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What Do We Know about Protein
quences arising from the use of the
Evolution of Breast Milk?
information contained herein.
Sagar K. Thakkar, Lausanne (Switzerland),
Peter Erdmann and Frédéric Destaillats,
Published by S. Karger AG, Switzerland, Vevey (Switzerland)
for Nestlé Nutrition Institute
Avenue Reller 22 Bioactive Proteins in Breast Milk –
CH–1800 Vevey Can Their Bioactivities Be Provided
Switzerland from Cow’s Milk Proteins?
Bo Lönnerdal, Davis, Calif. (USA)
© Copyright 2015 by
Nestlé Nutrition Institute, Switzerland Can Low-Protein Formulas Help
Solve the Obesity Problem?
ISSN 1270–9743 Ekhard E. Ziegler, Iowa City, Iowa (USA)

To learn more about the Nestlé Nutrition Institute

and its resources and fellowship opportunities visit:
www.nestlenutrition-institute.org

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the
nest What Do We Know about
Protein Evolution of Breast Milk?
Sagar K. Thakkara
Peter Erdmannb
Frédéric Destaillatsb
Nestlé Research Center, Nestec Ltd.
a 
Lausanne, Switzerland
Sagar.Thakkar@rdls.nestle.com
Nestlé Nutrition, Nestec Ltd.
b 
Vevey, Switzerland
Key Messages
Human milk is a comprehensive source
of nutrition for newborns.
The composition of human milk is
dynamic and evolves with time, imag-
inably, to adapt to the nutritional need
of the infants.
The concentration of protein in hu-
man milk follows a temporal pattern
and decreases with progressing stag-
es of lactation.
 during the first year of life [4]. As evi-
Human milk provides unparalleled nu-
trition to infants in early stages of life.
Consequently, the WHO/UNICEF, the
American Academy of Pediatrics and
other similar organizations recom-
mend exclusive breast-feeding for the
first 6 months of life, with the introduc-
tion of complementary food at the age
of 6 months and continuation of
breast-feeding for as long as mutually
agreeable by the mother and the infant
[1, 2]. Human milk is a comprehensive
source of energy, macronutrients as
well as bioactive micronutrients es-
sential for the growth and develop-
ment of an infant, with possible excep-
tions of iron and vitamin D [3]. It is also
a very dynamic fluid and may evolve
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with advancing stages of lactation to
reflect the nutritional need of the
growing infant. Various nutrients in hu-
man milk display such dynamism, and
indeed protein fraction is one of them.
We recently reviewed 34 publications
and examined data on true protein in
human milk at various lactation stages
from 26 publications to highlight the
evolution of proteins in human milk
dent from figure 1, there is a signifi-
cant decrease in true protein in human
milk from the very first week of lacta-
tion. The decline in the concentration
of the protein continues to 6 months
of life, after which it remains relatively
constant. It is also noteworthy that
during the early stages of lactation
there is a high content of whey fraction
in human milk compared to caseins
(approximately 90:10 to 80:20) that
transitions to 60:40 in transition milk
and to 50:50 in mature milk [5, 6]. Ad-
ditionally, colostrum protein is rich in
immune and growth factors. More-
over, there are also multitudes of bio-
