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An Opinion on “Staging” of Infant Formula

A Developmental Perspective on Infant Feeding

Lönnerdal, Bo; Hernell, Olle

Journal of Pediatric Gastroenterology and Nutrition: January 2016 - Volume 62 - Issue 1 - p 9–21
doi: 10.1097/MPG.0000000000000806
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Breast milk is a dynamic fluid with compositional changes occurring throughout the period of lactation. Some of these changes in nutrient concentrations reflect the successively slowing growth rate and developmental changes in metabolic requirements that infants undergo during the first year of life. Infant formula, in contrast, has a static composition, intended to meet the nutritional requirements of infants from birth to 6 or 12 months of age. To better fit the metabolic needs of infants and to avoid nutrient limitations or excesses, we suggest that infant formulas should change in composition with the age of the infant, that is, different formulas are created/used for different ages during the first year of life. We propose that specific formulas for 0 to 3 months (stage 1), 3 to 6 months (stage 2), and 6 to 12 months (stage 3) of age may be nutritionally and physiologically advantageous to infants. Although this initially may impose some difficult practical/conceptual issues, we believe that this staging concept would improve nutrition of formula-fed infants and, ultimately, improve outcomes and make their performance more similar to that of breast-fed infants.

*Department of Nutrition, University of California, Davis

Department of Clinical Sciences, Pediatrics, Umeå University, Umeå, Sweden.

Address correspondence and reprint requests to Olle Hernell, MD, PhD, Department of Clinical Sciences, Pediatrics, Umeå University, S-901 85 Umeå, Sweden (e-mail: olle.hernell@.umu.se).

Received 10 September, 2014

Accepted 25 March, 2015

Drs Lönnerdal and Hernell contributed equally to the article.

Dr Lönnerdal is consultant/scientific advisor for and has/have had research funding/travel grants/honoraria from Arla Foods, Semper, Hero, Valio, Nestlé, Mead Johnson Nutrition, and BiosTime. Dr Hernell is consultant/scientific advisor for and has/have had research funding/travel grants/honoraria from Swedish Orphan Biovitrum (Sobi), Arla Foods, Semper, Hero, and Valio.

See “Need for Infant Formula” by Baker and Baker on page 2.

The availability of nutritionally adequate infant formulas is a fairly recent achievement. Until the 20th century, there was no safe and reliable alternative to breast-feeding. It was not until the mid-19th century when chemical techniques were developed allowing analysis of the gross chemical composition of milk that it became clear that each species has a unique composition of its milk. Toward the end of the 19th century, pasteurization was adopted by the dairy industry, and several physicians attempted to develop adequate substitutes for breast milk. The first marketed preparation was patented in 1867 by a German chemist, Justus von Liebig, who named it “the perfect infant food.” It consisted of wheat flour, cow's milk, and malt flour, cooked with potassium carbonate to reduce acidity (1).

The development of infant feeding took a significant step forward at the end of the 19th century when Heubner and Rubner published their calorimetric method of feeding, which allowed feeding infants according to their energy requirements. This was the beginning of modern studies of infant metabolism (2). Eventually, substitutes for human milk were developed from milk of other mammals through numerous modifications into the complex formulas that are available today (Fig. 1). Successive improvements in the understanding of the chemical and nutrient composition of milks were the basis for these developments. The composition of human milk became the ultimate reference. The era of formulas of known composition as complete foods for infants began around 1915 when Gerstenberger et al (3) developed artificial milk, in which the fat content was adapted to mimic that of human milk. The fluid mixture (synthetic milk adapted) contained approximately 4.6% fat, 6.5% carbohydrate, and 0.9% protein. In 1961, the first whey-dominant formula was launched, and in 1972 came the first Codex Alimentarius standard for infant formulas. Today's infant formulas are by definition foods intended for particular nutritional use by infants during the first months of life and satisfying by themselves the nutritional requirements of such infants until the introduction of appropriate complementary feeding (5), and their composition is therefore strictly regulated.

FIGURE 1

FIGURE 1

Infant formulas on the market have thus been developed with the composition of human milk as the model and are generally safe and meet the nutritional needs of the vast majority of healthy term infants. There are still differences between formula-fed and breast-fed infants, however, and the composition of human milk is no longer considered the standard criterion for infant formulas. Instead, similar performance of formula-fed and breast-fed infants in terms of growth and various developmental and health outcomes has become the reference. This goal has not been met as there are still significant differences between these 2 groups of infants, both during infancy and with respect to long-term outcomes (6,4). We believe that part of the reason for these persisting differences is the present static approach to infant formula feeding.

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PERSISTING DIFFERENCES BETWEEN BREAST-FED AND FORMULA-FED INFANTS

In spite of the trend of decreasing the protein level in infant formulas, the growth pattern of formula-fed infants has been different from that of breast-fed infants with slower weight gain during the first months of life and then more rapid growth, which is particularly because of a gain in body fat. This is also the foundation for the revised growth standards by the World Health Organization (7), which are now based on the growth pattern of breast-fed infants from different populations. Even in resource-rich countries, breast-fed infants have fewer infections than formula-fed infants, and when they are infected, the severity is generally milder and the duration of the infections shorter (6,8–10). Although the causes for this are not fully understood, the presence of specific secretory immunoglobulin A (sIgA) and many bioactive substances such as oligosaccharides, gangliosides, cytokines, chemokines, some other proteins, and, possibly, nucleotides in breast milk are generally believed to contribute to this defence against infection as are differences in immune competence (11,12) and gut microbiota composition (13,14) between breast-fed and formula-fed infants. These differences are also reflected in clinical parameters such as plasma amino acid pattern, serum insulin, and blood urea nitrogen (BUN) (15).

