Current Nutrition & Food Science, 2008, 4, 305-320 305

Chemistry and Biological Properties of Human Milk

Mingruo Guo1 and Gregory M. Hendricks2,*
1
Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT 05405, USA; 2Department of Cell
Biology, Medical School, University of Massachusetts, Worcester, MA 01655, USA

Abstract: Human milk contains several solid components, water, and wide range of bioactive compounds. Over the
evolutionary course of human development, a number of adaptations have resulted in the production of nonnutritive
actions associated with breast feeding. In that regard, bioactive compounds in human milk appear not only to compensate
for developmental delays in the infants’ gastrointestinal tract but also encourage the symbiotic colonization of enteric
bacteria that inhibit the growth of bacterial and viral pathogens. Research has also linked these bioactive compounds to
certain aspects of the growth and proper development of brain and peripheral nervous system during early infancy. In this
review we have summarized what has been learned about the spectrum of nutritive and biological agents in human milk,
their physical distribution in human milk over the course of lactation, whether the physical structures and functions of
these ingested agents are modified by digestive processes; the precise chemical and cellular loci of their actions, and their
short- and long-term biochemical consequences. Although much is still unknown about human milk, and how to
optimize infant nutrition substitutes, new information is constantly being discovered. Human milk is and will remain the
one standard for infant nutrition by which all infant foods should be compared. It is our species-specific food.

INTRODUCTION against pathogenic bacteria and viruses. Prebiotic activity,

The idea that human milk supplies only nutrients; i.e.,
proteins, carbohydrates, fats and minerals and vitamins, to
the infant was still the accepted principle as little three to
four decades ago. Even though there was mounting
experimental and clinical evidence to the contrary that dated
back to the late 19th century [1]. The prevalence of this idea
spawned the infant formula industry and the notion that in
the “modern age” mother’s could and should nourish their
infants the new “scientific way”. During the last 30-40 years,
that misconception has been rectified by the following
discoveries: i) a wide spectrum of bioactive agents in human
milk [2, 3]; ii) potential targets in the gastrointestinal tract for
those agents; iii) modifications of some functions of the GI
tract by breast-feeding infants; and iv) may interact not only
with the GI tract, but may also be absorbed into the systemic
circulation of the infant, intact and appear to have profound
effects upon the function and integrity of many other organ
systems [1]. Human milk is now considered a biological
fluid or a tissue.
Human milk contains a wide variety of proteins that
contribute to its unique qualities. Many of these proteins are
digested and provide a well-balanced source of amino acids
to rapidly growing infants. Some of the proteins, such as bile
salt–stimulated lipase, amylase, -casein, lactoferrin,
haptocorrin, and
1-antitrypsin assist in the digestion and
utilization of micronutrients and macronutrients from the
milk. It is well known that milk contains a variety of pro-
teins, peptides, and steroids that possess biological activity
[4]. These components of human milk are also relatively
resistant against proteolysis in the infants developing
gastrointestinal tract and may, in an intact or partially
digested form, contribute to the defense of breastfed infants
*Address correspondence to this author at the Department of Cell Biology,
Medical School, University of Massachusetts, Worcester, MA 01655, USA;
E-mail: Gregory.Hendricks@umassmed.edu
such as the promotion of the growth of beneficial bacteria
such as Lactobacilli and Bifidobacteria, may also be
provided by human milk proteins [5]. This type of activity
can limit the growth of several pathogens by decreasing
intestinal pH [6].
Human milk has been shown to have many benefits for
infants, including a reduced risk of neonatal necrotizing
enterocolitis, gastroenteritis, respiratory infection and
immunologically based disease and also improved later
cognitive development [7-9]. Non-immune protection is
provided by many components in human milk that, contrary
to immune protection against specific antigens provided by
milk immunoglobulins, protect in a nonspecific way and,
thus, provide a broad spectrum of anti-infective activity. It is
speculated that these bioactive substances play important
roles in the non-nutritional effects of human milk on the
development of the infant [6]. These clinical findings
indicate that human milk may exert protective effects on
infants during neonatal maturation.
The newborn gastrointestinal tract undergoes
maturational changes in the first weeks after birth and human
milk has been shown to stimulate gastrointestinal mucosal
proliferation and maturation in animal models and is thought
to protect the neonatal infant from harmful environmental
factors by affecting and promoting the mucosal barrier [7,
10]. Further, the growth factors in human milk, such as
epidermal growth factor (EGF), transforming growth factor
alpha (TGF) and insulin-like growth factors (IGFs),
stimulate the proliferation of intestinal cells and the
formation of the mucosal barrier [7, 11-13]. Among them,
EGF is thought to have the most significant effect on the
proliferation of cells that line the intestine and the promotion
of the covering mucosal layer [4].
In this review, human milk chemistry will be discussed
as well as bioactive components present in human milk.

1573-4013/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

306 Current Nutrition & Food Science, 2008, Vol. 4, No. 4 Guo and Hendricks

HUMAN MILK CHEMISTRY Lactoferrin tightly binds iron, presumably limiting the
availability of iron to potentially pathogenic microflora.
Gross Composition
SIgA can bind specific antigens in the infant gastrointestinal
The gross composition of human milk compared to cow’s tract, preventing infection. Lysozyme is another human milk
milk and is shown in Table 1. The protein content of human protein that plays a specific role in host protection, by lysing
milk is approximately 1.0%, with approximately 70% of the the cell walls of potential pathogens, preventing infection.
protein being provided by whey proteins. Human milk has the Caseins contribute to the amino acid pattern of human milk,
highest level of lactose (7.0%) among mammals, providing and they are also highly digestible. Functionally, their most
40% of human milk's total energy. Fat provides approxi- important property is the ability to form stable aggregates
mately 50% of the gross energy of human milk, with an that include calcium and phosphorus, which allows for
average content of 3.8%. Ash content in human milk is only greater concentrations of these minerals in human milk than
0.2% compared with 0.7% in bovine milk. Water content in is possible by mineral solubility alone. Non-protein nitrogen
human milk is similar to bovine milk at about 87%. components consist of urea, peptides, nucleotides, nucleo-
sides, and free amino acids, and remain after milk protein
Table 1. Composition of Human Milk, Bovine Milk (%)
has been precipitated with 12% trichloroacetic acid. The
casein protein exists as micelles in colloidal dispersion.
Human Milk Bovine Milk Casein micelles of human milk range from 20 to 55 nm in
size compared with those from bovine milk, at 100 to 150
Protein 1.00 3.40 nm in diameter.
CN:WP 30:70 80:20 Proteins in human milk provide an important source of
amino acids to the growing infant, and also play a very
Fat 3.80 3.50
important role in facilitating the digestion and uptake of
Lactose 7.00 5.00 many other components in human milk. Lactoferrin, -
casein, and haptocorrin may enhance the absorption of iron,
Total Solids 12.40 12.50 calcium, and vitamin B12, respectively. Other activities of
Ash 0.20 0.70
human milk proteins include immune function enhancement,
defense against pathogenic bacteria, viruses and yeasts, and
gut development and function [6].
As lactation progresses the chemical composition of Table 2. Protein Components of Human and Bovine Milk (%)
human milk changes as a result of physiological and external
factors. Some external factors may contribute negatively to
Human Milk Bovine Milk
the quality of human milk, for example, some environmental
pollutants, such as heavy metals, can be detected in human Total Caseins 0.3 g/100 g 2.6g/100g
milk, as well as many drugs. It is difficult to fully measure
the impact of maternal diet on milk composition. Maternal
S1-Casein  40
malnutrition plays a role in many developing countries

S2-Casein  8
where the food supply is limited, infections are common due
to poor hygiene, and economic situations do not allow for -Casein 85 38
the choice of and proper used of infant formulas.
-Casein 15 12
Dehydration can affect water fluxes in the body and thus,
reduce the volume of milk produced [14]. Micelle size (nm) 50 150
Proteins in Human Milk Whey Protiens 0.7g/100g 0.8g/100g
The level of total protein in milk is approximately 0.9 -
-Lactalbumin 26 17
1.2%, of which approximately 70% is whey protein and 30%
is casein along with small amount of proteins associated -Lactoglobulin - 43
with the milk fat globules. There is no
-lactoglobulin in Lactoferrin 26 trace
human milk. The primary whey proteins are
-lactalbumin,
lactoferrin, and secretory IgA (SIgA). Human milk contains Serum albumin. 10 5
large amounts of -casein and -casein, but lacks
-caseins
Lysozyme 10 Trace
completely (Table 2).
-Lactalbumin is one of the major
whey proteins and required for the biosynthesis of lactose. Immunoglobulins 16 (IgA) 10 (IgG)
Human
-lactalbumin can bind both Ca and Zn. However,
only small part of the total calcium found in human milk is Others 12 24
bound to
-lactalbumin. It is possible that
-lactalbumin
may generate peptides that facilitate the absorption of
The contents of casein and whey proteins change
divalent cations, thus exerting a positive effect on mineral
profoundly in the early stages of lactation; whey protein
absorption. Supplementation of infant formula with bovine
concentration is very high and casein is virtually undetectable