active proteins in human milk, such as
lactoferrin, lysozyme, secretory IgA,
32
3.0
2.5
True protein in human milk (mg/100 ml)
haptocorrin, α-lactalbumin, bile salt-
stimulated lipase, κ-casein, and 2.0
β-casein [7]. Indeed, research over the
past decades has given us insights
into the dynamics of total and true pro-
tein in human milk; however, future 1.5
research is required to understand the
dynamics of bioactive proteins with
advancing stages of lactation along
1.0
with their contribution of biological ac-
tivities at various stages of an infant’s
growth.
0.5
0–5 6–15 16–30 31–60 61–90 91–360
References Time (days)
1. World Health Organization: Infant and young
child nutrition. Geneva, WHO, 2003.
2. American Academy of Pediatrics: Breast- Fig. 1. True protein in human milk over the first year of postpartum life.
feeding and the use of human milk. Section
on breastfeeding. Pediatrics 2012;129:e827–
e841.
3. Denne SC: Neonatal nutrition. Pediatr Clin
North Am 2015;62:427–438.
4. Destaillats F, Erdmann P, Thakkar SK,
Lönnerdal B: A developmental perspective of
the contents of total protein and bioactive
proteins in breast milk. In preparation.
5. Kunz C, Lönnerdal B: Re-evaluation of the
whey protein/casein ratio of human milk.
Acta Paediatr 1992;81:107–112.
6. Lönnerdal B: Nutritional and physiologic
significance of human milk proteins. Am J
Clin Nutr 2003;77:1537S–1543S.
7. Lönnerdal B: Bioactive proteins in breast milk.
J Paediatr Child Health 2013;49(suppl 1):1–7.
3
the
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Bo Lönnerdal
Distinguished Professor of Nutrition and
Internal Medicine
University of California, Davis, Calif., USA
bllonnerdal@ucdavis.edu
Key Messages
Breast-fed infants have a lower risk of
acute illness, diabetes, obesity and
cardiovascular disease than formula-
fed infants.
Bioactive proteins in breast milk
contribute to the health benefits of
breast-feeding.
Some proteins in cow's milk have
structures and functions similar to
those of their corresponding breast
milk proteins.
Dairy technology now allows the
enrichment of some of these proteins,
and they may be added to infant for-
mulas.
Clinical trials are needed to evalu-
ate whether supplementation of infant
formulas with such proteins will pro-
vide health benefits.
Breast-feeding has been shown to
result in both short- and long-term
benefits with regard to health and
cognitive development and a re-
duced risk of diabetes, obesity, and
cardiovascular disease. Many of
these benefits have been associated
with bioactive proteins in breast milk
[1]. Among these bioactive proteins
are lactoferrin, α-lactalbumin, and
milk fat globule membrane (MFGM)
proteins.
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Bioactive Proteins in Breast Milk – Can Their
Bioactivities Be Provided from Cow’s Milk Proteins?
Lactoferrin is an iron-binding pro-
tein and a major constituent (10–20%)
of the protein in breast milk. It is
known to inhibit the growth of, or kill,
potentially harmful bacteria and has
anti-inflammatory activity in the gas-
trointestinal tract of breast-fed infants
[2]. Lactoferrin has a very stable struc-
ture and is therefore relatively difficult
to break down; in fact, intact lactofer-
rin is found in considerable amounts