Differences observed between breast-fed and formula-fed infants during infancy are also believed to affect long-term health outcomes (6,10). Several meta-analyses have shown that breast-feeding has a small but significant effect on reducing the risk for obesity later in life (6,16). Several studies have also shown that having been breast-fed as compared with formula-fed results in slightly lower blood pressure (17) and possibly reduces the risk to develop both type 1 (18,19) and type 2 diabetes (20). Other studies have shown that although breast-fed infants have higher serum cholesterol while being breast-fed, they have slightly lower values than infants initially formula-fed later in life (17). This could possibly explain why some epidemiological studies have shown a reduced risk of later cardiovascular disease in previously breast-fed infants (17), although this is still an open question (21). It should be noted, however, that in resource-rich countries most of these effects are small, but on a population level they are likely to have significant health effects.

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DYNAMIC NATURE OF BREAST MILK COMPOSITION

Human milk is not static in its composition; it varies among mothers, during a feed, during the day, with gestational stage, to some extent with the mother's diet, and with lactation stage (22,23). Most of these variations have been taken into account when composing modern infant formulas. When compiling data in the literature, it is evident that there are significant changes in the composition of breast milk, however, during lactation, and it is clear that at least some of these changes reflect changes in requirements of the infant, which in turn reflect changing growth velocity and maturation of immunological and physiological functions. These changes have largely not been taken into account in formula development. In the United States, regular infant formulas are intended for all term infants throughout the first year of life, whereas in Europe they are intended to be used during the first 6 months of life, whereas follow-on formulas are intended to be used after that age, although infant formulas also can be used after the first half of infancy. Thus, the composition of infant formula is largely a compromise to meet the needs of this diverse group of infants. It is of note that the most profound changes in breast milk composition occur, however, during the first 4 to 6 months when breast milk (or formula) is intended to be the sole source of nutrition. Therefore, it would make nutritional and physiological sense to have infant formulas with different composition for these different stages in early life. The developmental changes in breast milk during lactation are described below and will then be used for discussing the concept of “staging” of infant formulas.

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Protein

The protein concentration of human milk during early lactation is considerably higher than later on (Fig. 2), which reflects a decreasing requirement for protein with age (Table 1), from 2.7 g · kg−1 · day−1 during the first month to 1.5 g · kg−1 · day−1 at 2 to 3 months, and 1.2 g · kg−1 · day−1 at 5 to 6 months and 1 g · kg−1 · day−1 toward the end of the first year (26). It is quite likely, though, that these protein requirements are overestimated because the mean adjusted protein intake of breast-fed infants is 1.95 to 2.04 g · kg−1 · day−1 at 1 month of age, 1.27 to 1.33 g · kg−1 · day−1 at 3 months of age, and 1.05 to 1.11 g · kg−1 · day−1 at 6 months of age (Table 2), that is, substantially lower than the estimated protein requirements. The protein concentration in early milk ranges from 12 to 18 g/L, whereas mature milk only contains 8 to 9 g/L. It is thus evident that infant formula with a protein concentration of 13 g/L (lowest level of most commercially available formulas) is sufficient during the first month of life but is unnecessarily high during the latter part of infancy, particularly in countries where complementary feeding will provide sufficient protein. The protein intake of infants fed formula with 13 g/L is ∼2.4 g · kg−1 · day−1 at 1 month of age, ∼2.0 g · kg−1 · day−1 at 3 months of age, and ∼1.6 g · kg−1 · day−1 at 6 months of age, all substantially higher (by 40%–60%) than the protein requirements described above. That the protein concentration of present infant formulas is likely to be excessive is demonstrated by findings of high plasma amino acid levels, particularly the branched-chain/insulinogenic amino acids, plasma insulin, urinary C-peptide, and serum urea in formula-fed as compared with breast-fed infants (15). That a lower protein content of formula (11 g/L) is adequate from 3 to 6 months of age was recently shown in a study by Inostroza et al (28). High protein intake during early life has in several studies been associated with increased obesity risk later in life (29–31). This is one of the reasons why formula manufacturers have successively reduced the protein content of formulas in both the United States and Europe. Even after these reductions, however, the metabolic differences mentioned above remain. Therefore, an alternative and more physiological approach would be to have an “early” infant formula with a comparatively high protein level and a “late” infant formula(s) with lower protein level (see “Nutrient Composition of Staged Infant Formulas: Protein,” below). Virtually all of the clinical studies to date show significantly higher levels of insulinogenic amino acids and insulin in infants fed formula with present protein levels up to 6 months of age; a successive lowering of the protein content of formula during infancy is likely to reduce these serum insulin values, possibly affecting long-term outcomes such as obesity and diabetes.

FIGURE 2

FIGURE 2

TABLE 1

TABLE 1

TABLE 2

TABLE 2

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Protein Utilization

The utilization of human milk proteins is another factor to consider when discussing protein requirements. Several human milk proteins are known to be relatively resistant to digestion, such as sIgA, lactoferrin, and lysozyme, and are actually found intact in significant quantities in the stool of breast-fed infants (32). Given the much higher concentrations of these proteins in human as compared with bovine milk and thus in infant formulas, utilizable protein will be even lower for breast-fed than for formula-fed infants. It should be noted, however, that although these proteins have structures rendering them some stability against proteolytic enzymes they are digested, even though slowly, and only a minor fraction will remain unutilized (32,33). The fraction of these proteins being undigested is higher during early life and decreases with increasing maturity of digestive functions. It has been shown in young developing pigs that proteolytic activity in intestinal contents is considerably lower during early life than at weaning, and that premature weaning induces maturation of exocrine pancreatic functions and also alters the pattern of pancreatic proteases secreted (34). This may suggest that immature pancreatic function is not really immature when the offspring is fed its own species’ milk but is so when formula is the main food. In fact, immature exocrine pancreatic function may be beneficial to the breast-fed offspring. This hypothesis receives some support from the fact that breast milk contains inhibitors of proteolytic enzymes, in particular α-1-antitrypsin (AAT), and it has been shown that AAT from breast milk at these concentrations has a significant limiting effect on pancreatic enzyme activity and digestion of infant formula (35). This may therefore contribute to a lower degree of protein utilization from breast milk in addition to being protective toward the many bioactive proteins in human milk that confer benefits to the newborn infant. This further strengthens the concept of adjusting the protein concentration of infant formulas with postnatal age.