-lactalbumin may increase the absorption of iron and zinc.
during the initiation of lactation. As lactation progresses,
casein synthesis in the mammary gland and milk increases,
Chemistry and Biological Properties of Human Milk
while the concentration of whey proteins decreases, in part
due to a larger volume of milk produced. Therefore, the
ratio of whey: casein is not constant, but fluctuates between
70:30 or 80:20 in early lactation, to 50:50 in late lactation
[6]. The amino acid profile of caseins and whey proteins are
different, thus, the amino acid profile of human milk varies
during lactation.
Historically, the protein content of human milk was
overestimated due to the large proportion of non-protein
nitrogen (NPN) in human milk relative to the milk of other
species. In the milk of most species, NPN makes up a small
fraction (~5%) of the total nitrogen (TN); therefore, it is
fairly accurate to estimate the protein content by total
nitrogen analysis. Thus the true milk protein content is
estimated by multiplying the nitrogen content of the milk by
a conversion factor of 6.38, which takes into account the
fraction of NPN in dairy products. In human milk, NPN
accounts for approximately 20 - 25% of TN, thus, the use of
the 6.38 conversion factor with the total nitrogen in milk
yields an overestimate of total protein [6]. To obtain a more
accurate estimate, it is best to determine the TN content,
subtract the NPN, then multiply the remaining nitrogen by
the conventional Kjeldahl factor of 6.25[6].
The protein content in human milk ranges from 1.4-1.6
g/100 ml during early lactation, 0.8-1.0g/100ml by 3 - 4
months of lactation, and 0.7-0.8 g/100 ml after 6 months.
The levels of protein and corresponding intake may not
accurately represent the amount of utilizable amino acids
supplied to infants, as intact breast-milk proteins have been
found in the stool of the breastfed infant, indicating that they
are incompletely digested, and that available amino acids do
not represent utilized amino acids. Undigested, biologically
active proteins may have physiological benefits for the
breastfed infant, therefore, the nutritional loss of the amino
acids in these proteins may be insignificant, depending on
the quantity lost.
It is commonly understood that nutrients in human milk
are exceptionally well utilized by the breastfed infant.
Human milk proteins play many roles in the absorption of
these nutrients. Proteins bind essential nutrients, aid in
maintaining their solubility, and facilitate their uptake by
intestinal mucosa. Protease inhibitors may assist in this
process by limiting proteolytic enzyme activity, which can
preserve the physiological function of some relatively stable
binding proteins. In addition, some enzymes in human milk
affect the digestion and utilization of some micronutrients.
Human milk proteins involved in digestive function
include bile salt-stimulated lipase, amylase, and
1 -
antitrypsin [6]. Bile salt-stimulated lipase may aid in the
digestion of lipids in newborns, especially in premature
infants, who experience reduced lipase activity and poor
lipid utilization. Bile salt-stimulated lipase hydrolyzes di-
and triacylglycerols, cholesterol esters, diacylphosphatidyl-
glycerols, and micellar and water-soluble substrates. Human
milk has a significant amount of
-amylase, although there is
not a substrate for amylase in human milk. It has been
suggested that the amylase in human milk may compensate
for low salivary and pancreatic amylase activity in new-
borns, and may aid in the digestion of complex carbohy-
Current Nutrition & Food Science, 2008, Vol. 4, No. 4 307
drates when complementary foods are fed close to the
breastfeeding session.
The protease inhibitors
1-antitrypsin and antichy-
motrypsin are both present in human milk. It is thought that
they may collaborate to limit the activity of pancreatic
enzymes in breast fed infants. Proteins such as
1-antitrypsin
may escape complete digestion, and can be found in the stool
of breastfed infants. In in vitro experiments, the addition of

1-antitrypsin results in a larger amount of lactoferrin
resisting proteolytic degradation, although data on total
nitrogen balance of breastfed infants are not substantially
affected. This suggests that the protease inhibitor effect of

1-antitrypsin and antichymotrypsin may simply delay
breakdown of these proteins, rather than preventing it
completely [6].
-Casein is the major form of caseins in
human milk, and it is a highly phosphorylated protein.
Phosphopeptides formed during digestion have been shown
to keep Ca soluble, thus enhancing calcium absorption.
Clusters of phosphorylated threonine and serine residues are
located close to the N-terminal end of
-casein, and can
complex Ca ions. Thus, phosphopeptides formed from
-
casein contribute to the high bioavailability of calcium in
breast milk [6]. Casein phosphopeptides may also affect the
absorption of zinc and other divalent cations.
Lactoferrin, a major iron-binding protein capable of
binding two ferric irons, binds a major portion of the iron in
human milk. It facilitates human intestinal cell iron uptake in
cultured cells, which is most likely mediated by a specific
enterocyte lactoferrin receptor [15]. Studies investigating the
addition of bovine lactoferrin to infant formula have not
revealed an enhancing effect on either iron uptake or iron
status. Therefore, it appears that bovine lactoferrin does not
bind to a human lactoferrin receptor, or that lactoferrin only
exerts a benefit in human milk, and that when added to
infant formula, other constituents interfere with iron
utilization from lactoferrin. Heat treatment processing in
formula after lactoferrin is added may contribute to the lack
of effect observed when lactoferrin is added to human milk
[6].
Haptocorrin, previously known as vitamin B12 binding
protein, binds nearly all the vitamin B12 found in human milk.
Haptocorrin exists at a much higher level than vitamin B12 in
human milk, which results in this protein being found most
commonly in the unsaturated form. This may have
important antimicrobial benefits, as research indicates that
haptocorrin may inhibit bacterial growth. Holohaptocorrin,
the complex of vitamin B12 and haptocorrin, appears to bind
in a saturable manner to human intestinal brush border
membranes, and human intestinal cells in culture take up
haptocorrin-associated vitamin B12 [16]. This collectively
suggests a role for haptocorrin in vitamin B12 absorption
early in life. Intrinsic factor is a substance secreted by the
gastric mucosa that facilitates vitamin B12 absorption.
Although intrinsic factor is present in the stool of breastfed
infants at a young age, it may not be present in amounts
adequate to facilitate the uptake of vitamin B12 via the
intrinsic factor receptor; therefore, haptocorrin is the main
route for vitamin B12 absorption [16].
Folate-binding protein (FBP) has been found in human
milk, both in particulate and soluble forms. When found as
308 Current Nutrition & Food Science, 2008, Vol. 4, No. 4 Guo and Hendricks

the soluble form, FBP is ~22% glycosylated which may aid Table 3. Fatty Acid Profiles of Human and Bovine Milks (%,
this protein in resisting proteolytic digestion. In newborn w/w)
goats, FBP has been found to resist proteolysis and tolerate
low gastric pH, and it is possible that it behaves in a similar Human Bovine
manner in human infants. Experiments performed with rat
intestinal cells observed an increased uptake of folate when Saturated
it was complexed with FBP than when it was provided in the
Butyric (4:0) - 3.5
free form [17]. It has been theorized that FBP may slow the
release of folate in the small intestine, allowing for a slow Caproic (6:0) - 1.9
absorption of folate, which could increase tissue use. Caprylic (8:0) - 1.3