in the stool of breast-fed infants [3]. It
4 5
can be taken up by human intestinal
cells and is capable of binding to the
nucleus of the cell [4]. There, it can
affect the expression of various genes,
which most likely explains its effects
on the immune system and on cellular
growth and proliferation.
α-Lactalbumin is also a major pro-
tein (15–20%) in human milk. During
its digestion in the gastrointestinal
tract of breast-fed infants, peptides
are released that, in turn, have been
shown to have biological activity.
Among these peptides are an im-
mune-stimulating peptide, peptides
enhancing the absorption of essential
micronutrients such as iron and zinc,
and peptides that have prebiotic ac-
tivity, i.e. that stimulate the develop-
ment of a beneficial gut microflora [5].
MFGM proteins are associated
with the lipids in milk and are very di-
verse in composition and function [6].
They have been shown to have both
anti-bacterial and anti-viral activities.
Human milk MFGM proteins have
been shown to bind to various rotavi-
rus strains and prevent replication, an
ability that was associated to a spe-
cific protein, namely lactadherin [7].
Further, the concentration of lactad-
herin in breast milk was shown to be
negatively correlated to rotavirus in-
fection in Mexican infants [8].
Breast milk contains a multitude of
bioactive proteins, but the concentra-
tions of the above-mentioned proteins
are particularly high. They are, how-
ever, also present in cow’s milk, al-
beit at considerably lower concentra-
tions. Thus, if they could be enriched
and concentrated, they can possibly
be added to infant formulas. However,
as they are somewhat different in
structure from their human milk coun-
terparts, it is important to evaluate if
they can provide any of the bioactivi-
ties of the breast milk proteins. Such
tests need to be done in the research
laboratory, and if successful, clinical
trials on human infants will be re-
quired.
References
1. Lönnerdal B: Bioactive proteins in breast milk.
J Paediatr Child Health 2013;49(suppl 1):1–7.
2. Lönnerdal B: Nutritional roles of lactoferrin.
Curr Opin Clin Nutr Metab Care
2009;12:293–297.
3. Davidson LA, Lönnerdal B: Persistence of
human milk proteins in the breast-fed infant.
Acta Paediatr Scand 1987;76:733–740.
4. Liao Y, Jiang R, Lönnerdal B: Biochemical
and molecular impacts of lactoferrin on small
intestinal growth and development during
early life. Biochem Cell Biol 2012;90:476–484.
5. Lönnerdal B, Lien EL: Nutritional and
physiologic significance of alpha-lactalbumin
in infants. Nutr Rev 2003;61:295–305.
6. Liao Y, Alvarado R, Phinney B, Lönnerdal B:
Proteomic characterization of human milk fat
globule membrane proteins during a 12
month lactation period. J Proteome Res
2011;10:3530–3541.
7. Yolken RH, Peterson JA, Vonderfecht SL,
Fouts ET, Midthun, K, Newburg DS: Human
milk mucin inhibits rotavirus replication and
prevents experimental bacteriostasis. J Clin
Invest 1992; 90;1984–1991.
8. Newburg DS, Peterson JA, Ruiz-Palacios GM,
et al: Role of human-milk lactadherin in
protection against symptomatic rotavirus
infection. Lancet 1998;351:1190–1194.
the
nest
Can Low-Protein Formulas Help
Solve the Obesity Problem?
E k h a r d E . Z i e g l e r, M D

