the infant's host defense, provide protection against infections, and decrease the risk of necrotizing enterocolitis (NEC). When mother's own milk is unavailable or insufficient, donor human milk is considered the best alternative, especially for preterm VLBW infants. However, to assure biological safety, donor milk must be pasteurized. This results in partial or complete inactivation of some of the immunomodulatory constituents of human milk, which confer host defense. This review summarizes the current evidence regarding the effects of pasteurization on the different immunological constituents of donor milk, and their clinical significance, especially in relation to prevention of NEC.
Introduction
Mother's own breast milk is the ideal source of nutrition for infants, especially preterm infants.1–3 When mother's milk is not available, donor's milk is considered the best alternative, especially for very-low-birth-weight (VLBW) (≤1,500 g) preterm infants.1–3 However, to avoid transmission of infectious bacterial and viral agents, milk banks use Holder pasteurization that includes heating to 62.5°C for 30 minutes followed by rapid cooling.1–3 The problem is that such pasteurization completely or partially inactivates some of the immunomodulatory constituents of human milk, which confer host defense to the nursing infant, and are extremely important for VLBW premature infants who are immune-compromised to start with, and are at increased risk of infections and necrotizing enterocolitis (NEC). Mother's milk is much more than a nutrient rich compound, it is a reactive biological system, which constitutes a rich and complex source of bioactive and immunological compounds with antimicrobial properties.4,5 In the following paragraphs we shall review the major immunomodulatory components of human milk, and the effects of donor milk pasteurization on them.
Milk Immuno-Reactive Cells
Many of the immune components of the maternal immune system are secreted into the breast milk she produces. Many of these are immune cells (∼108 per day 6 ), the majority of them are T cells (>80%). 7 In addition to T cells, B cells, 8 neutrophils, 9 and macrophages 10 are also secreted into the mother's milk. Activated T cells from the mother can traverse the intestinal wall, and promote maturation of the newborn's immature endogenous T cells. 11 Pasteurization completely inactivates the cellular components of milk, including B and T cells, macrophages, and neutrophils.4,12–16
Immunoglobulins
Pasteurization also partially inactivates other milk immunological constituents, especially biologically active proteins (e.g., immunoglobulins 17 ), by causing heat denaturation with disturbance to their three-dimensional structure that is central to their immune function and recognition of foe. After birth human milk is the main source for different kinds of antibodies to the newborn infant. IgA and secretory IgA (sIgA), the dimeric form of IgA, and the most abundant immunoglobulin in human milk, are moderately reduced by Holder pasteurization (20–30% [range: 0–48%] reduction in concentration and 33–39% reduction in activity).4,13–35 sIgA prevents micro-organisms' attachment to the gut mucosa, increases viral excretion and neutralizes microbial toxins.4,36 IgM concentrations are significantly decreased by pasteurization to the point that they are virtually abolished.4,14,16,17,21,23,25,27–29 IgG concentrations are also significantly decreased (34% reduction) by heat pasteurization.4,14–16,20,21,23,25,27–29 Of IgG subclasses, IgG1 is not affected and IgG4 is reduced. IgG2 and IgG3 are undetectable in either fresh or pasteurized milk.4,23,28
Lactoferrin and Lysozyme
Lactoferrin and sIgA are the most abundant immunological components of human milk (∼30% of its protein content).4,37 Lactoferrin, along with lysozyme, comprises the bacteriostatic immune-reactive proteins in human milk.4,23,38 Lactoferrin binds free iron, thus reducing its availability for iron-dependent pathogens, thus inhibiting their growth. In addition, lactoferrin has direct cytotoxic effect by binding to the lipid-A-portion of the lipopolysaccharide component of the bacterial cell membrane, thus disrupting it.39–41 Lysozyme hydrolyzes linkages in the outer cell wall of gram-positive bacteria, 42 thus working synergistically with lactoferrin. Both lactoferrin and lysozyme protein concentrations and activities are considerably reduced after Holder pasteurization (by 35–90% and 20–85%, respectively).4,15–17,19–23,27–30,32,33,35,43,44 Lactoferrin-iron-binding capacity is also reduced after pasteurization (by 57–80%).4,17,19,20 In general, most studies demonstrated a significant reduction in lactoferrin and lysozyme biological activity as well. However, it is possible that some of their bactericidal activity is still retained in pasteurized human milk despite the reduction in their protein content, for example, by bactericidal peptides that form during lactoferrin digestion, 38 or by the action of other antibacterial enzymes and factors that co-act with lysozyme. 23
Cytokines