Insulin-like growth factors (IGFs) I and II are also Capric (10:0) 1.4 2.5
present in human milk, most commonly associated with IGF- Lauric (12:0) 6.2 2.8
binding proteins. These IGF-binding proteins may protect Myristic (14:0) 7.8 10.7
against IGF being digested, prolong their half-life, and
Palmitic (16:0) 22.1 27.8
control their interaction with intestinal receptors.
Stearic (18:0) 6.7 12.6
Lipids in Human Milk
Total 48.2 65.6
Lipids play a diverse role in human nutrition and
development (e.g., energy source, energy storage, vehicles Monounsaturated
for the absorption and transport of fat-soluble compounds). Palmitoleic (16:1) 3.1 2.5
Fat is the most variable component of human milk and
Oleic (18:1) 35.5 26.5
although the fat content in human breast milk is markedly
influenced by lactational stage, but fatty acid composition Gadoleic (20:1) 0.96 Trace
remains relatively stable. Normal growth and weight gain of Cetoleic (22:1) Trace Trace
infants is dependent on an adequate supply of fats in the diet. Total 39.8 30.3
Especially the essential fatty acids, a group of naturally
occurring unsaturated fatty acids with chain lengths of 18, Polyunsaturated
20, and 22 carbon atoms and containing between two and six Lineoleic (18:2) 8.9 2.9
methylene interrupting double bonds [18]. Of these, Oleic
(18:1), Palmitic (16:0), Linoleic (18:2
-6), and -linolenic Linolenic (18:3) 1.2 1.6
acid (18:3
-3) are most abundant in mature breast milk. Parinaric (18:4) - Trace
With the latter two generally recognized as dietary essential Arachidonic (20:4) 0.72 Trace
fatty acids because of the inability of tissues to introduce the
Eicosapentenoic (20:5) Trace Trace
necessary double bonds in the carbon chains before carbon 9.
Total 10.82 4.5
Human milk contains about 3 to 5% total lipid, existing
as emulsified globules, 1-4
m in diameter, covered with a
phospholipid-protein membrane derived from the mammary
cells that line ducts of the glands and are released with the The major sterol in both human and bovine milk is
milk during lactation. The main function of the cholesterol. Trace amounts of other sterols are present also,
phospholipids in milk is as emulsifying agents and stabilizers e.g., lanosterol in bovine milk and desmosterol and some
of the milk fat globule membrane. They readily bind cations phytosterols in human milk. The amount of cholesterol
like calcium, sodium and magnesium, and possibly interact present in human milk is 10 to 15
g/100ml. Since the role
with digestive enzymes. of dietary cholesterol is still not fully defined, an intake
similar to that obtained through breast feeding is generally
Bovine milk contains substantial quantities of C4:0 to C10:0 recommended.
short chain, saturated fatty acids, about 2% (w/w of fat) C18:2
(linoleic), and almost no other long-chain polyunsaturated EPA and DHA are the predominant long chain
fatty acids (Table 3). The fatty acid composition is not polyunsaturated fatty acids in human milk, and are known to
altered by ordinary changes in diet. In contrast, human milk be essential to normal development of infants. These fatty
contains very little short chain fatty acids (C4:0 to C10:0), 10 to acids may also be formed from precursors, even in preterm
14% (w/w of fat) linoleic (18:2
-6), and small quantities of infants.
other polyunsaturated fatty acids. The triacylglycerol The studies indicated that DHA supplementation
structure differs as well, with much of the sn-2 position in increased DHA levels and there are correlations between
human milk lipids occupied by C16:0 (palmitic). Human milk DHA levels in maternal plasma and human milk, and
also contains the long chain polyunsaturated fatty acids between milk and infant plasma phospholipids. Until
docosahexanoic (DHA) (22:6
-3) and eicosapentaenoic recently, infant formulas did not contain any significant
(EPA) (20:5
-3) which have been shown to be important in levels of DHA, even though it is present in human milk.
the development of retinal and brain tissue. DHA can be synthesized from linoleic acid, but high intakes
of linoleic acid can also inhibit this process. Thus, a
Chemistry and Biological Properties of Human Milk
preformed source of DHA in the infant diet may be more
efficient in assuring the supply of an adequate amount [19].
Carbohydrates in Human Milk
Carbohydrate in human milk is comprised of mono-
saccharides, such as glucose and galactose; disaccharides,
such as lactose and lactulose; oligosaccharides; and some
more complex carbohydrates, such as glycoproteins. Lactose
is the primary carbohydrate the most likely to contribute to
malabsorption and intolerance syndromes resulting from
metabolic disturbances, such as lactose intolerance, lactose
malabsorption, and galactosemia. An exclusively breastfed
baby receives approximately 10 - 14 g of lactose per day/kg
body weight.
Monosaccharides in milk are primarily made up of
glucose and galactose, and are found at levels of
approximately 100 mg/100 ml in human milk. Lactose is the
nutrient least likely to be affected by maternal nutrition,
including malnutrition or energy supplementation. The
concentration of lactose in human milk is relatively stable at
about 7%. Total oligosaccharide levels comprise of up to
10% of total carbohydrates. Lactose together with the
mineral constituents, are crucial to maintaining a constant
osmotic pressure in milk.
Lactose in human milk has been reported to exert a
beneficial effect on the absorption of minerals, most notably
calcium, which is most likely due to its conversion to lactic
acid by intestinal flora, which lowers the pH, causing
increased solubility of calcium salts. This is also possible
because human milk has a low buffering capacity and a low
content of protein and phosphorus. Neither lactose nor
lactulose are hydrolyzed in the upper GI tract, and only to a
very small extent in the proximal intestinal tract. Lactulose,
a disaccharide of galactose and fructose, is a growth
promoting factor and energy source to Lactobacillus bifidus
and Lactobacillus acidophilus.
Oligosaccharides in human milk, ranged from tri- to
octasaccharides at levels of 0.8-1.4%. At least 21 different
types of oligosaccharides have been identified in human
milk, composed of many different molecules, including
simple sugars and sugar derivatives such as uronic acid, and
these can be acidic, neutral, linear, or branched. Oligo-
saccharides in human milk have been divided into nitrogen-
free oligosaccharides, or oligosaccharides containing either
N-acetylglucosamine or N-acetylneuraminic acid (sialic
acid). Small oligosaccharides are common in human milk,
and there also exists a high content of complex and
fucosylated and sialylated oligosaccharides. More than 130
components have been characterized in human milk. Some
components are thought to be involved with the immune
system, while others may be involved with the development
of a specific intestinal microflora. The oligosaccharide
component of human milk is thought to be the main energy
source for the intestinal flora of the breast-fed infant, which
is rich in bifidobacteria and lactobacilli. Lactobacilli ferment
lactose to lactic acid which, along with a low pH, promotes
the growth of Lactobacillus bifidus, as well as the bifidus
factors lactulose, oligosaccharides, glycoproteins, and
glycopeptides. The bifidus factor is most likely found in the
nitrogen-containing oligosaccharides. Oligosaccharides
Current Nutrition & Food Science, 2008, Vol. 4, No. 4 309
added to cow's milk based infant formula include galacto-
oligosaccharides and inulin, and have been shown to
stimulate the growth of bifidi and lactobacilli.
Vitamins in Human Milk
All of the water-soluble and fat-soluble vitamins are
found in human milk. Human milk contains more vitamin A,
E, C, nicotinic acid, and inositol than bovine milk, however,
it has a lower content of vitamins B1, B2, B6, B12, K, biotin,
pantothenic acid, and choline (Table 4). Human milk appears
to contain adequate amounts of most vitamins to support
infant normal growth, with the exception of vitamin D and
possibly, vitamin K. Exclusively breastfed infants of
mothers on a total vegetarian diet may require vitamin B12
supplementation to prevent a deficiency, which results in
severe and permanent neurologic damage.
Fat-Soluble Vitamins
Vitamin A (trans-retinol) is comprised of a family of
compounds having differing levels of Vitamin A activity.
Compounds with vitamin A activity present in human milk
include retinyl esters, retinol, and
-carotene. Vitamin A is
required for a large number of life processes and a deficiency
has been associated with clinical disorders unique to infants.
When maternal nutritional status is good, human milk
supplies adequate amounts of vitamin A. Although vitamin
A content of milk decreases as lactation progresses, milk
ingestion volumes increase, therefore the infant receives
adequate amounts of vitamin A. Poor maternal nutritional
status results in milk with a low vitamin A content, which
can place the infant at risk for deficiency. Mechanisms
regulating storage, mobilization, and secretion of retinoids
from mammary cells have yet to be determined; although,
there is indication that the concentration of retinal binding
protein in serum determines the amount of retinol delivered
to milk. Research indicates that vitamin A supplementation
just preceding or following parturition can significantly
increase vitamin A levels in human milk, especially in
situations of low intake levels.
Vitamin D plays an essential role in bone metabolism and
may also be implicated in immune system regulation. The
serum concentration of 25-OH-D (25-OH cholecalciferol),
the active metabolite of vitamin D, is generally used to
measure vitamin D status. Dietary ergocalciferol (D2) and
cholecalciferol (D3) are converted to the active metabolite,
25-OH-D, in the body. Infants can synthesize vitamin D
endogenously in the epidermis upon exposure to sunlight, or
they can receive it through dietary intake. Seasonal
variations in vitamin D synthesis in infants have been
observed, and light-skinned infants are more likely to benefit
from sunlight exposure than dark-skinned infants. The level
of 25-OH-D in human milk is low, corresponding with both
maternal serum 25-OH-D levels and maternal dietary
vitamin D intake, and can also be affected by race, season,
and latitude. Infants who are exclusively breastfed receive
below the minimum recommended intake of vitamin D, and
much lower than the recommended dietary intake, and as
such, are at risk for deficiency, rickets, and improper bone
mineralization, especially if sunlight exposure is poor.
Normal vitamin D stores present at birth are depleted
within 8 weeks, and formula-fed infants have higher serum
310 Current Nutrition & Food Science, 2008, Vol. 4, No. 4 Guo and Hendricks

