Peptides 73 (2015) 101105

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Peptides
journal homepage: www.elsevier.com/locate/peptides

Bioactive peptides released by in vitro digestion of standard and

hydrolyzed infant formulas
Yasuaki Wada a,b , Bo Lnnerdal a,
a
b

Department of Nutrition, University of California, Davis, One Shields Ave., Davis, CA 95616, USA
Nutritional Science Institute, Morinaga Milk Industry Co., Ltd., 5-1-83, Higashihara, Zama, Kanagawa Pref. 252-8583, Japan

a r t i c l e

i n f o

Article history:
Received 14 July 2015
Received in revised form 5 September 2015
Accepted 12 September 2015
Available online 16 September 2015
Keywords:
Bioactive peptide
Infant formula
In vitro digestion
Milk protein
Peptidomic technique

a b s t r a c t
Hydrolyzed infant formulas serve as appropriate nutritional sources for infants aficted with cows milk
allergy, and milk proteins in hydrolyzed formulas are industrially hydrolyzed extensively or partially.
To investigate whether industrial hydrolysis may modulate the digestive trajectory of milk proteins,
thereby releasing different proles of bioactive peptides compared with standard formulas, both standard
and hydrolyzed formulas were subjected to in vitro digestion and formation of bioactive peptides were
compared. One standard, one extensively hydrolyzed, and one partially hydrolyzed infant formula were
digested in vitro with pepsin and pancreatin, taking into account the higher gastric pH of infants, and the
digesta were subjected to peptidomic analysis. The standard formula released a larger variety of bioactive
peptides than from the hydrolyzed formulas, indicating that industrial hydrolysis of milk proteins may
generally attenuate their indigenous bioactivities such as antibacterial, immuno-regulatory, and antioxidative activities. Conversely, industrial hydrolysis may facilitate the formation of bioactive peptides
from hydrophobic proteins/regions such as -LG and the strategic zone of -CN, which encrypt bioactive peptides including a dipeptidyl dipeptidase-4-inhibitory, hypocholesterolemic, and opioid peptides.
Infants fed hydrolyzed infant formulas may be inuenced by milk protein-derived bioactive peptides in
a manner different from those fed standard formula.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Infant formulas (IFs) are frequently used when availability of
mothers milk is limited. Most IFs are formulated using cows milk
protein because of its high nutritional value. However, for infants
allergic to cows milk protein, hydrolyzed IFs (HIFs) are used instead
of standard IF [1]. Furthermore, there is increased use of HIFs
for infants without allergic issues, as treatment of non-specic
gastrointestinal problems as well as for prophylaxis of milk protein allergy [2]. Milk proteins in HIFs are hydrolyzed extensively
or partially using industrial proteases such as alcalase, pronase,
and papain, as well as gastrointestinal digestive enzymes such as
pepsin, trypsin and chymotrypsin [3]. Industrial proteases may
hydrolyze milk proteins in a way different from gastrointestinal
digestive enzymes. The degree of hydrolysis and the composition

Abbreviations: IF, infant formula; HIF, hydrolyzed IF; HM, human milk; CN,
casein; WP, whey protein; MS, mass spectrometry; -LA, -lactalbumin; -LG, lactoglobulin; eHIF, extensively-hydrolyzed IF; pHIF, partially-hydrolyzed IF; ACE,
angiotensin I-converting enzyme; DPP4, dipeptidyl dipeptidase-4.
Corresponding author. Fax: +1 530 752 3564.
E-mail address: bllonnerdal@ucdavis.edu (B. Lnnerdal).
http://dx.doi.org/10.1016/j.peptides.2015.09.005
0196-9781/ 2015 Elsevier Inc. All rights reserved.

of peptides formed also depends upon the digestive conditions such

as temperature, digestion time, enzyme/protein ratio, etc [3]. Thus,
industrial hydrolysis may modify the digestive trajectory of milk
proteins in the gastrointestinal tract in different ways. As milk proteins encrypt a variety of bioactive peptides [4], it is likely that
infants fed HIFs will be inuenced by milk protein-derived bioactive peptides differently from those fed standard IFs. To the best of
our knowledge, there are no studies comprehensively comparing
gastrointestinal release of bioactive peptides from standard IFs and
HIFs.
The objective of this study was to compare the release of bioactive peptides after in vitro gastrointestinal digestion of 3 types
of infant formulas, using the same peptidomic technique as we
recently reported for bioactive peptides released from human milk
(HM) [5]. A standard formula, an extensively hydrolyzed casein
(CN) formula, and a partially hydrolyzed whey protein (WP) formula were subjected to in vitro digestion, taking into account the
higher gastric pH of infants, using porcine pepsin and pancreatin,
followed by liquid chromatography conjugated with tandem mass
spectrometry (MS), and released bioactive peptides from major
milk proteins, such as -lactalbumin (-LA), -lactoglobulin (LG), and CNs, were probed using established databases.