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Fat

Early human milk is low in fat but soon reaches a concentration of 3% to 4% and stays at this level, accounting for on average 50% of the energy content of milk (36). Both the concentrations of total fat in the milk and the fatty acid pattern vary among mothers. Furthermore, mother's diet has considerable influence on the fatty acid composition of breast milk (22). The fat concentration is higher in the milk of preterm mothers than in that of term mothers (37).

The observation that human milk contains not only the essential fatty acids (linoleic acid and α-linolenic acid) but also their longer chain derivatives arachidonic acid (ARA) and docosahexaenoic acid (DHA) contributed to research on the requirement of these fatty acids, particularly for preterm infants, and the classification of them as conditionally essential, at least for preterm infants. In spite of the concentration of DHA in milk being dependent on the DHA status of the mother, the concentration in the milk was reported to be higher in preterm milk than in term milk (37), although this was not confirmed in a 2007 study (38), and decreases with duration of lactation, which, if so, may reflect the decreasing need for preformed DHA.

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Carbohydrate

The major part of carbohydrate in human milk is lactose (5%–6 %), but the complex mixture of oligosaccharides constitutes the third largest fraction of human milk, or 1 to 2 weight% (39). Similar to fat, the lactose content is lower in the early milk than in mature milk, produced after the first weeks of lactation, but thereafter remains relatively constant (23). The milk oligosaccharides, however, exhibit pronounced changes during lactation not only in concentration but also in composition, complexity, and structure (39,40).

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Energy Density/Volume

It is a general opinion that an infant's average intake of formula is considerably higher than that of breast milk. For example, at 3 to 4 months of age, formula intake is usually approximately 1000 mL/day, whereas the average intake of breast milk is approximately 700 to 800 mL/day (41). This underscores that with a higher protein content of infant formulas, the daily protein intake of formula-fed infants is much higher than that of breast-fed infants. Even if the protein content in formulas needs to cover the requirement for infants consuming volumes similar to those of breast-fed infants, a protein content of 13 to 15 g/L is at least 50% higher than that required to satisfy the needs of such infants. It should also be noted that there is considerable variation among breast-fed infants with intakes ranging from 500 to 1000 mL/day, but it is not clear if the protein concentration of milk consumed by infants at the lower end of volume intakes is higher than in milk consumed by infants receiving larger volumes.

The energy density of human milk has conventionally been considered to be 670 kcal/L, and to this date this has been the level of energy used in virtually all of the infant formulas. Studies in 2005, however, suggest that this represents an overestimate and that the actual energy content is closer to 550 kcal/L (42). Thus, as discussed above for protein, the higher energy density of the formula will be further accentuated by the higher volume intake of formula-fed infants, leading to considerably higher energy intakes. This is most likely a major reason for the higher accumulation of body fat in formula-fed infants (43).

To date, there have been very few studies comparing different energy densities of formulas intended for healthy term infants (44,45). This may seem surprising but can be explained by the relatively high lower limit set by regulatory authorities. This is a circular argument, however, because clinical studies on formulas with lower energy density are needed to provide the scientific background for changing present limits. We have recently conducted a randomized control trial comparing the effects of feeding formulas with different energy densities (600 vs 660 kcal/L) and protein concentrations (12.0 and 12.7 g/L) on formula intake and growth, including body composition, from 4 to 8 weeks until 6 months of age (15). Interestingly, this study showed that there was no difference in growth between the groups, and infants in the group consuming the formula with lower energy density compensated the difference in energy density by increasing their daily intake (volume) resulting in very similar energy and protein intakes. Because differences in protein levels of infant formulas have not been associated with effects on daily intake, it is highly likely that the difference in energy density was the cause of the observed difference in intake.

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Iron

Term infants with normal birth weight are born with relatively large stores of iron, which are mobilized and used during the first part of infancy. There is general consensus that these stores are adequate to meet infants’ iron requirement for the first 6 months of life. After this age, iron requirement is difficult to meet, and iron fortification of infant formulas and supplemental iron (as iron drops) are often recommended as are iron-rich complementary foods. The level of iron fortification used in infant formulas is quite generous ranging from 4 to 12 mg/L, which is 20 to 40 times higher than in breast milk, and the major reason for this is to meet the iron requirements during the latter part of infancy (46,47). If different infant formulas would be available for different periods of infancy, it would be logical that early infant formula would contain a lower level of iron whereas formula for the second half of infancy should contain a higher level of iron as is recommended for follow-on formulas in Europe. Iron is a known strong prooxidant, and excess iron given as supplements has been shown to have adverse effects on linear growth of infants in both developed (48,49) and developing countries (49,50–52). A recent study also suggests that a formula with higher iron content fed to iron-replete infants results in poorer cognitive development than a formula with lower iron content (53) (see below). Although it may be argued that the provision of iron early in life may contribute to improved iron status, we have shown that additional iron given between 4 and 6 months of age does not result in larger iron stores that can be used when the requirements increase during the second half of infancy (54). We have also shown that an iron level of 1.8 mg/L in infant formulas is sufficient to result in adequate iron status at 6 months of age in healthy term infants (55), whereas other studies suggest that a higher level of iron is needed after 6 months. Thus, infant formula during early life could contain a lower level of iron fortification and formula(s) intended for older infants should contain higher levels.