concentrations of vitamin D metabolites than breast fed
infants.
Maternal supplementation with 400 - 2000 IU of
vitamin D daily increases the vitamin D content of human
milk, however, only the 2000 IU dose achieves satisfactory
levels of 25-OH-D in the infant. Adequate sunlight exposure
levels have not been clearly established, and due to the low
level of vitamin D in human milk, vitamin D supple-
mentation is recommended for breast-fed infants in Europe
and the northern United States.
Vitamin E is comprised of a group of compounds with
different degrees of biological activity, with the most active
compound being 
-tocopherol. Vitamin E is an antioxidant,
acting as a free radical scavenger and protecting against
PUFA peroxidation in cell membranes. The transport of
vitamin E across the placenta is limited, thus, neonatal
tissues have low levels of vitamin E. Hemolytic anemia can
result in neonates with a vitamin E deficiency. The vitamin
E content of human milk is adequate for a term infant, but
may not be sufficient for a preterm infants, that have even
lower levels of vitamin E at birth than the term infants.
Decreased vitamin E levels in preterm infants maybe related
to PUFA, iron, and selenium concentrations, and hemolytic
anemia is observed more frequently in preterm infants than
term infants, presumably due to lower vitamin E levels in
infants fed formula supplemented with PUFAs and iron.
Therefore, preterm infants who are not breastfed should
receive formula enriched with vitamin E as well as LC-
PUFA, although these fatty acids may provoke early
postnatal decreases in both serum vitamin E and total lipids
ratio.
Table 4. Vitamin Contents in Human and Bovine Milk
(mg/L)
Vitamin Human Milk Bovine Milk
Vitamin A 0.53 0.37
Carotene 0.24 0.21
Cholecalciferol (D) 0.001 0.0008
Tocopherol (E) 5.4 1.1
Vitamin K 0.015 0.03
Thiamin (B1) 0.15 0.42
Riboflavin (B2) 0.37 1.72
The vitamin E content of human milk is dependent on
many factors, including individual variation, stage of
lactation, and amounts of maternal dietary vitamin E.
However, maternal supplementation with vitamin E has not
been shown to affect the vitamin E content of human milk in
mothers with moderate vitamin E intake. In addition, in
populations with low vitamin E status, adequate vitamin E
content in human milk has been observed, suggesting that
maternal stores of vitamin E can be mobilized during
lactation to ensure adequate supply in human milk.
Vitamin K activity is provided by several different
naturally occurring compounds, including vitamin K1, which
is found in the diet as phylloquinone, and vitamin K2,
menaquinones, which are synthesized by bacteria in the
gastrointestinal tract. Vitamin K is essential to the proteins
involved in blood coagulation, and some plasma proteins and
organs have been shown to be dependent on vitamin K,
including proteins that are involved in the maintenance of
bone structure. The transfer of vitamin K across the placenta
is very limited, thus, newborn infants generally exhibit
extremely low concentrations of vitamin K. However,
vitamin K levels remain constant in human milk over 6
months of lactation. Vitamin K is localized in the lipid core of
the milk fat globule, and not the membrane. Even in
situations where maternal vitamin K consumption exceeds
the recommendations, exclusively breastfed infants do not
receive the recommended dietary intake, and their plasma
concentrations are low compared with formula-fed infants.
In addition, breast-fed infants more frequently report the
development of hemorrhagic disease. Due to the low content
of vitamin K in human milk, and the low concentration of
vitamin K in neonates, vitamin K supplementation is recom-
mended after birth. Studies investigating the relationship
between maternal vitamin K intake and content in human
milk have mixed results, as some indicate no correlation,
while another observed that maternal supplementation with
vitamin K appeared to increase maternal plasma and breast
milk concentrations, unless the supplemental dose of
vitamin K was low. Preterm infants may require vitamin K
supplementation as they tend to develop deficiencies more
easily than term infants.
Water-Soluble Vitamins
Water-soluble vitamins are not effectively stored;
therefore, it might be expected that maternal dietary intake
would affect the contents of water-soluble vitamins in
human milk more readily than fat-soluble vitamins.

Pyridoxine (B6) 0.10 0.48

Thiamin content in human milk is on average 0.15 mg/L.
Six weeks of supplementation with thiamin from 1.3 to 3.4
Cobalamin (B12) 0.0003 0.0045 mg/day did not increase milk thiamin levels in women who
were adequately nourished. Urinary excretion of thiamin
Niacin 1.7 0.92
was higher in women who were taking a thiamin
Folic acid 0.043 0.053 supplement, suggesting that a limit exists in the transfer of
this vitamin into human milk. Early studies indicated that a
Ascorbic acid (C) 47 18 maternal thiamin deficiency could lead to low levels of
Biotin 0.007 0.036 thiamin in milk.

Pantothenic acid 2.1 3.6

Low maternal intake of riboflavin can produce low
concentrations of riboflavin in breast milk. Supplementation
Inositol 300 160 with a modest amount of riboflavin (2 mg/day) increased
milk riboflavin levels. A maternal intake of 2.5 mg/day was
Chemistry and Biological Properties of Human Milk
considered sufficient to maintain riboflavin status during
lactation.
The concentration of biotin in human milk from women
in the U.S. was reported to be between 5-12 mg per liter.
Supplementation with higher levels of other B vitamins does
not appear to affect biotin levels in milk. Supplementation
with biotin increases biotin level in milk when its level is
initially low, and has no effect when biotin levels are in the
normal range.
Vitamin B6 concentration in milk of mothers with vitamin
B6 intakes around the RDA (2.5 mg/day) appears to be
approximately 210 μg/L. Vitamin B6 level in the milk of
women in the U.S. with low socioeconomic status and low
vitamin B6 intake was 120 μg/L. Supplementation of vitamin
B6 at levels above the RDA (5.3 mg/day) did not alter the
vitamin B6 level in milk. However, it is important to note
that supplementation of vitamin B6 at high levels to lactating
women should be avoided as this can suppress lactation.
Folate concentration in human milk increases with
lactation time, ranging from 15 - 20 ug/L in early lactation, to
40 - 70 ug/L in mature milk. Supplementation of 0.8 mg/L of
folate to well-nourished women in the U.S. did not change
milk folate concentration. However, when women of lower
socioeconomic status and concomitant low folate intake
(60% of the RDA) were supplemented with folate, the folate
level in their milk was increased.
Low intakes of vitamin B12, cyanocobalamin, are most
likely reflected in lower milk concentration of this vitamin.
Mean vitamin B12 levels in well nourished women in the U.S.
range between 0.97 - 1.10 ng/mL, and women of low
socioeconomic status averaged 0.55 μg/mL. Maternal
supplementation for 40 days with additional vitamin B12
raised the milk levels to 0.79 ng/mL, suggesting that long-
term impaired maternal vitamin B12 status may not be
completely alleviated in a relatively short time period.
Supplementation of well-nourished women with additional
vitamin B12 does not appear to augment milk concentration.
Vitamin B12 levels can be low in the milk of some women,
especially when the mother follows a vegetarian diet, and
can cause a vitamin B12 deficiency in the infant. Vitamin B12
in human milk is found as a protein-bound vitamin.
Women in the US typically have Vitamin C levels in the
range of 50 mg/L. In well-nourished women, neither short-
term nor long-term (6 months) supplementation of high levels
(800 mg/day) of vitamin C affected the concentration of this
vitamin in their milk. Therefore, there appears to be an
upper limit on the transfer of vitamin C into human milk,
past which additional supplementation will not further
augment levels in human milk.
The level of niacin in Human milk is 1.96 mg/L with a
daily intake between 15 - 23 mg, and this level could be
increased to 3.9 mg/L with 120 mg niacin/day for 6-14 days.
Like niacin, pantothenic acid level in human milk
appears to be influenced by maternal daily dietary intake.
The level of pantothenic acid in human milk is strongly
correlated with the maternal intake of pantothenic acid over
the previous 24 hours.
Current Nutrition & Food Science, 2008, Vol. 4, No. 4 311
Minerals in Human Milk
Minerals exist in the body in several chemical forms,
including inorganic ions and salts, or as constituents of other
organic molecules, including proteins, fats, and nucleic
acids. They contribute to a variety of physiological
functions, including structural components of body tissues to
essential parts of many enzymes and biologically important
molecules. Sodium, potassium, chloride, calcium,
magnesium, phosphorus, and sulfate make up the macro
minerals found in human milk. Citrate is not a mineral, but
it is found as a water-soluble portion of human milk that
can bind some minerals. The primary determinant of macro
mineral concentration in human milk is the duration of
lactation, during which sodium and chloride decrease, and
potassium, calcium, magnesium, and free phosphate
increases over time. However, the mineral content of human
milk is also influenced by nutritional status of the mother,
environmental and other factors. The concentrations of
macro- and microelements in human milk and bovine milk
are compared in Table 5.
Microelements
Sodium is the main cation of extracellular fluid, and it is
also the main controller of extracellular volume. It is
involved in the regulation of osmolarity, acid-base balance,
active transport across cells, and the membrane potential
across cells. Potassium is the primary intracellular cation,
with a concentration 30 times greater inside the cell than in
the extracellular fluid. Potassium in the extracellular fluid is
involved in the transmission of nerve impulses, maintenance
of blood pressure, and control of skeletal muscle contraction.
Chloride is also essential in the maintenance of fluid and
electrolyte balance, as it is the principal extracellular anion
[20, 21]. Under normal circumstances, a dietary deficiency
of sodium, potassium, or chloride does not occur. However,
depletion of sodium and chloride can occur during extreme
conditions, such as chronic diarrhea, heavy perspiration, or
renal disease, and depletion of potassium can occur in
situations where there are large alimentary or renal losses.
The concentrations of sodium, potassium, and chloride in
breast milk decrease with duration of lactation, from a
reported 480, 740, and 850 mg/L in colostrum, respectively,
to 160, 530, and 400 mg/L respectively. No relationship has
been identified between maternal intake and the
concentration of these electrolytes in human milk. Sodium,
potassium, and chloride in human milk are present almost
entirely as free ions.
Calcium comprises about 1.5 - 2% of body weight in an
adult, which has accrued approximately 1200 g of calcium.
About 99% of this calcium is found in teeth and bones,
providing structure and strength as calcium phosphate. The
remaining calcium is found in extracellular fluids,
intracellular structures, and cell membranes, and is involved
in several regulatory functions, such as maintenance of a
normal heart beat, hormone secretion, blood coagulation,
nerve conduction, muscle contraction, activation of
enzymes, and integrity of membranes. Human milk supplies
approximately 200 mg of calcium in an average daily milk
secretion of 750 ml, which appears to be sufficient for the
312 Current Nutrition & Food Science, 2008, Vol. 4, No. 4 Guo and Hendricks