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Y. Wada, B. Lnnerdal / Peptides 73 (2015) 101105

Table 1
Bioactive peptides present in undigested hydrolyzed infant formulas.
Parental protein

Residue

Amino acid sequencea

eHIFb

pHIFb

Functionc

Ref.

-LA

7580
104108
1520
7882
7883
92100
102105
142148
9197
91100
174179
174181
125
5966
5967
5968
6063
6065
6068
6368
132139
132140
133139
134138
134139
170176
177183
193201
193202

(ISCDKF)
WLAHK
VAGTWY
IPAVF
IPAVFK
VLVLDTDYK
YLLF
ALPMHIR
YLGYLEQ
YLGYLEQLLR
FALPQY
FALPQYLK
RELEELNVPGEIVESLSSSEESITR
VYPFPGPI
VYPFPGPIP
VYPFPGPIPN
YPFP
YPFPGP
(YPFPGPIPN)
PGPIPN
(NLHLPLPL)
NLHLPLPLL
(LHLPLPL)
HLPLP
HLPLPL
VLPVPQK
AVPYPQR
(YQEPVLGPV)
(YQEPVLGPVR)

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
Y
N
Y
N
N
N
Y
N
N
N
Y
N

(Y)
Y
Y
Y
(Y)
Y
Y
Y
(Y)
(Y)
(Y)
(Y)
(Y)
Y
Y
Y
N
N
Y
Y
(Y)
Y
Y
N
Y
Y
(Y)
N
Y

Antibacterial (6168/7580)
ACEi (104108)
Antibacterial (1520)
DPP4i (7882)
Antibacterial (7883)
Antibacterial (92100)
OP (102105)
ACEi (142148)
Anxiolytic (9197)
Anxiolytic (9197, 91100)
ACEi (174179 or 181), AO (174181)
ACEi, AO (174181)
CPP (125)
PEPi (5966)
PEPi (59-66 or 67)
ACEi (5968), PEPi (5966 or 67)
OP (6063, 64, 65, 66, 67, or 70)
OP (6063, 64, 65, 66, 67, or 70)
OP (6063, 64, 65, 66, 67, or 70)
Immuno-regulatory (6368)
ACEi (133, or 134138), PEPi (134139)
ACEi (133, or 134138), PEPi (132-140, 134139)
ACEi (133, or 134138), PEPi (134139)
ACEi (134138)
ACEi (134138), PEPi (134139)
AO (170176)
AO (177183)
AO (193196)
AO (193196)

[14]
[15]
[14]
[14]
[14]
[14]
[14]
[16]
[17]
[17]
[18,19]
[18,19]
[20]
[21]
[21]
[16,21]
[22]
[22]
[22]
[23]
[21,24]
[21,24,25]
[21,24]
[24]
[21,24]
[26]
[26]
[27]
[27]

-LG

S1 -CN
S2 -CN

-CN

a
Peptides in parentheses indicate possible precursors for bioactive peptides, which include bioactive sequences. As for the amino acid sequences of caseinophosphopeptide
(CPP), letters underscored indicate phosphorylated sites, and bold letters represent the acid motif.
b
The abbreviations eHIF and pHIF denote extensively hydrolyzed infant formula and partially hydrolyzed infant formula, respectively. Ys in parentheses show the
bioactive peptides/precursors that were not detected after in vitro digestion.
c
The abbreviations ACEi, AO, CPP, DPP4i, OP, and PEPi denote angiotensin I-converting enzyme-inhibitory, anti-oxidative, caseinophosphopeptide, dipeptidyl peptidase4-inhibitory, opioid peptide, and prolyl endopeptidase-inhibitory, respectively.