Department of Pediatrics Pickwick USA

University of Iowa
Iowa City, Iowa, USA
ekhard-ziegler@uiowa.edu
Key Messages
Protein intakes of formula-fed infants
are higher than those of breast-fed in-
fants. Efforts to lower protein intakes
Requirement Safe CTRL EXPL
4.50
does not change with the age of the ers. They were enrolled in the feeding
infant, as indicated in figure 1, protein study at 3 months of age and fed the
4.00
intakes of formula-fed infants exceeds study formulas up to 1 year. Although
their protein requirements by an in- infants in both formula groups showed
3.50
creasing margin. That is indicated in what was considered normal growth,
figure 1 by the green line representing infants fed the low-protein formula 3.00

Protein (g/100 kcal)

of formula-fed infants are under way.
Protein requirements are defined
as intakes that must on average be
met in order to enable normal growth
of the infant. Safe intakes are defined
as requirements of those infants who
have the highest needs [1]. As figure
1 illustrates, both requirements and
safe intakes of normal infants decline
markedly during the first 4–5 months
of life. The protein content of breast
milk (not shown) also declines during
the first few months of lactation, re-
sulting in the situation where protein
intakes of breast-fed infants match
the protein requirements of normal
infants closely. As requirements and
intakes of breast-fed infants are nearly
identical, they are represented by the
same symbol (blue) in figure 1. The
fact that protein intakes of breast-fed
infants follow requirements closely is
important because it ensures there is
neither a shortfall nor an excess of
protein intake relative to protein re-
quirement.
Protein intakes of infants fed for-
mula follow a very different course.
Since the protein content of formulas
the protein content of a typical formu-
la. As the protein requirement of the
infant continues to fall, the excess of
protein becomes larger and larger. It
has been documented in several lo-
calities [2] that protein intakes of older
infants and toddlers typically exceed
requirements for protein by a consid-
erable margin. Since it is evident from
epidemiological studies that exces-
sively high protein intakes during in-
fancy are associated with increased
adiposity in childhood [3], the protein
content of formulas fed to older in-
fants has come under scrutiny. Impor-
tantly, in prospective studies, high
protein intakes during infancy have
been shown to lead to increased adi-
posity lasting into childhood [4, 5].
Lower-protein formulas have been
developed in recent years as new
dairy technologies have made it pos-
sible to improve the biological quality
of formula protein. In a recently com-
pleted study [6], a formula with a low
protein content of 1.61 g/100 kcal was
compared to a standard formula with
a protein content of 2.15 g/100 kcal
(fig. 1). The study involved infants
born to overweight and obese moth-
grew significantly less rapidly than in-
fants fed the high-protein formula.
The growth-slowing effect of the low-
protein formula was particularly
strong in infants of obese mothers
and in infants who already were on a
fast growth trajectory.
A similar study in infants of normal
(as opposed to overweight) mothers
showed a milder effect on growth after
3 months of a low-protein formula [7].
Importantly, however, it also showed
that the low-protein formula supported
normal growth, suggesting that the pro-
tein content of formulas could safely be
reduced, thereby contributing to a re-
duction of overall high protein intakes in
late infancy and bringing protein intakes
closer to those of breast-fed infants.
Now that the obesogenic potential of
high protein intakes has been recog-
nized, an overall reduction of intakes of
protein appears desirable from an obe-
sity prevention point of view. Reduction
of the formula protein content offers a
contribution to the fight to prevent obe-
sity, in particular since low-protein for-
mula represents an intervention at an
age at which preventive efforts may be
particularly effective.
2.50
2.00
1.50
1.00
0.50
0
1 2 3 4
Fig. 1. Requirement and safe intakes of protein in infants. CTRL = Normal protein; EXPL = low protein.
References
1. Dewey KG, Beaton G, Fjeld C, Lönnerdal B,
Reeds P: Protein requirements of infants and
children. Eur J Clin Nutr 1996;50:S119 –S150.
2. Alexy U, Kersting M, Sichert-Heller W, Manz
F, Schöch G: Macronutrient intake of 3- to
36-month-old German infants and children:
results of the DONALD study. Ann Nutr
Metab 1999;43:14–22.
3. Rolland-Cachera MF, Deheeger M, Akrout M,
Bellisle F: Influence of macronutrients on
adiposity development: a follow up study of
nutrition and growth from 10 months to 8
years of age. Int J Obes Relat Metab Disord
1995;19:573–578.
2.1
2.15
1.61
5 6 7 8
Age (months)
4. Koletzko B, von Kries R, Closa R, Escribano J,
Scaglioni S, Giovannini M, et al: Lower
protein in infant formula is associated with
lower weight up to age 2 y: a randomized
clinical trial. Am J Clin Nutr 2009;89:1837–
1845.
5. Weber M, Grote V, Closa-Monasterolo R,
Escribano J, Langhendries J-P, Dain E,
Giovannini M, et al: Lower protein content in
infant formula reduces BMI and obesity risk
at school age: follow-up of a randomized trial.
Am J Clin Nutr 2014;99:1041–1051.
9 10 11 12
6. Inostroza J, Haschke F, Steenhout P,
Grathwohl D, Nelson SE, Ziegler EE:
Low-protein formula slows weight gain in
infants of overweight mothers. J Pediatr
Gastroenterol Nutr 2014;59:70–77.
7. Ziegler EE, Fields DA, Nelson SD, Chernau-
sek SD, Steenhout P, Grathwohl D, Jeter JM,
Nelson SE, Haschke F: Adequacy of infant
formula with protein content of 1.6 g/100 kcal
for infants between 3 and 12 months: A
randomized multicenter trial. J Pediatr
Gastroenterol Nutr, in press.

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