Table 5. Mineral Composition of Mature Human and Bovine of approximately 30-35 mg/L. With a normal range of
Milk dietary magnesium intake, there is no relationship between
maternal magnesium consumption and concentration of
Mineral Mature Human Milk Bovine Milk magnesium in human milk. It has been reported to be about
30% higher in colostrum than in mature milk. Some
Sodium (mg/L) 207±94 580 magnesium associates with phosphate and caseins in human
milk.
Potassium (mg/L) 543±78 1400
Phosphorus is a nutrient essential to humans, as it
Chloride (mg/L) 453±53 1040 serves a number of important biological functions. It exists
Calcium (mg/L) 259±59 1180 as organic and inorganic phosphates in all tissues and fluids,
and is essential to many body components, including lipids,
Magnesium (mg/L) 31.4±5.9 120 proteins, carbohydrates, and nucleic acids, and also plays an
important role in metabolism. It is an important part of
Phosphorus (mg/L) 142±25 930
calcium phosphate, a major structural component of teeth
Iron (μg/mL) 0.4-0.76 0.2-0.6 and bones. Dietary deficiencies of phosphorus do not usually
occur as phosphorus is contained in a wide variety of foods
Zinc(μg/mL) 1-3 4
of plant and animal origin. In human milk, phosphorus
Copper (μg/mL) 0.2-0.4 0.05-0.2 content increases from 100 mg/L on day 1 to 170 mg/L by
day 8, and decreases to 130 mg/L by day 36 of lactation.
Manganese (ng/mL) 3-6 21
Trace Elements
Iodine (ng/mL) * 12-178 70-219
Trace elements, also known as micro minerals, are
Fluoride (ng/mL) 4-15 19 substances that make up less than 0.01% of the body mass.
Selenium (ng/ml) ** 15-20 10 In human milk these include iron, zinc, copper, manganese,
selenium, iodine, fluorine, molybdenum, aluminum, cobalt,
Aluminum (ng/mL) 4-14 27 chromium, and nickel.
Chromium (ng/mL) 0.2-0.4 5-15 Iron is an essential component of heme in hemoglobin,
myoglobin, cytochromes, and other proteins; therefore, it
Molybdenum (ng/mL) 1-2 22
plays a role in the transport, storage, and utilization of
(Adapted from Picciano 2000) oxygen. Iron deficiency anemia affects about 30% of the
* Selenium content in Bovine and Human milk varies; in selenium deficient areas, world's population, including Western and underdeveloped
values for bovine and human milks are 2-7 ng/mL and 3-8 ng/Ml. In China, where countries. The mean iron concentration in human milk is 0.3
wide spread toxicity is prevalent, values for human milk have been reported as high mg/L. The iron content of human milk decreases over the
as 283 ng/mL. duration of lactation; colostrum iron level is about 1mg/L,
** The concentration of iodine in both bovine and human milk is dependent on intake. and decreases to 0.3 - 0.6 mg/L in mature milk. Dietary
In the U.S. where iodinated salt is common the levels tend to range on the higher side. intake of iron has no relationship with iron concentration in
human milk, and supplementation with iron at levels up to
term infant, this may not be adequate for a preterm infant. 30 mg/day does not affect milk iron concentration. Human
Supplementation of 1000 mg calcium/day in lactating milk iron is bound to three main components: lactoferrin, a
women does not affect milk calcium or lactation-associated low molecular weight compound, and a component of the
bone mineral changes. The calcium content of human milk milk fat globule membrane. Lactoferrin is the primary iron-
increases in early lactation, from 250 at day 1, to 320 mg/L binding protein in human milk, possessing a high affinity
by day 5, and remains constant at approximately 300 mg/L for the ferric ions, which bind two sites together with
up to day 36 of lactation. Studies on calcium concentration bicarbonate or carbonate ions. Lactoferrin concentration in
during lactation reveal an approximate 30% decrease human milk is much higher than iron concentration, so
between the first and ninth months of lactation. There is no although one-third of iron is bound to lactoferrin, only 3-
correlation between maternal dietary consumption of 5% of lactoferrin is saturated with iron. However, iron
calcium and its concentration in human milk. Calcium binds released from other components during digestion may
with phosphate and casein in human milk to produce become bound to lactoferrin, especially when bicarbonate
calcium phosphate linkages in casein micelle subunits, and from pancreatic fluid is present. Citrate in the low
it can also bind to citrate, or be found in the ionized form. molecular weight fraction and xanthine oxidase in the fat
The calcium in human milk is more available for absorption. globule membrane may be among these other iron-binding
components. Very little iron in human milk is bound to
Magnesium plays an essential role in a variety of casein (Lönnerdal 1989 [22]).
physiological processes, including neuromuscular trans-
mission, muscle contraction, protein and nucleic acid Zinc is essential to proper growth and development,
metabolism, and as a cofactor for many enzymes. Mag- sexual maturation, wound healing, and it may play a role in
nesium, along with calcium and phosphate, supports skeletal immune system function and other physiological processes.
growth. A deficiency of magnesium is not common except in Zinc assists several hormones involved in reproduction, it is
conditions of severe malnutrition and certain disease states. required for DNA, RNA, and protein synthesis, and is a
Mature human milk contains magnesium at a concentration cofactor for many enzymes involved in most major
Chemistry and Biological Properties of Human Milk
metabolic processes (Flynn 1992, Picciano 2000, Guo and
Hendricks 2007 [5, 20, 21]). Human zinc deficiency was first
reported in the 1960s in the Middle East, resulting in
dwarfism, impaired sexual development, and anemia. It is
difficult to detect mild deficiencies of zinc, although they
have been shown to occur in Western countries, especially in
infants and children, and give rise to suboptimal growth,
poor appetite, impaired taste acuity, and low hair zinc levels.
Mean zinc concentration in mature human milk during the
first six months of lactation is about 2 mg/L, although large
variations in zinc have been reported at 0.65 - 5.3 mg/L.
Dietary zinc intake has no correlation to zinc content of
human milk, and zinc supplementation of a zinc adequate
diet does not significantly affect human milk zinc
concentration. Zinc in human milk is found in three major
components: serum albumin and citrate in the whey, and in
alkaline phosphatase in the fat globule membrane.
Copper is required for iron utilization and is a cofactor for
enzymes involved in glucose metabolism, as well as the
synthesis of hemoglobin, phospholipids, and connective
tissue. Copper deficiency is rare except in conditions of
severe malnutrition. Mature human milk contains copper at
a concentration of 0.3 mg/L. Copper concentration decreases
with advancing lactation, from 0.6 mg/L in weeks one and
two of lactation, to 0.36 mg/L by 6 - 8 weeks, and 0.21 - 0.25
mg/L by 20 weeks of lactation. No significant correlation
exists between milk copper concentrations and dietary
copper intake. Copper in human milk is bound to serum
albumin and citrate. Copper has also been found in the fat
globule membrane, however, the ligand has not yet been
identified.
Manganese is a cofactor for glycosyl transferases, which
play a role in mucopolysaccharide synthesis, and is a non-
specific cofactor for many other enzymes. Two manganese
metalloenzymes have been identified: mitochondrial super-
oxide dismutase and pyruvate carboxylase [23] (Hurley &
Keen 1987). As manganese is widely distributed in foods, a
dietary deficiency is not known to occur in humans (Flynn
1992, Picciano 2000, Guo and Hendricks 2007 [5, 20, 21]).
In mature human milk, the mean concentration of
manganese is approximately 10 μg/L and manganese is
known to decrease with duration of lactation. No cases of
manganese deficiency in human infants have been reported,
thus, fully breastfed infants appear to receive adequate
manganese [24] (Lönnerdal et al. 1983). Manganese in
human milk is mainly bound to lactoferrin, however, it exists
at such a low concentration that approximately 2000 times
more iron is bound to lactoferrin than manganese. There-
fore, very little of the metal-binding capacity of lactoferrin is
occupied by manganese (Lönnerdal 1989 [22]).
Selenium is an important component of the enzyme
glutathione peroxidase. Glutathione peroxidase is present in
many tissues, where it works with vitamin E, catalase, and
superoxide dismutase as an antioxidant, protecting cells
against oxidative damage. Selenium concentration in the
human milk is approximately 16 μg/L. Selenium concen-
tration is higher in colostrum, at 41 μg/L. A correlation was
observed between human milk selenium content and both
maternal plasma selenium concentration and plasma
glutathione peroxidase activity, suggesting that milk
Current Nutrition & Food Science, 2008, Vol. 4, No. 4 313
selenium content is influenced by maternal selenium status
[25]. The average selenium content of milk of North
American women is considered more than sufficient for
breastfed infants.
Iodine is essential to the thyroid hormones, thyroxine and
triiodothyronine, which play an important role in the
regulation of basal energy metabolism and reproduction.
Iodine deficiency causes the thyroid gland to enlarge and
form a goiter, while excess iodine in the diet reduces uptake
of iodine by the thyroid gland, mimicking signs of thyroid
deficiency. In the United States, mean iodine concentrations
in human milk have been reported as 142 μg/L (range: 21 - 281
μg/L). A correlation between milk iodine concentration and
dietary iodine intake has been observed, therefore, the use of
iodized salt can augment milk iodine content [26] (AAP
1981). North American women have an elevated iodine
intake, and thus, the amounts of iodine in their milk are
adequate.
Molybdenum is a crucial component of several enzymes,
including aldehyde oxidase, xanthine oxidase, and sulfite
oxidase, where it exists in the prosthetic group molyb-
dopterin. It has yet to be determined whether the human
requirement is specifically for molybdenum, or whether it is
for molybdopterin or a precursor. Dietary deficiency has not
been observed in humans, except for a patient on long-term
total parenteral nutrition. Molybdenum content of human
milk is strongly correlated with stage of lactation,
decreasing from 15 μg/L on day 1, to 4.5 μg/L by day 14, and
finally to a concentration of approximately 2 μg/L by one
month and thereafter.
Chromium is considered essential to human health, and
the earliest sign of a deficiency is impaired glucose tole-
rance. Chromium deficiency has been observed exclusively
in patients receiving long-term total parenteral nutrition,
who respond to intravenous trivalent chromium with
amelioration of glucose intolerance. The mean chromium
content of mature human milk is 0.27 μg/L.
The only function of cobalt identified in humans is its
presence as an essential part of vitamin B12. Vitamin B12 is
synthesized from bacteria. Therefore, inorganic cobalt is
essential for all animals that rely completely on their
bacterial flora for vitamin B12 supply. Mature human milk
contains cobalt at a concentration of approximately 0.1 μg/L.
Dietary supplementation of cobalt increases the vitamin B12
level of human milk only when the maternal diet is cobalt
deficient.
Fluoride is considered a beneficial element, rather than
an essential element to human health, as it protects against
dental caries and accumulates in bones and teeth. However,
excessive fluoride intake leads to fluorosis, which causes
mottling of the teeth, and also affects bone health and
kidney function. In mature human milk, the mean fluoride
content is about 16 μg/L. Infants who are breast fed or
consuming concentrated or powdered formula prepared
with nonfluorinated water have a low fluoride intake, and
should receive fluoride supplements [27].
Substantial evidence exists to establish the necessity of
nickel, silicon, arsenic, aluminum and boron in animals, and
it is most likely that these trace elements are also essential to
314 Current Nutrition & Food Science, 2008, Vol. 4, No. 4
humans. However, the nutritional functions of these
elements are yet to be determined [27]. Nickel is found in
mature human milk at a level of 1.2 μg/L, silicon is found at 700
μg/L, arsenic is found at 0.2 - 0.6 μg/L [28].
BIOACTIVE COMPONENTS IN HUMAN MILK
Bioactive Proteins and Peptides
The protein content of human milk is approximately
1.0%, with approximately 70% of the protein being provided
by a variety of growth factors and whey proteins. Many of
these proteins are known to have beneficial effects on the
growth and development of the micro-flora found in the
intestine. Bifidus factor, one of the oldest known disease-
resistance factors in human milk, promotes the growth of a
beneficial organism bifidobacteria, a popular addition to
many yogurt products and probiotic supplements. B12
binding protein, as its name suggests, binds vitamin B12 in
the intestinal tract and thus deprives harmful microorganisms
of vitamin B12. Even -casein, a 162-amino-acid, highly
glycosylated human milk protein has been shown to inhibit
adherence of H. pylori to human gastric mucosa and of
Streptococcus pneumoniae and Haemophilus influenzae to
human respiratory-tract epithelial cells. The C-terminus
proteolysis product of -casein is a strong growth-promoting
factor for Bifidobacterium bifidium, an acid-producing
anaerobe that reduces the growth of intestinal pathogenic
microorganisms in breastfed infants.
The primary whey proteins are
-lactalbumin,
lactoferrin, and secretory IgA (SIgA).
-Lactalbumin is one
of the major whey proteins in human milk, accounting for
more than 25% of the whey protein in human milk and is
required for the biosynthesis of lactose. Human
-
lactalbumin, the other dominant whey protein can bind both
Ca and Zn. However, only a small part of the total calcium
found in human milk is bound to
-lactalbumin. It is possible
that
-lactalbumin may generate peptides that facilitate the
absorption of divalent cations, thus exerting a positive effect
on mineral absorption. Lactoferrin tightly binds iron and
facilitates it uptake, limiting the availability of iron to
potentially pathogenic microflora. Each molecule of
lactoferrin can bind to two atoms of iron. Because many
pathogenic bacteria thrive on iron, lactoferrin halts their
spread by making iron unavailable [5]. It is especially
effective at stalling the proliferation of organisms that often
cause serious illness in infants, including Staphylococcus
aureus. Lactoferrin also disrupts the process by which
bacteria digest carbohydrates, further limiting their growth.
Lysozyme, an anti-infective agent, is a 130-amino-acid-
containing glycoprotein that hydrolyzes the 1-4 linkage
between N-acetyl glucosamine and N-acetylmuramic acid in
bacterial walls; gram-negative species appear to be
particularly susceptible [29]. Lysozyme, like lactoferrin, is
present in other exocrine secretions and lyses mostly gram-
positive and few gram-negative bacteria. Contrary to the
other protective proteins in human milk (i.e., antibodies and
lactoferrin), the concentration of which decreases during
lactation, lysozyme steadily increase with prolonged
lactation. Concentrations in milk are higher than in serum,
and lysozyme concentrations are several-fold higher in
Guo and Hendricks
human than in bovine milk [30]. Bioactive functions or non-
nutitive functions of proteins are listed in Table 6.
Table 6. Non-Nutritive Functions of Proteins in Human Milk
Protein Component Proactive (non-nutritive) Function
-Casein Ion carrier, inhibits microbial adhesion to
mucosal membranes