2. Materials and methods

2.1. Samples
A standard IF (CN + WP-based, Similac Advance; Abbott Nutrition, Abbott Park, IL), an extensively hydrolyzed CN-based IF
(Similac Expert Care Alimentum; Abbott Nutrition), and a partially
hydrolyzed WP-based IF (Gerber Good Start Gentle; Nestl Infant
Nutrition, Florham Park, NJ) were purchased at a local supermarket.
They were designated as standard IF, eHIF, and pHIF, respectively.
2.2. In vitro digestion of formula samples
Formula samples were subjected to in vitro digestion as reported
previously [5]. Conditions for the in vitro digestion in general [6],
digestion time [7], and pH of the gastric digestion [711], were
reviewed carefully and selected accordingly. Shorter digestion
times were chosen here than we routinely employ to investigate
protein digestibility [12], aiming to obtain the prole of bioactive
peptides present in the mid-course of gastrointestinal digestion.
Powdered standard IF, eHIF, and pHIF were reconstituted with
Milli-Q water (Millipore, Billerica, MA), with reference to instructions on product labels. Reconstituted formulas were centrifuged
at 8500 g for 30 min twice, and the upper fat layer was removed
(we have previously conrmed that no protein loss is accompanied by this step [12]). The skimmed samples were subjected to
in vitro digestion. Simulated gastric digestion was started by adjusting the pH to 4.0 (mimicking the stomach pH in infants) with 1 M
HCl. Porcine pepsin (SigmaAldrich, St. Louis, MO; 2% in 1 mM HCl)
was added to the sample in a 1:12.5 ratio (pepsin:protein). Samples
were placed in an incubating shaker (New Brunswick Scientic,
Edison, NJ) at 140 rpm at 37 C for 15 min. Then, after the pH of

the samples was adjusted to 7.0 with 0.1 mol/L NaHCO3, simulated
intestinal digestion was performed. Pancreatin (SigmaAldrich;
0.4% in 0.1 mol/L NaHCO3 ) was added to the samples in a 1:62.5
ratio (pancreatin:protein), and the samples were placed in the incubator shaker at 140 rpm at 37 C for 5 min. After the incubation, the
enzymes were inactivated in a water bath at 85 C for 3 min.
2.3. Peptidomic analysis
Undigested eHIF and pHIF, and the 3 digested IFs were subjected to peptidomic analysis as described previously [5]. Briey,
undigested eHIF & pHIF, and the 3 digested formulas were spun
with a 10 kDa lter (Millipore). The collected ltrates were subjected to solid phase extraction using Aspire Chromatography Tips
(Thermo Scientic, San Jose, CA). The desalted peptides were reconstituted in 2% acetonitrile/0.1% triuoroacetic acid, and each sample
was loaded onto a 100 m 25 mm Magic C18 100 5U reverse
phase trap before being separated on a 75 m 150 mm Magic C18
200 3U reverse phase column. Peptides were eluted using a gradient of 0.1% formic acid and 100% acetonitrile. All MS/MS samples
were analyzed using X! Tandem [The GPM, thegpm.org; version
CYCLONE (2013.02.01.2)]. X! Tandem was set up to search the
Uniprot Bos taurus reference database (June, 2013; 23860 entries)
plus an equal number of reverse sequences and 60 common laboratory contaminant proteins, assuming a non-specic digestion
enzyme. Scaffold (version Scaffold 4.2.1, Proteome Software Inc.,
Portland, OR) and SEQUEST (Proteome Discoverer 1.1; Thermo Scientic) were used to validate MS/MS based peptide and protein
identications. Peptides derived from major bovine milk proteins
of interest were compared with the database of BIOPEP [13], and
the search results were further corroborated by browsing reference
articles.

Y. Wada, B. Lnnerdal / Peptides 73 (2015) 101105

103

Table 2
Bioactive peptides in in vitro digested infant formulas.
ParentalProtein

Residue

Amino acid sequencea

IFb

eHIFb

pHIFb

Functionc

Ref.