Cytokines are immune-modulatory components, which serve as signaling molecules that can exert multiple effects on the neonatal immune system, including chemoattraction of other cells, development and priming of the intestinal immune cells, angiogenesis, and suppression of inflammation.4,45–47 Many of the cytokines found in human milk exert anti-inflammatory effects, thus decreasing the response to infections in the newborn.23,38 There are different degrees of thermal resistance for the different cytokines, yet the biological significance of these differences is not clear.23,48 Interleukin (IL)-2, IL-4, IL-5, IL-12, and IL-13 are not significantly affected by pasteurization in both colostrum and mature donor milk.15,23,28 Although IL-10, IL-6, IL-1β, interferon-γ and tumor necrosis factor (TNF)-α are not significantly affected by pasteurization of colostrum,23,28 their concentrations are decreased after pasteurization of mature donor milk.15,23,49,50 Surprisingly, the immunosuppression of T cell proliferation by human milk that is attributed to IL-10, persists after Holder pasteurization. 50 IL-8 is not affected in pasteurization of colostrum, 28 and after pasteurization of mature donor milk it is not only preserved but may even be increased, possibly after being released from another compartment by the heat.15,48,49 IL-17 and Monocyte Chemotactic Protein (MCP)-1 are preserved after pasteurization of colostrum, whereas IL-7 is increased, possibly due to release from cellular or fat compartments into the aqueous fraction, and Macrophage Inflammatory Protein-1β (MIP-1β) is reduced.23,28 Erythropoietin is also significantly reduced after pasteurization. 50
Growth Factors
Some growth factors are decreased after pasteurization, including insulin-like growth factor (IGF)-1, IGF-2, IGF-Binding Proteins (IGF-BP) 2 and 34,23,51 (7–39% reduction), and hepatocyte growth factor, 15 whereas others such as epidermal growth factor, heparin-binding epidermal-like growth factor, and transforming growth factor (TGF)-β1, TGF-β2, and TGF-α withstand heat treatment.4,15,23,50–52 Granulocyte-macrophage colony-stimulating factor concentrations increase after pasteurization’ probably due the release of that factor from the cellular or fat compartments. 23 TGF-β2 was found to be stable also in pasteurized colostrum. 28 Granulocyte-colony-stimulating factor was not detected before or after Holder pasteurization in mature or colostrum donor milk.15,23,28
Oligosaccharides
Oligosaccharides are abundant in human milk and provide protection by binding pathogens53,54 and by their prebiotic activity stimulating the growth of beneficial bacteria, 55 that is, giving human milk its bifidogenic characteristics. 56 Oligosaccharides are not affected by Holder pasteurization. 57 This property of pasteurized donor milk is probably responsible for some of its beneficial effects on prevention of NEC, since NEC is known to be associated with both delayed colonization of Bifidobacterium and abnormal fecal microflora (Brook 2008). 58 Glycosaminoglycans in human milk, which also have anti-infective, antioxidant, and prebiotic effects, 56 are stable in pasteurization. 59
Gangliosides
Gangliosides are glycosphingolipids present in human milk, which function as target receptors for bacterial adhesion, enhance bifidogenesis, and have other effects on immune cells for prevention of infection. 60 They reduce bowel necrosis and inflammatory reactions related to it, thus decreasing the risk for NEC. 61 They are not affected by pasteurization. 15
Lipids
Milk fats, particularly medium-chain fatty acids and monoglycerides, possess antimicrobial properties, particularly lauric acid 62 and caprylic acid. 63 Long-chain polyunsaturated fatty acids contribute to prevention of NEC.64,65 Total lipid content and fatty acid profile are unaffected by pasteurization.49,66,67 Free fatty acid content is significantly increased after pasteurization.4,23,68 There are some changes in the relative profile of medium-chain fatty acids. Although some of the medium-chain saturated fatty acids are increased by pasteurization, oleic acid is slightly decreased.15,69 Long-chain polyunsaturated fatty acids are not affected by pasteurization. 15
Effect of Pasteurization on the Immunological Properties of Human Milk in Clinical Practice and on Its Effects in Prevention of NEC
Although some of the bioactive components of human milk are decreased or even abolished by pasteurization of donor milk, its ability to exert beneficial effects is not fully lost. Pasteurized donor milk maintains much of its capacity to induce T cell proliferation, 50 and some of its ability to inhibit the growth of Escherichia coli in vitro. 70 Cochrane meta-analysis that compared the effect of formula milk compared with donor human milk on growth and development of preterm or low-birth-weight infants concluded that although formula feeding resulted in short-term faster rates of weight gain, head, and linear growth, it also resulted in a significant increase in the risk of NEC (risk ratio: 1.87, 95% confidence interval: 1.23–2.85), without any long-term effects on growth or neurodevelopment. 71 Only few trials examined the issue of nutrient-fortified donor breast milk,72,73 and based on them it is evident that the rates of NEC and its severity are significantly decreased in infants receiving an exclusively human-based diet (including human milk-based fortification) compared with infants who get any bovine-based milk (either as preterm infant formula or as human milk fortifier). 73 The reason why donor human milk maintains some of the immunological advantages of fresh human milk, including prevention of NEC, 74 despite that the effects of Holder pasteurization is not fully clear. It may be related to the relative reduction in the different cytokines shifting the balance of pro-(TNF-α, IFN-γ, IL-1β) and anti-inflammatory (IL-10) effects toward the latter, thus decreasing the risk of NEC, 15 or to the increase in some immune-modulatory factors after pasteurization, such as that seen for IL-8.15,48,49 Alternatively, this may be related to the fact that long-chain polyunsaturated fatty acids and gangliosides, both known to have positive effects on prevention of NEC,61,75 were unaffected by pasteurization. 15 In summary, mother's own milk is the gold standard, but if unavailable or insufficient—donor human milk is the best alternative even after pasteurization. 76 Newer methods of pasteurization or treatments for donor milk that will assure biological safety, but could better preserve its biologic immunological properties are being explored.16,19,22,24,26,43,51,70,77–80
Footnotes
Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received.