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STAGING WOULD ALLOW MORE TAILOR-MADE COMPOSITION OF INFANT FORMULAS

Infants born at term have a high growth rate that declines during infancy, and it seems logical that infant formulas intended for term infants should reflect their changing needs. Staging of term infant formulas would also allow consideration of the issues discussed above, such as higher protein, energy, and DHA needs during the first months of life and lower requirements later during infancy, and, conversely, less iron during early life and more during late infancy (56) Although such staging will need clinical studies for evaluation of the concept, it seems reasonable to suggest that there at least should be an infant formula for early infancy (0–2 or 3 months, stage 1), another one for 3(4) to 6 months of age (stage 2), and a third one after the first half of infancy (6–12 months) when the diet is becoming diversified (stage 3) (Fig. 3). This latter formula would according to the European Union Directive be either an infant formula or a follow-on formula. Hypothetically, even more stages may be considered, if this could be solved practically.

FIGURE 3

FIGURE 3

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NUTRIENT COMPOSITION OF STAGED INFANT FORMULAS

Energy and Macronutrients

Protein

Several clinical studies have shown that a protein concentration of 13 g/L of a formula containing 60% whey protein and 40% casein meets the needs for normal growth even during the first 2 months of life, when the protein requirement of term, appropriate weight for gestational age infants is highest. It seems more than likely that a level of 12 g/L would supply enough protein, but there are fewer studies at this level, and some are difficult to evaluate as the formula was fortified with some amino acids (57). Thus, it is likely that a level of 13 g/L in stage 1 formulas would include a considerable safety margin, particularly if the protein source is enriched in α-lactalbumin to resemble the whey composition of human milk. α-Lactalbumin is relatively rich in tryptophan, the first limiting amino acid when reducing the protein content in infant formulas based on cow's-milk protein, and clinical studies have shown that a reduced protein content in infant formula enriched in α-lactalbumin results in satisfactory growth and plasma tryptophan levels similar to those of breast-fed infants and infants fed standard formula (with a higher protein level) (58,59). As the whey to casein ratio is >60/40 in early breast milk (60) (Fig. 4), it may be advantageous to also consider higher whey to casein ratio in stage 1 infant formula. This would also increase the tryptophan level of the formula, although the proportion of β-lactoglobulin, a protein absent in breast milk, would also increase, and the amino acid profile of this protein is less than optimal for infants. Based on estimated protein requirements and intakes in breast-fed infants, it seems that a protein content of 12 g/L would be sufficient in stage 2 formula. We verified this in a blinded randomized clinical trial comparing 2 formulas, one with 12.7 g protein/L and the other 12.0 g/L. There was no difference in growth or BUN, nor was there an indication of too-low plasma amino acid levels (15). Inostroza et al (28) concluded in their study on infants of overweight mothers that an even lower protein concentration (11 g/L) would suffice from 3 months of age. Because complementary foods are recommended from 6 months of age (but often introduced between 4 and 6 months) and generally contain relatively high levels of protein, at least in resource-rich countries; it appears prudent to use a protein level of approximately 11 g/L in stage 3 formulas (follow-on formulas), which is also in agreement with the lower limit of 10.8 g/L as recommended within the European Union (5).

FIGURE 4

FIGURE 4

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Energy

Energy requirements per kilogram body weight decreases with increasing age and decreasing growth rate (42). There are no pronounced changes in the energy density of breast milk, however, during lactation. Although the protein concentration is high in early milk, lipid and carbohydrate concentrations are lower making the energy density similar. Thus, from this perspective, one might argue that staging of the energy density would be advisable. As we discussed above (“Dynamic Nature of Breast Milk Composition: Energy Density/Volume”) a reduction in the energy density of formula, however, resulted in a compensatory increase in daily volume intake making the energy intake very similar. Thus, it seems that the infant itself is capable of controlling the energy intake from formula at least within the range 60 to 67 kcal/L (15).

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Long-Chain Polyunsaturated Fatty Acids

Similar to iron, long-chain polyunsaturated fatty acids (LCPUFAs), particularly DHA, are needed for optimal brain development. After this was first recognized, several studies showed a beneficial effect of DHA on neurodevelopment, particularly on visual function in preterm infants. Several subsequent studies also showed positive effects, both short term and longer term, of a supply of preformed DHA in term infants. Recent meta-analyses and systematic reviews have not supported some of these earlier results (61). Campoy et al (62) wrote in a recent systematic review: “We conclude that based on present evidence there is still no clear evidence of long-term beneficial or harmful effect of LCPUFA supplementation on neurodevelopment or visual function in term infants.” It should be noted, though, that there are positive effects, but at present these seem to be limited to certain groups of preterm infants (63) and term infants with special needs (64). Today, many infant formulas intended for term infants are supplemented with DHA and ARA, but until more substantial evidence is available it seems justified to question whether formulas used after the first months of life (stage 1) need to contain DHA and ARA, given that proper levels and balance of their respective precursors, α-linolenic acid and linoleic acid, are provided, particularly as the former fatty acids add a substantial extra cost to the product.

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Oligosaccharides

The structures of human milk oligosaccharides depend partly on the mother's blood group and partly on the developmental pattern of the various glycosyltransferases involved in their synthesis (65). The physiological impact of the oligosaccharides on the recipient infant is not yet fully understood, and even less is known about the significance of their temporal patterns. There is, however, considerable evidence supporting a role for these oligosaccharides acting as decoy receptors for various pathogens and as prebiotics assumed to promote the development of a bifidogenic microbiota. A major function, however, is as a source of indigestible carbohydrate, or soluble dietary fiber, which is the most likely reason why breast-fed infants rarely become constipated, whereas formula-fed infants often are. Although recent studies have suggested that some specific oligosaccharides may be used as substrate for some specific bifidobacteria (66,67), this is likely a minor part of the total fraction. Hence, most of the oligosaccharide fraction is still regarded as nonutilizable energy. It is interesting, though, that the oligosaccharides have been shown to interact with intestinal cells and affect gene transcription (68), suggesting that this fraction may be involved in intestinal cross talk with the immune system. A major interest for formula manufacturers today is to add oligosaccharides to their formulas aiming at making formula-fed infants more similar to breast-fed infants. To date, however, there are few oligosaccharides with structures similar to those in human milk, not even the simplest structures, commercially available, although this is an area of rapid development. There have been attempts to add oligosaccharides to formulas to mimic at least part of the functions of breast milk oligosaccharides, such as, microbiota composition and stool consistency, but these carbohydrates, primarily fructooligosaccharides and galactooligosaccharides, are very different in structure and most likely in most functions. Given the present lack of knowledge, it is hard to predict at what age oligosaccharides would be beneficial if added to formulas and what composition and concentration would be ideal at various stages.