-Lactalbumin Ion carrier (Ca2+ ), part of lactose synthase
Lactoferrin Anti-infective, iron carrier
Lysozyme Anti-infective
Bile salt dependent lipase Production of FFA with antiprotozoan and
antibacterial activity
Glutathione peroxidase Anti-inflammatory (prevents lipid
oxidation)
PAF: acetylhydrolase Protects against necrotizing enterocolitis
(hydrolysis of PAF)
Cytokines Modulate functions and maturation of the
immune system
SIgA Immune protection
IgM Immune protection
IgG Immune protection
IgD Immune protection
IgE Immune protection
Antibodies, also called immunoglobulins take five basic
forms, denoted as IgG, IgA, IgM, IgD and IgE. All have
been found in human milk, but by far the most abundant type
is IgA, specifically in the form known as secretory IgA
(SIgA). In human milk, these antibodies consist of two
joined IgA molecules and a so-called secretory component
that seems to shield the antibody molecules from being
degraded by the gastric acid and digestive enzymes in the
stomach and intestines [31]. The secretory IgA molecules are
passed to the suckling child during nursing. These antibodies
are helpful in ways that go far beyond their ability to bind
and kill microorganisms. This collection of antibodies
transmitted to the infant is highly targeted against pathogens
in the infant’s immediate surroundings [32]. The mother
synthesizes these antibodies when she inhales or ingests a
disease-causing organism. The antibodies she makes are
specific to the organisms or infectious agents she encounters,
i.e., it binds to a single protein, or antigen on the infectious
agent. Because the mother makes antibodies to pathogens in
her environment, her infant receives the protection it most
needs against the infectious agents it is most likely to
encounter. Thus, breast feeding provides targeted immunity
[33].
Because these antibodies are passed to the infant in a
joined and shielded form they are inactive until absorbed and
ignore useful bacteria normally found in the gut [34]. This
helps the infant build up a bacterial flora of "good bacteria"
Chemistry and Biological Properties of Human Milk
that serves to crowd out the growth of pathogenic organisms,
thus providing another measure of resistance [35].
Secretory IgA further keep an infant from harm in that,
unlike most other antibodies, they ward off disease without
causing inflammation; a process in which various chemicals
destroy microbes but potentially hurt healthy tissue [36]. The
infant's developing digestive system is extremely delicate
and an excess of these chemicals can do considerable
damage to the developing mucosal membranes. Interestingly,
it seems SIgA can probably protect mucosal surfaces other
than those in the gut. In many developing countries,
particularly in the Middle East, South America and northern
Africa, women put drops of their milk in their infants' eyes to
treat infections. It has never been scientifically tested, but
there are theoretical reasons to believe it could be beneficial.
It probably does work at least some of the time, or the
practice would have died out [35].
Bioactive Lipids Components
Human milk contain about 3 to 5% total lipid, existing as
emulsified milk fat globules 2 to 4 micrometers in diameter
with a protein membrane derived from the secretory cells of
the mother’s mammary tissue. The lipids in human milk can
be divided into four types: triacylglycerol (triglycerides),
fatty acids, sterols and phospholipids. About 98% or more of
the lipid in human milk is in the form of triacylglycerol.
Fresh human milk contains only trace amounts of free fatty
acids (FFA), diglycerides (DG), and monoglycerides (MG).
Free fatty acids in the milk are the end product of lipolysis;
the breakdown of triacylglycerol to free fatty acids and
glycerol. Phospholipids account for about 0.5 to 1 % of total
lipids while sterols; including cholesterol and various esters,
make up about 0.2 to 0.5% of the milk fat; the later are
mostly found in the milk fat globule membrane along with
trace amounts of carotenoids, retinyl esters and squalene
[18].
Lipids are the most variable components of human milk
and although the lipid content of human milk is markedly
influenced by lactational age, the overall lipid composition
remains relatively constant. These lipids play a diverse role
in human nutrition and development. They constitute a major
energy source, provide an almost unlimited form of energy
storage, and they act as vehicles for absorption and transport
of lipophilic compounds such as fat soluble vitamins (i.e.,
vitamins A, D, E and K). Phospholipids act as functional as
well as structural components of cell membranes. Human
milk contains primarily fatty acids containing 10 to 20
carbon atoms; oleic (C18:1), palmitic (C16:0), linoleic (C18:2, -
6), and -linolenic acid (C18:3, -3) are most abundant in
human milk. The latter two are generally recognized as
dietary essential fatty acids because we lack the enzymatic
pathways to introduce the necessary double bonds in the
carbon chains before carbon 9 [18].
Some unsaturated fatty acids have been shown to provide
protective effects against microorganisms. A possible
mechanism for this action is the disruption of viral envelopes
by these unsaturated fatty acids [37]. However, the
contribution of fatty acids in the defense against enteric
parasites such as Giardia lamblia is well documented [38].
Other fatty acids, such as docosahexaenoic (DHA) and eico-
Current Nutrition & Food Science, 2008, Vol. 4, No. 4 315
sapentaenoic (EPA) acids, also essential fatty acids, are
critical for normal growth and development of the central
nervous system and retina [39].
Normal growth and weight-gain of infants is dependent
on an adequate supply of essential fatty acids, a group of
naturally-occurring unsaturated fatty acids with a chain
length of 18, 20 or 22 carbon atoms and containing between
two and six methylene-interrupted double bonds in the cis-
configuration. Human infants and adults, are unable to
synthesize de novo, these compounds and they must
therefore be supplied in the diet after birth and through the
maternal plasma in utero. These polyunsaturated fatty acids
are an essential component of lipid membrane structures and
act as precursors in the formation of prostaglandins and
related compounds, as well as providing precursors of other
fat-soluble hormones.
The cream fraction of human milk is composed of milk
fat globules that consist of encapsulated fat droplets in a
membrane derived form the epithelial cells of the mother’s
mammary glands. The milk fat globule membrane is
composed of three major glycoproteins; mucin (MUC1),
lactadherin, and butyrophilin [40]. MUC1 and lactadherin
have both been shown to prevent infection by various
microorganism; MUC1 prevents the attachment and invasion
of S-frimbriated E. Coli to buccal epithelial cells in the gut,
preventing the initial step in the development of systemic
infections such as septicemia and meningitis [41].
Lactadherin has been shown to prevent symptomatic
rotavirus in breast fed infants but the mechanism is unclear
[41, 42]. The anti-infective property of lactadherin against
rotavirus induced diarrhea appears to involve its ability to
bind to the virus thus interfering with the virus’ ability to
bind to cells in the infant’s digestive track. However,
Peterson et al. (1998) suggest that the effect may also be due
to a mediated indirect mechanism; promoting the develop-
ment of the intestinal mucosa [43]. Since lactadherin is
resistant to degradation in the stomach and is abundant
throughout the digestive track of the breast fed infant,
lactadherin in human milk may actually provide protection
against other pathogenic microorganisms besides rotaviruses.
Butyrophilin is thought to be a structural component of the
milk fat globule membrane but it appears to have no anti-
infective properties [44].
Carbohydrate-Based Bioactive Compounds
Human milk contains a number of carbohydrate-based
bioactive compounds, including oligosaccharides, mucin,
gangliosides, and other N-acetylneuraminic acid-containing
components (Table 7). These complex sugar containing com-
pounds are usually attached to lactose. Human milk oligo-
saccharides play an important role, as pre-biotic soluble
fibers, in the postnatal development of the intestinal flora.
Besides 7% lactose, human milk contains approximately 1%
neutral oligosaccharides and about 0.1% acidic oligo-
saccharides. These oligosaccharides make up a large portion
of human milk composition, similar to the level of proteins.
The biological function of these complex oligosaccharides is
not yet fully understood. Principally because they are
thought to pass undigested through the infant’s intestine
[45]. However, there is evidence that human milk oligo-
saccharides are important for the pre-biotic effect (essentially
316 Current Nutrition & Food Science, 2008, Vol. 4, No. 4 Guo and Hendricks