-LA

104108
109114
1520
2540
7175
7882
92100
102105
142148
17
127
414
1016
2334
9196
9197
143149
144149
146149
157164
180193
2532
174182
4956
5966
5967
5968
6063
6065
6068
6368
98105
129138
130139
130142

WLAHK
(ALCSEK)
VAGTWY
AASDISLLDAQSAPLR
IIAEK
IPAVF
VLVLDTDYK
YLLF
ALPMHIR
RPKHPIK
(RPKHPIKHQGLPQEVLNENLLRFFVAP)
(HPIKHQGLPQE)
(GLPQEVL)
FFVAPFPEVFGK
YLGYLE
YLGYLEQ
(AYFYPEL)
YFYPEL
YPEL
DAYPSGAW
SDIPNPIGSENSEK
NMAINPSK
(FALPQYLKT)
IHPFAQTQ
VYPFPGPI
VYPFPGPIP
(VYPFPGPIPN)
YPFP
YPFPGP
(YPFPGPIPN)
PGPIPN
VKEAMAPK
(DVENLHLPLP)
(VENLHLPLPL)
(VENLHLPLPLLQS)

Y
N
N
N
N
N
N
N
N
Y
Y
Y
N
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
N
N
N
N
Y
N
Y
Y

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
N
N
N
Y
Y
Y
Y
Y
Y
Y
N
N
N
N

Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
Y
Y
Y
N
Y
N
N
Y
Y
N
N
N
Y
Y
Y
N
N
Y
Y
N
Y
Y
N

[15]
[14]
[14]
[14]
[14]
[14]
[14]
[14]
[16]
[28]
[29]
[30]
[29]
[16]
[31]
[17]
[16]
[16,26]
[26]
[32]
[30]
[18]
[18,19]
[21]
[21]
[21]
[16,21]
[22]
[22]
[22]
[23]
[26]
[24]
[21,24]
[21,24,25]

132139
133138
133139
134138
134139
169176
170176
191209

(NLHLPLPL)
LHLPLP
(LHLPLPL)
HLPLP
HLPLPL
KVLPVPQK
VLPVPQK
LLYQEPVLGPVRGPFPIIV

Y
N
Y
N
Y
Y
Y
Y

N
N
N
Y
N
N
N
N

Y
Y
Y
N
Y
N
Y
N

192209
193197
193209
1824
2530
3139
3340
3541
4249
5660
6175
106111
106112
162169

LYQEPVLGPVRGPFPIIV
(YQEPV)
YQEPVLGPVRGPFPIIV
FSDKIAK
YIPIQY
(VLSRYPSYG)
(SRYPSYGL)
YPSYGLN
YYQQKPVA
LPYPY
(YAKPAAVRSPAQILQ)
MAIPPK
(MAIPPKK)
VQVTSTAV

Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y

N
N
N
N
N
N
N
N
N
N
N
N
N
N

N
N
N
N
N
N
N
N
N
N
N
N
N
N

ACEi (104108)
Antibacterial (1731/109114)
Antibacterial (1520)
Antibacterial (2540)
hypocholesterolemic (7175)
DPP4i (7882)
Antibacterial (92100)
OP (102105)
ACEi (142148)
Antibacterial (17)
Immuno-regulatory (123)
Antibacterial (614)
Antibacterial (1014)
ACEi (2334)
OP (9196)
Anxiolytic (9197)
ACEi (144149)
ACEi (144-149), AO (144146 or 149)
AO (146149)
ACEi (157164)
Antibacterial (180193)
ACEi (2532)
ACEi, AO (174181)
PEPi (4956)
PEPi (5966)
PEPi (5967)
ACEi (5968), PEPi (5966 or 67)
OP (6063, 64, 65, 66, 67, or 70)
OP (6063, 64, 65, 66, 67, or 70)
OP (6063, 64, 65, 66, 67, or 70)
Immuno-regulatory (6368)
AO (98105)
ACEi (133, or 134-138)
ACEi (133, or 134138) PEPi (134139)
ACEi (133, or 134138),
PEPi (130, 131, or 132140, 134139)
ACEi (133, or 134-138), PEPi (134139)
ACEi (133, or 134138)
ACEi (133, or 134138), PEPi (134139)
ACEi (133, or 134138)
ACEi (133, or 134138), PEPi (134139)
AO (169176)
AO (170176)
ACEi (191209),
Immuno-regulatory (192 or 193209)
Immuno-regulatory (192 or 193209)
AO (193196)
Immuno-regulatory (193209)
Antibacterial (1824)
ACEi (2530)
OP Antagonist (3338)
OP Antagonist (3338)
OP Antagonist (3541)
Antibacterial (4249)
ACEi (5660)
Antibacterial (6475)
Anti-thrombotic (106111)
Anti-thrombotic (106111)
Antibacterial (162169)