It should be noted, however, that the oligosaccharides may exert their function as decoy receptors for pathogens in both free form and bound to protein. For instance, breastfeeding-associated protection against calicivirus diarrhea is associated with high levels of 2-linked fucosylated oligosaccharides in the milk, and human calicivirus strains, including Norwalk virus, use gut 2-linked fucosylated glycans as receptors. In 2006, it was shown that milk of mothers who are nonsecretors, and therefore lack 2-fucosylated oligosaccharides in their milk, had little inhibitory activity against binding of the virus to mucosal biopsies (69). Interestingly, the same was true for free oligosaccharides from milk of secretor mothers, having 2-linked fucosylated glycans, whereas the milk proteins bile salt–stimulated lipase (BSSL) and mucins MUC1 and MUC4 accounted for virtually all of the inhibitory activity. These proteins have in common O-glycosylated tandem repeat sequences offering multiple binding sites (69), which these viruses obviously need. Thus, the backbone to which the decoy receptors are attached is also important. In analogy, we recently showed that BSSL is the main or only glycoprotein in human milk that potently binds dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN) and blocks DC-SIGN mediated transinfection of CD4+ lymphocytes with human immunodeficiency virus type 1, probably by offering multiple Lex binding sites (70). Another example may be κ-casein, a heavily glycosylated casein subunit in human milk, which was shown to be a strong inhibitor of Helicobacter pylori attachment to human gastric mucosa via its Lewis b structures (71). The possibility of adding protein-bound oligosaccharides to infant formula has not yet been pursued.

It seems reasonable to assume that the antimicrobial effect of the oligosaccharides as well as their prebiotic effect should be most important during the first few months of life and therefore the need, particularly in stage 2 and stage 3 formulas, should be questioned when discussing staging of formulas.

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MINERALS AND TRACE ELEMENTS

Calcium and Phosphorus

The concentration of both calcium and phosphorus in breast milk decreases with length of lactation, probably reflecting decreasing needs with decreasing growth rate. Because the calcium:phosphorus ratio in breast milk is assumed to be optimal for the growing infant, infant formulas need to be discussed from these perspectives. Present infant formulas have a calcium concentration approximately 100% higher than that of breast milk, primarily because of lower bioavailability of calcium from cow's milk and assumed lower bioavailability from formulas. Data on requirements during infancy are scarce, and no studies have been performed on concentrations of calcium and phosphorus lower than those presently used, but it seems likely that a stage 1 formula should contain more of these minerals than a stage 2 formula. The phosphorus level in formulas is also considerable higher in formulas than in human milk, and therefore the same argument should be applicable to the phosphorus concentration, given that the ratio between calcium and phosphorus is kept at the present recommendation, that is, 1.1:1 to 2:1. With increasing efficiency of fat absorption during the first months of life (72,73), the risk for formation of insoluble calcium soaps may decrease, which, theoretically, might be a further reason why the calcium concentration in stage 2 formula could be lower than in stage 1 formula.

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Iron

The iron requirement in early life is very low as term infants are born with considerable hepatic iron stores as well as high hemoglobin iron concentrations, which are both successively reduced and mobilized during the first 4 to 6 months of life (see “Dynamic Nature of Breast Milk Composition: Iron”). Thus, there is no need for iron fortification of starting infant formulas, which normally would contain approximately 1 mg/L when unfortified, which is 2 to 3 times the concentration in human milk. In stage 2 formulas, most infants would not require additional iron. Some infants with high growth rates, lower stores from birth, and early clamping of the umbilical cord, however, may require some additional iron, possibly making it reasonable to have an iron level of 2 to 4 mg/L. During the second half-year of life (stage 3), iron requirements increase considerably, and as many weaning foods may have low iron content or bioavailability, a higher level of iron in such formulas is recommended, for example, 4 to 8 mg/L depending on the iron fortification tradition of complementary food in each country or area. An even higher level of iron fortification in infant formulas may have negative consequences. Lozoff et al (53) showed that iron-replete Chilean infants as defined by hemoglobin >128 g/L fed an infant formula with 12.7 mg iron/L had significantly poorer cognitive development at 10 years of age than infants fed a formula with a lower level of iron fortification (2.3 mg/L). Jaeggi et al (74) recently showed that the gut microbiota was adversely affected when 6-month-old infants were given home-fortified porridge with a micronutrient powder (MNP) with 12.5 mg iron for 4 months as compared with MNP without iron. In addition, fecal calprotectin, a marker of intestinal inflammation, was significantly higher in the group given additional iron. Thus, provision of iron to term infants may have adverse effects, warranting some caution. It is of note, though, that preterm infants and also moderately preterm infants have higher iron requirements and lower iron stores than term infants and therefore need a stage 1 formula or a postdischarge formula with higher iron level than a stage 1 formula intended for term infants. Alternatively, they need iron supplementation (75).

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Zinc

Zinc is required for optimal growth. The concentration in breast milk steadily decreases during lactation from approximately 4 to 5 mg/L to 0.5 to 1 mg/L at 6 months, and there is no indication that breast-fed infants need extra zinc during the first half of infancy (76). Hence, the requirements of iron and zinc move in opposite directions during infancy. This is not reflected in present formulas. The bioavailability of zinc varies considerably from complementary foods depending on the contents of enhancers and inhibitors of zinc absorption. Therefore, the level of zinc in formulas after 6 months may need to be adjusted depending on the type of weaning foods used in different populations; with primarily plant-based weaning foods, a relatively higher level should be used because of the relatively high phytate content of the diet, whereas consumption of weaning foods containing meat would require lower levels of zinc fortification (77).