Table 7. Carbohydrate-Based Protective Factors in Human Milk

Proactive Compound Organism Mechanism

Oligosaccharides Clostridia, Escherichia coli, various pathogens Prebiotics; promotes the growth of bifidobacteria,
inhibits the growth of pathogens

Oligosaccharides Rotavirus Prebiotics; promotes the growth of bifidobacteria,

improves immune response

Oligosaccharides Campylobacter jejun Binds bacterium

Oligosaccharides Streptococcus pneumoniae Binds bacterium

Fucosylated oligosaccharides Enterotoxin Escherichia coli Binds stable toxin

Mucin Escherichia coli Binds bacterium

GM1 ganglioside Vibrio cholera Binds toxin

GM1 gangliosides Campylobacter jejuni Binds toxin

GM1 gangliosides Enterotoxigenic Escherichia coli Binds labile toxin

Mannosylated glycoprotein Enterohaemorrhagic Escherichia coli Binds toxin

bifidogenic) as well as the anti-infective and allergy- absorption. Lactulose is used as a promoter of probiotic bacteria;
preventive properties of human milk. the bacteria that are necessary for normal digestion and intestinal
function. Lactulose is now commonly used in commercially
One characteristic of human milk oligosaccharides is the
available infant formulas. Lacto-oligosaccharides are also used
large amount of galactose. The backbone structure of human
as probiotic growth promoters. The oligosaccharides in human
milk oligosaccharides is based on lactose (galactose-glucose,
milk appear to inhibit the binding of enteropathogenic E. coli,
see Fig. 1) plus a further galactose residue forming the three
Campylobacter jejuni, and Streptococcus pneumoniae to the cells
different galactosyl-lactoses, namely 3'-galactosyl-lactose,
4'-galactosyl-lactose and 6'-galactosyl-lactose. Larger of the intestinal wall; thus warding off many serious diarrheal
diseases [48]. Mucin is another long macromolecule in human
oligosaccharides are formed by repeated units of galactose-
milk and links with oligosaccharides. Human milk mucin
N-acetylglucosamine added to the core lactose. The
complex (i.e., mucin-associated glycoprotein called lactadherin)
backbone is further modified by the specific addition of
binds to rotavirus and inhibits the rotavirus from binding to target
fucose and sialic acid residues. Roughly 130 different neutral
tissues by surrounding and coating the viral particles rendering
and acidic oligosaccharides have been characterized so far.
The pattern of human milk oligosaccharides depends largely them harmless [49].
on the mother's Lewis blood group [46]. Human breast milk contains hundreds of complex
oligosaccharides that are involved in growth-promotion of
Glucose
6
OH Bifidogenic bacteria. These compounds act as receptor
OH
analogues for epithelial cells to prevent the adhesion of
HO HO 1 OH pathogens, or can inactivate toxins. Human milk glycopeptides
5 O 2
4 3
and glycoproteins are also thought to stimulate the growth of
3 1 4
2 5 O bifidobacteria. Protective effects of fucosylated oligosaccharides and
HO O
6
glycoproteins and glycolipids against enterotoxigenic E. coli
HO have been reported [50]. This inhibition appears to be associated
Galactose HO
with acidic glycolipids that contain sialic acid gangliosides.
Fig. (1). Lactose is a disaccharide composed of one galactose and Growth Factors
one glucose monosaccharide. Lactose is the predominant carbo-
hydrate in breast milk. The main function of some important human milk
proteins is to provide antimicrobial activity against patho-
The concentration of oligosaccharides in human milk can genic bacteria, viruses, and fungi. It has been well
range from 10 to 20 g/1. Human milk contains more types documented that human milk proteins are involved in the
and higher amounts of oligosaccharides than cow’s milk. The immune system function of breastfed infants. Human milk
oligosaccharides in human milk can also comprise the also contains many cytokines, including tumor necrosis
carbohydrate portion of glycoconjugates, such as glycolipids and factor a, transforming growth factor b, and interleukins (IL)
glycoproteins. Glyco-protein in human milk inhibits the binding 1 b, IL-6, IL-8, and IL-10. All of these cytokines are
of enterohaemorrhagic E. coli [47]. immunomodulatory, and most of them are anti-inflam-
matory, which may mitigate the effect of infections. The
Lactose can be converted to lactulose and lacto- cytokines are found in free form, and also may be released
oligosaccharides and has been reported to enhance calcium from cells in breast milk [6]. Human milk also contains
Chemistry and Biological Properties of Human Milk Current Nutrition & Food Science, 2008, Vol. 4, No. 4 317