-LG

S1 -CN

S2 -CN

-CN

-CN

[21,24]
[24]
[21,24]
[24]
[21,24]
[26]
[26]
[33,34]
[34]
[27]
[34]
[35]
[36]
[22]
[22]
[22]
[35]
[36]
[35]
[37]
[37]
[35]

a
Peptides in parentheses indicate possible precursors for bioactive peptides, which include bioactive sequences. As for the amino acid sequences of caseinophosphopeptide
(CPP), letters underscored indicate phosphorylated sites, and bold letters represent the acid motif.
b
The abbreviations eHIF and pHIF denote extensively hydrolyzed infant formula and partially hydrolyzed infant formula, respectively. Ys in parentheses show the
bioactive peptides/precursors that were not detected after in vitro digestion.
c
The abbreviations ACEi, AO, CPP, DPP4i, OP, and PEPi denote angiotensin I-converting enzyme-inhibitory, anti-oxidative, caseinophosphopeptide, dipeptidyl peptidase4-inhibitory, opioid peptide, and prolyl endopeptidase-inhibitory, respectively.

3. Results
Hydrolyzed formulas were applied to peptidomic analysis without in vitro digestion, in order to investigate bioactive peptides
potentially present in these formulas (Table 1; [1427]). eHIF contained only peptides derived from CNs in accordance with its
composition. Conversely, both CN- and WP-derived peptides were

observed in pHIF although this formula uses partially hydrolyzed

WP concentrate as the sole protein source, suggesting inclusion
of CNs in the raw material. When focusing on CN-derived bioactive
peptides in undigested eHIF and pHIF, among all 21 peptides, only 5
peptides were detected in undigested eHIF while 17 peptides were
found in undigested pHIF, presumably reecting the fact that protein hydrolysis of pHIF is partial while it is extensive in eHIF.

104

Y. Wada, B. Lnnerdal / Peptides 73 (2015) 101105

All of the 5 bioactive peptides found in undigested eHIF were also

conrmed to be present after in vitro digestion with pepsin and
pancreatin, but 9 of the 25 peptides found in undigested pHIF were
not detected in pHIF after the digestion. We therefore infer that
peptides having strong resistance to enzymatic digestion (including gastrointestinal digestion) are likely to survive the extensive
hydrolysis used for eHIF, while the partial hydrolysis used for pHIF
may generate peptides that are still vulnerable to further digestion,
as well as hydrolysis-resistant peptides.
In vitro digestion of the standard and hydrolyzed IFs released
a multitude of bioactive peptides, and the standard IF released a
larger variety of bioactive peptides than the hydrolyzed IFs (Table 2
[1419,2137]). Among 57 peptides identied after in vitro digestion of the three IFs, the standard IF released as many as 39 peptides,
while only 9 and 27 were detected in digested eHIF, and pHIF,
respectively. Among the bioactive peptides identied, angiotensin
I-converting enzyme (ACE)-inhibitory peptides (residues 2334 of
S1 -CN and 134138 of -CN [16,24]) and opioid peptides (residues
6063 and 6065 of -CN [22]) should be noted because they have
previously been extensively investigated in terms of in vivo bioactivity and bioavailability [24,3846].
4. Discussion
A considerable larger number of bioactive peptides were
released from the standard IF and their bioactivities were more
diverse than those from the HIFs. We therefore suggest that
industrial hydrolysis of milk proteins, regardless of extensive or
partial, will attenuate their encrypted bioactivities, which was
notable for antibacterial, immuno-regulatory, and anti-oxidative
peptides. However, there appears to be some exceptions; several
several other bioactive peptides were preferentially released from
the HIFs, and this was prominent at the entire part of -LG, and
at residues 6070 of -CN called a strategic zone, which encrypts
opioid peptides (called -casomorphins) and immuno-regulatory
peptides [23]. -LG is known for higher resistance to digestion [47],
and the strategic zone in -CN is situated in a hydrophobic part of
the molecule and considered to have low accessibility to proteolytic
enzymes [23]. It can therefore be assumed that fragmenting LG and -CN by industrial hydrolysis would allow gastrointestinal
enzymes easier access to digest these proteins, thereby facilitating
the release of bioactive peptides.
The major limitation of this study is that this in vitro digestion
assay employs only digestive enzymes in gastrointestinal tract and
it does not simulate other digestive enzymes such as brush border and cytoplasmic peptidases, which are inevitably involved in
the trajectory of post-absorptive peptides, such as ACE-inhibitory
peptides, opioid peptides, etc. While it is not known whether most
of the post-absorptive peptides identied here can survive further
digestion by these peptidases and reach target organs, 2 ACEinhibitory peptides (residues 2334 of s1 -CN and 133138 of
-CN) and an hypocholesterolemic peptide (residues 7175 of LG) likely do; oral ingestion of these peptides reportedly exhibited
antihypertensive/hypocholesterolemic actions in rats [3842,48],
and it has been conrmed for residues of 133138 of -CN (LHLPLP)
that this peptide was hydrolyzed to the minimum active form
HLPLP, when transported across the intestinal epithelium in monolayers of Caco-2 cells, and showed high stability in human plasma
[24]. Further, immuno-reactive materials of -casomorphins have
been identied in larger forms in plasmas of newborn calves
and dogs fed cows milk [43,44], and it can be extrapolated that
the longer fragments identied here, (i.e., residues 5967, 5968,
and 6068 of -CN), may serve as precursors of -casomorphins,
absorbed into the circulation and subsequently digested into active
forms such as residues 6063 and 6065. Nevertheless, it needs to
be addressed that physiological benets of these peptides in infants