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Sodium, Chloride, and Potassium

Sodium and chloride concentrations are considerably higher in early human milk than later during lactation. The concentration of sodium decreases from an average of 300 to 400 mg/L during the first 2 weeks to 200 mg/L at 1 month and 125 mg/L at 6 months and chloride from 600 to 900 mg/L during the first 2 weeks to 400 to 450 mg/L between 1 and 6 months of lactation (78). Given the potential negative effects of high salt intakes and the general recommendations to limit these intakes also in infants and children, it seems reasonable not to use more sodium and chloride in infant formulas than necessary. Therefore, we suggest that the sodium and chloride concentrations could be lower in stage 2 than in stage 1 formula. The concentration of potassium also declines to some extent with the duration of lactation, with concentrations between 600 and 700 mg/L during the first 2 weeks and 400 to 500 mg/L from 1 to 6 months. Presently, most infant formulas contain approximately 180 mg sodium, 270 mg potassium, and 220 mg chloride per liter.

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VITAMINS

Water Soluble

Generally, our knowledge of the requirements of water-soluble vitamins in infancy is limited. As surplus intakes are generally considered rather harmless, the practice has been to use generous levels in formulas. Collectively, these vitamins are higher in breast milk during the first 5 days of lactation than in mature milk, with the possible exception being vitamin B12. It should be noted that it is difficult to analyze vitamin B12 in breast milk, which also impacts the assessment of requirements in early life. Collectively, there is no detailed information on how the concentrations vary with lactation duration in mature milk. The mother's diet seems to have little effect of their concentrations in breast milk, however, unless the mother is initially deficient (22). Based on the available literature, it is difficult to conclude whether the levels should be different in stage 1 and stage 2 formulas.

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Fat Soluble

It is evident that breast milk concentrations of vitamins A and D cannot be used as a reference for the concentrations to be used in formulas. It is, however, of note that the concentrations of vitamins A and E decrease in milk during lactation and that high intakes of vitamin A in adults may be associated with decreased bone mineralization and fracture risk (79). Whether higher intakes of vitamin A than required in infants have an impact on bone mineralization has to our knowledge not been studied. In many countries, complementary foods are fortified with vitamins A and D. To avoid the risk of bleeding, infants should be given vitamin K immediately after birth to meet their needs until colonization of the gut secures sufficient endogenous synthesis. As human milk is low in vitamin D, supplements are recommended for all infants in most European countries independent of whether they are breast-fed or formula-fed. It is not possible to conclude whether there should be different concentrations of fat-soluble vitamins in stage 1 and stage 2 formulas.

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BIOACTIVE PROTEINS

Some of the proteins in human milk confer benefits to the breast-fed infant in addition to being a source of amino acids, which can be used for protein synthesis. Collectively, they are often referred to as bioactive proteins and have in common that they are totally or partially resistant to digestion, at least in the proximal part of the gastrointestinal tract (80). This means that these proteins or fragments of them can exert bioactivities in the intestinal lumen or interact with the intestinal mucosa and thereby affect cell signaling and gene transcription. That these proteins resist digestion has been shown by both in vitro digestion experiments as well as findings of intact proteins in the stool of breast-fed infants.

sIgA is the major immunoglobulin (Ig) in human milk comprising 5% to 10% of total milk protein with the highest concentration during the first weeks of lactation (23). This protein will transfer maternal immune competence to the recipient infant, and intact sIgA is found in the stool of breast-fed infants. Although it would be desirable to add sIgA to formulas, this is a difficult endeavor. The primary Ig in cow's milk is IgG, which in contrast to IgA is easily digested. Attempts have been made to “hyperimmunize” dairy cows with pathogens that infants are commonly exposed to and adding the bovine Ig fraction to infant formula (81) or given it orally to infants and children with Escherichia coli diarrhea (82), but the very minor sIgA fraction would most likely need to be enriched to achieve at least some of the biological benefits of human sIgA. If such attempts would become successful, then the need would certainly be the greatest in a stage 1 formula, that is, before the infant's own production of IgA has developed.

Lactoferrin is another major protein in human milk with the highest concentration in early milk. Lactoferrin is also present in cow's milk but in much lower concentration. Bovine lactoferrin is, however, already commercially available in large quantities and can therefore be added to infant formulas. In fact, its antimicrobial effect has been documented in both preterm and term infants (83,84). Both human and bovine lactoferrin have been shown to be present in the stool of breast-fed infants and infants fed formula fortified with bovine lactoferrin, respectively, but the capacity of lactoferrin to resist digestion is larger in early life. Lactoferrin would therefore be a suitable candidate for fortification of particularly stage 1 formula when the susceptibility for infections is the most pronounced. Lactoferrin can also stimulate intestinal growth by affecting both cell proliferation and differentiation, and development of immune functions (85). These functions are also of particular importance during early infancy.

Some of the antimicrobial effects of lactoferrin seem to be exerted in combination with lysozyme, an enzyme present in high concentrations in human milk, particularly during early lactation (23). Lysozyme has been shown to break down the proteoglycan matrix of Gram-positive bacteria and to have a synergistic effect with lactoferrin to also effectively kill Gram-negative bacteria (86). Although lysozyme is present in cow's milk in low concentrations, to our knowledge there is no commercial source of bovine lysozyme.

Enriched fractions of bovine α-lactalbumin are already added to some infant formulas to improve their amino acid composition, thereby allowing a reduction of the total protein content of the formulas (see “Nutrient Composition of Staged Infant Formulas: Protein”). α-Lactalbumin, however, has also been suggested to have biological functions such as promoting immune development and trace element absorption (87). Because α-lactalbumin is relatively easily digested, these biological functions are more likely to be exerted during early life when intermediary peptides may survive for at least some time in the upper gastrointestinal tract. Although the latter functions would support adding α-lactalbumin to stage 1 formula, the former effect allowing reduced protein concentration would support the use in stage 2 formula.