lactoferrin, which has been shown to increase the production
and release of cytokines such as IL-1, IL-8, tumor necrosis
factor a, nitric oxide, and granulocyte-macrophage colony
stimulating factor, which may also affect the immune system
[51]. When lactoferrin binds to its receptor in the small
intestine, this may either cause signaling events that affect
cytokine production downstream, or it is possible that the
internalized lactoferrin can bind to the nucleus, which could
affect nuclear transcription factor B, and subsequently,
cytokine expression. Lactoferrin was recently shown to
activate the transcription of IL-1b in mammalian cells,
which indicates that lactoferrin may interact directly with the
nucleus.
Several proteins are also implicated in the development
of the infant gut and its functionality, including growth
factors, lactoferrin, and casein-derived peptides. Research
has shown that IGF-I and IGF-II stimulate DNA synthesis
and promote the growth of many types of cells in culture;
therefore, they may play a role in the development of the
infant gastrointestinal tract.
Several peptides that possess physiological activity have
been generated from human casein, and especially from
-
casein [6]. Although these proteins have been generated in
vitro, they have also been detected from intestinal contents,
suggesting that they are formed in vivo as well [6].
Weight gain has been higher in infants who are fed
formula supplemented with bovine lactoferrin than infants
who are fed unsupplemented formula [52]. And adminis-
tration of lactoferrin has been shown to enhance cell
proliferation in the small intestine of experimental animals,
and also to affect crypt cell development. The rapid
development of intestinal mucosa in suckling newborns has
been hypothesized to be due in part to the mitogenic effect
of lactoferrin. Breastfed premature infants excrete intact
lactoferrin in their urine, demonstrating that functionally
intact lactoferrin is absorbed by the infant gut [1].
Milk fat has been shown to promote bone growth [53].
Weinsier and Krumdieck (2000) found that this effect may
be due to the increase in calcium associated with milk fat
intake [54]. These results point to the possibility that milk fat
itself has a small (but measurable) effect on bone health.
Nucleotides, Neuropeptides and Other Bioactive Factors
Nucleotides (naturally present in breast milk) seem to be
essential nutrients for rapidly dividing tissues such as the
intestinal epithelium and lymphoid cells. In addition to their
effect on the gastrointestinal tract, nucleotides have also been
implicated to be important for the immune system (see Table
8). Nucleotides and their metabolic products are present in
human and animal milk. Their presence, together with
nucleic acids, accounts for up to 20% of the non-protein N
content in human breast milk. The possibility that these
exogenous nucleotides may play a significant role in the
development and modulation of normal immune response
has been studied for the last 15 to 20 years [55, 56].
Laboratory and clinical responses to dietary nucleotides
include three areas of investigation: immune function,
intestinal function, and lipoprotein metabolism. Experi-
mental investigations relating immune function to dietary
nucleotides have demonstrated that T-lymphocyte function is
diminished in dietary nucleotide deprivation, yet the mecha-
nism responsible remains unknown [57, 58]. However,
supplementation of the diet with nucleotides during weaning
increases killer cell activity and macrophage activation [59].
Nucleotides derive from the bases purine and pyrimidine
and can be acquired through the diet or synthesized in
humans de novo from amino acid precursors. While these
synthetic pathways are present in term infants, de novo
synthesis of one molecule of adenine requires six ATP's,
where as the salvage pathway requires only two [55, 60].
Thus, the salvage and recycling of nucleic acids is compa-
ratively more energy efficient. The intracellular nucleotide
pools are maintained by the salvage pathway or by de novo
synthesis. The particular pathway utilized is based on
availability of preformed bases, either absorbed from the diet
or through salvage of nucleosides.

Table 8. Nucleotides and other Bioactive Factors in Human Milk

Bioactive Factor Effect

Nucleotides: Five bases—adenine, guanine, Besides serving as precursors of RNA and DNA the nucleotides participate in a wide variety of biological
cytosine, uracil, and thymine processes. They serve as the bases for the high energy source ATP, as regulatory signals (cyclic AMP and
In combination with a pentose sugars and a cyclic GMP), as components of coenzymes, and as important methyl donors
phosphate they form nucleotides.

casomorphins Opioid-like substances that may affect infant behavior and mood in addition to a range of other functions

-sleep peptides Sleep-inducing peptide

Galanin, neuropeptide Y, neurotensin, Neuropeptide galanin is widely distributed in the nervous and endocrine systems. It appears to facilitate the
substance P, somatostatin and vasoactive growth and repair of sensory neurons in the peripheral nervous system and gut. Some of these agents
peptide appear to potentiate certain immune responses. In addition, substance P induces interleukin (IL)-12
production by macrophages. Many cells in the immune system display receptors for these neuropeptides

thyrotropin-releasing hormone. These various growth factor releasing hormones have been shown to act directly on the developing gut
gonadotropnin-releasing hormone, growth tissue, lengthening of the villi, enhancing the activities of lactase, maltase and sucrase. There is evidence
hormone-releasing hormone that certain agents may interact not only with the GI tract, but may also be absorbed into the systemic
circulation on act on other target tissues
318 Current Nutrition & Food Science, 2008, Vol. 4, No. 4
Free nucleotides in human milk range from 5 to 8
mg/100ml. Thus, assuming that these are all metabolically
available, they would contribute as much as 25% of the daily
nucleotide needs at a significant energy savings over de novo
synthesis [55].
Delta sleep-inducing peptide (DSIP) is a naturally
occurring substance, which was originally isolated from
rabbit brain. This curious substance is a non-peptide, an
eligopeptide containing nine amino acid residues (e.g.,
exyteein that is normally synthesized in the hypothalamus
and targets multiple sites including some within the
brainstem. As its name suggests DSIP promotes sleep and
this has been demonstrated in rabbits, mice, rats, cats and
human beings. In fact DSIP promotes a particular type of
sleep which is characterized by an increase in the delta
rhythm of the EEG [61].
DSIP is normally present in minute amounts in the blood.
However, DSIP is present in relatively high concentrations
in human milk colostrum (30 ng/ml) and decreases to about
10 ng/ml after 2 months of lactation [61]. Brain and plasma
DSIP concentrations exhibit a marked diurnal variation and
there has been shown to be a correlation between DSIP
plasma concentrations and circadian rhythm in humans.
Concentrations are low in the mornings and higher in the
afternoons. When compared with most other peptides, DSIP
is unusual in that it can freely cross the blood-brain barrier
and is readily absorbed from the gut without being denatured
by enzymes, even in adults. However, it is not known
whether DSIP concentrations are related to the sleep-wake
cycle in human neonates [62, 63].
There are also numerous neuropeptides, growth factors,
and releasing factors present in human milk (see Table 8).
Galanin is widely distributed in the nervous and endocrine
systems and in the intestine [64]. It appears to facilitate the
growth and repair of sensory neurons in the peripheral
nervous system and gut. Along with thyrotropin-releasing
hormone, gonadotropnin-releasing hormone, growth
hormone-releasing hormone, these various growth factor
releasing hormones have been shown to act directly on the
developing gut tissue, lengthening of the villi, enhancing the
activities of lactase, maltase and sucrase [12, 65]. There is
evidence that certain agents may interact not only with the
developing gastrointestinal tract but may also be absorbed
into the systemic circulation on act on other target tissues
[30].
It appears that a number of evolutionary adaptations have
resulted in numerous nonnutritive actions associated with
breast feeding. In that regard, bioactive compounds in human
milk appear to have the following effects: i) compensate for
developmental delays in the production of bioactive agents
by the infant; ii) alter gastrointestinal functions from those
adapted to intrauterine life to those suited to extrauterine life;
iii) encourage the colonization of enteric bacteria that inhibit
the growth of bacterial pathogens or stimulate certain
biological activities by the gastrointestinal tract; iv) provide
antimicrobial and anti-inflammatory protection; or v)
program cells and tissues in the infant for augmented or new
responses. In respect to the last point, bioactive compounds
in human milk may affect certain aspects of the growth,
development or functions of the gastrointestinal tract, brain
Guo and Hendricks
and peripheral nervous system during early infancy.
However, there is much to be learned about the spectrum of
the programming agents in human milk, their physical
distribution in human milk, how their quantitative patterns
and physical distributions change as lactation proceeds;
whether the physical structures and functions of these
ingested agents are modified by digestive processes in the
gastrointestinal tract of the recipient infant; the precise
anatomic, cellular and molecular loci of their actions, and the
short- and long-term molecular-biological and biochemical
consequences of their actions. As more sophisticated and
noninvasive imaging procedures are developed, perhaps we
will be able to measure organ functions such as blood flow,
activities of metabolic pathways, phagocytosis and other
immune functions, the effects of these bioactive factors in
human milk may be analyzed more completely.
SUMMARY
Human milk contains wide range of bioactive com-
ponents and it is recognized as the "gold" standard for infant
nutrition and the preferred food for infants due to its
nutrient balance, immunological protection, and other
growth-promoting substances. It is the single reference by
which all infant nutritional products are compared, and it has
always been considered a species-specific food. Although
much is still unknown about human milk, and how to
optimize infant nutrition substitutes, new information is
constantly being discovered. Research on bioactive com-
ponents or biological functions of human milk will remain
one of the hot topics in human milk chemistry and the infant
nutrition industry.
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