remain to be demonstrated. Both blood pressure and lipid status may be involved in metabolic programming in infants [49,50],
and ACE-inhibitory and hypocholesterolemic peptides might modulate it. -Casomprhins may be of biological signicance in control
and development of the gastrointestinal tract and development of
learning and memory [51,52], while they have sometimes been
blamed for adverse effects such as sudden infant death and delay
in psychomotor development [45,53].
It is interesting to compare the release of peptides from HM
with those released from IFs. We recently investigated the release
of bioactive peptides from HM using the same methods as in this
study, with an emphasis on the effects of pasteurization [5]. Only
1 ACE-inhibitory peptide and 1 precursor, WLAHK from -LA and
HLPLPL from -CN, are found in common among the bioactive peptides in our two studies. There is some homology in the amino
acid sequences of major milk proteins in HM and IF, and some
of the released bioactive peptides are also homologous, e.g., casomorphins and immuno-regulatory peptides from the strategic
zone in -CN (residues 5158 and 5459 of human; residues 6068
and 6368 of bovine), and anti-thrombotic peptides from -CN
(residues 100103 and 100104 from human; residues 106111
and 106112 from bovine). However, bioactivities and bioavailability of these peptides are likely different from each other as
there are variations in amino acid sequences, and they may not be
functionally equal. Furthermore, many IFs contain -LG as a major
protein, and this protein, which is absent in HM, released peptides
with unique bioactivities such as dipeptidyl dipeptidase-4 (DPP4)inhibitory and hypocholesterolemic peptides, which are absent in
digested HM. Thus, the proles of bioactive peptides released from
HM and IFs are considerably different, and they may possibly exert
different biological effects on neonatal physiology. It would therefore be a long way to bridge the gap between HM and IFs in terms
of bioactive peptides.
5. Conclusions
Bioactive peptides released after in vitro digestion of standard
and hydrolyzed IFs were quite different. It may be extrapolated
that infants fed HIFs may be provided bioactivities that are different from those fed standard IFs in terms of peptides released
from milk proteins. However, these peptides have less been investigated in vivo and there has been no clear demonstration that any
of them function in human infants. Our in vitro digestion assay is
a simplied, static simulation, missing dynamic factors, such as
different gastric emptying rates between CNs and WPs, and continuous changes in gastric pH. Conditions for the digestion were
carefully reviewed and selected, but discrepancies may still remain
between our in vitro digestion approach and digestion in vivo. It
should also be noted that the peptidomic results obtained cannot
simply be extrapolated to similar types of formulas; the formulas used in our study were randomly chosen and, in particular,
the 2HIFs were selected to reect 2 different extents of hydrolysis but the degree of hydrolysis varies among products. Thus,
further in vivo and clinical studies are needed to establish which
of the observed bioactive peptides are of biological signicance for
infants, and to substantiate their clinical relevance.
Acknowledgments
We thank Dr. Brett Phinney and Darren Weber, University of
California, Davis, for performing the proteomic analysis.
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