There are many other proteins with biological activities in human milk such as cytokines, chemokines, enzymes, and hormones, the effects of which on the breast-fed infants still are relatively unknown. One cytokine of particular interest is transforming growth factor β (TGF-β), which seems to affect immune development and, particularly, the development of oral tolerance of breast-fed infants (88). Its concentration is particularly high in early breast milk, and it is resistant to digestion in vitro under conditions similar to those in vivo in newborn infants (89). Interestingly, some bovine whey protein fractions have been found to be comparatively rich in TGF-β, and infant formulas can therefore be made with TGF-β concentrations similar to that of human milk (89). Beneficial effects of such formulas, however, have not yet been documented, and it is too early to recommend fortification of formulas with TGF-β. Furthermore, heat treatment may diminish the activity of TGF-β, which is similar to several other bioactive proteins, and novel ways of adding these components to infant formula without excessive heat treatment need to be considered. Another growth factor present in high concentrations in human milk is osteopontin, a highly glycosylated phosphoprotein, originally isolated from bones. It has an integrin-binding motif (90) and is involved in cell-mediated immunity and inflammation, triggering activation and recruitment of leucocytes and the production of cytokines. Therefore, osteopontin has been classified both as a matricellular protein and as a cytokine (91). Osteopontin is also present in bovine milk, even though at much lower concentrations, and osteopontin fractions are now commercially available and may be added to infant formulas. Interestingly, studies in infant Rhesus monkeys have shown that feeding infant formula with osteopontin added to the concentration of human milk resulted in a gene expression pattern in intestinal biopsies considerably closer to that of breast-fed infants than to infants fed the formula without added osteopontin (92). Thus, osteopontin could be an interesting future component to be added to formulas, perhaps particularly to stage 1 formula.

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Bile Salt–Stimulated Lipase

BSSL is a lipolytic enzyme that facilitates digestion and absorption of dietary fat. It has broad specificity and hydrolyzes a variety of different substrates, for example, tri-, di- and monoglycerides, cholesteryl- and retinyl esters, phospholipids, and ceramides (74,93) and also has antimicrobial effects (see above). BSSL is expressed in the exocrine pancreas and secreted into the intestinal lumen in all of the mammalian species. Exocrine pancreatic function, however, is not fully developed at birth, particularly not in preterm infants (73,74). In some species, though, notably the human, BSSL is expressed by the lactating mammary gland and secreted in the milk. Milk-derived BSSL, once activated by endogenous bile salts in the upper small intestine, may contribute to the efficient utilization of milk fat in breast-fed infants (94,95). Pasteurization of mother's milk inactivates BSSL, which results in reduced fat absorption and growth in preterm infants (95–97). In fact, together with pancreatic lipase–related protein 2, BSSL is a key enzyme in neonatal fat digestion (98,99). It is of note that BSSL is not expressed by the mammary gland of the cow (74); as a result, BSSL is not present in infant formulas based on cow's milk. The use of recombinant human BSSL (rhBSSL) was recently evaluated in 2 blinded placebo-controlled clinical phase II trials using a crossover design in preterm infants. One compared the effect versus placebo of adding rhBSSL to preterm formula and the other to add it to pasteurized human milk. rhBSSL treatment resulted in greater mean weight gain with significantly higher growth velocity over placebo and reduced the proportion of infants with suboptimal growth (<15 g · kg−1 · day−1). rhBSSL significantly increased absorption of DHA and ARA (100).

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Milk Fat Globule Membranes

A small, but significant fraction of proteins in human milk (1%–4%) consists of the membrane proteins surrounding the fat globules (101). This milk fat globule fraction (MFGM) is usually discarded during processing of bovine milk because mixtures of vegetable oils instead of milk fat have been used as the fat source in infant formulas. The MFGM fraction contains several proteins known to have antibacterial and antiviral activities. Some of them have been demonstrated to have broad activities against pathogens, and a bovine whey protein concentrate enriched in MFGM may therefore help to prevent diarrhea of bacterial and viral origin (102). This protein concentrate contains several bioactive components including mucin (MUC1), lactadherin, folate-binding protein, lactoferrin, sialic acid, sphingomyelin, and gangliosides (102). A bovine milk fraction containing MUC1 has been shown to inhibit hemagglutination of Vibrio cholera and E coli(103). In addition, purified mucin, a MFGM constituent, was demonstrated to decrease the adherence of Yersinia enterocolitica to intestinal membranes (104). Human milk mucin components were able to bind to various rotavirus strains and prevent replication, and the ability was correlated to lactadherin (105). Furthermore, the content of lactadherin in breast milk was shown to be negatively correlated to symptomatic rotavirus infection in Mexican infants (106). The MFGM fraction has also been found to reduce rotavirus infection in vitro (105). Sphingolipids, particularly gangliosides, have been shown to inhibit enterotoxins both in vitro and in vivo (107). Infant formula with added sphingolipids (gangliosides) has been shown to reduce E coli counts in the stool and to increase beneficial bifidobacteria (108). It is also a good source of sialic acid, both in bound and free form (109).

Most studies on individual components of the MFGM fraction have been conducted in vitro and in animal models. Interestingly, studies on the entire MFGM fraction suggest several bioactivities. Addition of bovine MFGM to complementary foods given to 6- to 12-month-old Peruvian infants showed a reduction in the prevalence of diarrhea, particularly bloody diarrhea (110). More recently, a small study on the ganglioside fraction, assumingly extracted from the MFGM fraction, suggested a positive effect on cognitive development in Indonesian infants given this fraction in infant formula from 2 to 8 weeks until 24 weeks of age (111). This appears to be an experimental product, however, because it is not commercially available, and it is thus unlikely that it will be applicable to regular infant formula. We have conducted a randomized controlled trial on Swedish infants fed regular infant formula and infant formula with added MFGM fraction from 4 to 8 weeks until 6 months of age. Evaluation by the Bayley III test at 12 months of age showed significantly better mental cognitive development in infants fed formula with MFGM fraction than those fed regular formula. Interestingly, the MFGM group did not differ from the breast-fed reference group (15). Infants in the MFGM group also had fewer acute otitis media infections and less use of antipyretics during the intervention (112). Furthermore, supplementing the infant formula with MFGM modified the fat composition of the formula and narrowed the gap between breast-fed and formula-fed infants with regard to lipid status at 12 months (113). This MFGM fraction is commercially available and can thus possibly be incorporated into regular formula with potentially positive outcomes.

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PROBIOTICS

Under normal conditions, the gut microbiota and the host thrive in symbiosis, that is, in close mutuality. The healthy fetal intestine has been considered “sterile” (or with few bacteria present), and an intense colonization process starts during delivery. This is a highly dynamic process recently suggested to begin already in utero, and it takes years to develop an adult-type gut microbiota with mostly harmless bacteria—the commensal microbiota. Over the last decades, our modern way of living has contributed to a shift in gut microbial colonization patterns and composition. Not only intestinal but also extraintestinal disorders have been proposed to be linked to aberrations in the gut microbiome (114,115). This has in turn led to intense interest in the manipulation of the gut microbiome by nonpathogenic microorganisms, such as probiotics, in the treatment and prevention of various clinical conditions.

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Breast Milk Microbiome

That human milk contains bacteria and viruses is common knowledge and a reason why donor milk and even mother's own milk is pasteurized in milk banks to avoid transmission of pathogenic bacteria and viruses, for example, cytomegalovirus. It is also known that although breast milk may contain high numbers of bacteria, it is rare that such milk causes infection in recipient infants (116). It has been suggested that breast milk contains a collection of bacteria more diverse than previously reported (117), some of which have probiotic effects and even that this is not because of contamination from the skin but because of active transport from the mother's intestine (118). Moreover, it has been shown that the human milk microbiome changes over lactation and is affected by factors such as maternal weight and mode of delivery (119,120).

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Oral Microbiome

The microbiota in the oral cavity and other parts of the gastrointestinal tract develop from sterility, or almost sterility at birth into the most heavily colonized parts of the human body. Different environmental conditions lead to distinct bacterial communities at the different anatomical niches, with >700 taxa identified in the mouth and 700 taxa identified in the colon, with minimal species overlap between the 2 sites, and significantly fewer taxa detected in the small intestine (120,121).

Breast milk provides nutrition for the infant and is a source of lactobacilli, bifidobacteria, and streptococci (122). Furthermore, breast milk components may inhibit growth and attachment of bacteria, such as the caries pathogen Streptococcus mutans(123). Therefore, breast milk likely affects establishment of the microbiota in the mouth as well as in the gut. In a recent study comparing breast-fed and formula-fed infants, we (124) indeed demonstrated that the observed differences in the gastrointestinal tract microbiota composition because of feeding mode extend to the oral cavity; lactobacilli colonized the oral cavity of breast-fed infants significantly more frequently than in formula-fed infants. The dominant Lactobacillus strain was Lactobacillus gasseri, which was detected at higher levels in breast-fed than in formula-fed infants and displayed characteristics consistent with probiotic traits in vitro in as much as viable lactobacilli detected in saliva from breast-fed, but not formula-fed infants, had an inhibitory effect on oral streptococci (125). The finding that the oral microbiota differs between breast-fed and formula-fed infants, with a potentially more health-associated oral flora in breast-fed infants, strongly suggests that prospective longitudinal studies in which health outcomes, primarily early childhood caries, but possibly also gut microbiota and associated health conditions, should be carried out.

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Gut Microbiome

Most of the studies on the microbiome of the intestine are based on analyses of the fecal microbiome, which most likely is a proxy for the colonic microbiome. The microbiota, however, varies both in number and in composition along the entire gastrointestinal tract (114,121). Although the cross talk between the bacteria and the immune system is most important in the small intestine, much less is known about the exact composition in this compartment than in the large intestine and how the composition varies with environmental factors. The gut microbiome is known to differ between breast-fed and formula-fed infants, a difference that has been further substantiated with modern molecular techniques. The gut microbiome of the breast-fed infant is likely shaped by a combination of bacteria ingested during delivery, in saliva, and in breast milk, and by an array of prebiotic factors in breast-milk, for example, oligosaccharides and lactose. The extent to which the gut microbiome of formula-fed infants is affected by prebiotic components and/or probiotic interventions is uncertain but is likely to vary depending upon the prebiotic as well as the probiotic chosen, but also to region, mode of feeding, and the early commensal microbiota (126–128).

Whether probiotics should be added to infant formula, and at what ages, is difficult to predict. Although some infant formulas today do contain probiotics of different strains, there are very few comparative studies evaluating their relative efficacy with regard to various outcomes, and no studies have evaluated the true metabolic consequences of feeding infants probiotics by using metabolomics methods.

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CONCLUSIONS

Staging of infant formulas would allow meeting infant nutrient requirements at each given age/developmental stage more closely and would also avoid providing excesses of several nutrients during periods of less need. We therefore believe this would be a major step forward in formula-feeding of infants. We have suggested a few time periods/stages during infancy when this would be most beneficial; even shorter time intervals will be possible once this concept becomes more commonly accepted. Clinical trials evaluating this staged approach of infant feeding will be needed, but we believe they are likely to result in improved outcomes. Finally, if benefits are shown and the staged approach will be introduced, it will be important that this change is accompanied by parent/caregiver/pediatrician education.

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    Keywords:

    formula-feeding; human milk; infant nutrition

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