International Dairy Journal 21 (2011) 286e293

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International Dairy Journal

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The effect of storage at 25
C on proteins in human milk

Claire E. Molinari*, Ylenia S. Casadio, Peter G. Arthur, Peter E. Hartmann
School of Biomedical, Biomolecular and Chemical Sciences, Faculty of Life and Physical Sciences, The University of Western Australia, Crawley, WA 6009, Australia

a r t i c l e i n f o a b s t r a c t

Article history: The effect of storage of milk at 25
C upon the major human milk proteins was investigated. Milk samples
Received 10 September 2010 (n ¼ 8) were collected and stored at 25
C for 72 h. The degree of proteolysis in the skim milk and fat
Received in revised form globule protein fractions, protein oxidation and milk lipase activity were measured at regular time
14 December 2010
intervals. b-casein was rapidly hydrolyzed, with 38  5% (mean  SEM) remaining after 72 h. The
Accepted 20 December 2010
immunological proteins, secretory IgA and lactoferrin, appeared to be resistant to proteolysis. The lipase
activity was reduced after 2 h of storage (p < 0.001). Protein oxidation was not observed. These results
highlight the importance of appropriate milk handling when analyzing the protein composition of milk
in a laboratory. Further investigation is required to determine whether the observed effects of storage
upon milk proteins are of clinical importance.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction major milk proteins qualitatively using SDS-PAGE and reported no

The effect of storage conditions upon the quality of human milk
is an important consideration. In developed countries, many
mothers now return to paid work whilst breast-feeding, and
express and store milk for later use in infant feeding. Milk storage is
also important for mothers who deliver prematurely. Fortified
mothers’ own milk is the optimal source of nutrition for preterm
infants (Schanler, 2001) and thus these mothers are encouraged to
express and store milk for enteral feeding of their infants. In both of
these instances, milk may be held for variable periods of time at
ambient temperature, prior to infant feeding or longer-term storage
at either 4
C or 20
C.
The effect of storage on the microbiological content (Hamosh,
Ellis, Pollock, Henderson, & Hamosh, 1996), lipid composition
(Bitman, Wood, Mehta, Hamosh, & Hamosh, 1983), cellular
components (Pittard & Bill, 1981), anti-bacterial properties
(Lawrence, 1999) and antioxidant capacity (Hanna et al., 2004) of
breast milk has been well documented. Milk proteins also merit
attention with regard to storage. There are many proteins in human
milk that perform a range of immunological, nutritional and
developmental functions (Alvarez, 2007). However, protein func-
tion may be lost during storage, through mechanisms such as
proteolysis or oxidative damage.
There have been conflicting findings in the literature regarding
proteolysis in human milk. Hamosh et al. (1996) examined the
* Corresponding author. Tel.: þ61 8 6488 4428; fax: þ61 8 6488 7330.
E-mail address: molinc01@student.uwa.edu.au (C.E. Molinari).
proteolysis of these proteins after 24 h of storage at a range of
temperatures, including at 25
C. However, a number of studies
have reported that the casein fraction of human milk is highly
susceptible to plasmin-mediated proteolysis during storage
(Chtourou, Brignon, & Ribadeau-Dumas, 1985; Ferranti et al., 2004).
Furthermore, there have been no reports on whether the proteins
associated with the milk fat globule undergo proteolysis under
these conditions.
Other milk components including lipids are oxidized during
storage (van Zoeren-Grobben, Moison, Ester, & Berger, 1993) and it
has been shown that protein oxidation in general can result in
altered protein function, increased protein breakdown, and struc-
tural changes that affect protein digestibility (Grant, Jessup, & Dean,
1993; Nystrom, 2005). It is not known if protein oxidation is
functionally important in human milk, but the antioxidant capacity
and the levels of glutathione in human milk are reduced after as
little as 2 h storage at 25
C, rendering stored human milk less
protected against protein oxidation (Ankrah, Appiah-Opong, &
Dzokoto, 2000; Hanna et al., 2004). Furthermore, lactoperoxidase,
an anti-bacterial enzyme, is capable of oxidizing proteins in bovine
milk (Ostdal, Bjerrum, Pedersen, & Andersen, 2000) and, although
less active in human milk (Gothefors & Marklund, 1975), provides
a potential mechanism for protein oxidation.
Consideration of milk storage conditions is also important
within a laboratory setting. During sample collection, transport,
and preparation, there are significant practical advantages to being
able to hold milk at ambient temperatures for short periods of time.
However, in order to gain meaningful experimental information, it
is critical that the structure and function of the proteins of interest

0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2010.12.001
 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293
are not affected by these processes. There have been indications
that different laboratory storage and handling conditions may
affect the outcomes of studies examining human milk proteins
(Chandan, Shahani, & Holly, 1964; Ferranti et al., 2004), and
therefore further research in this area is warranted.
In the present study, the effect of storage at ambient tempera-
ture (25
C) on the protein quality of human milk after 2, 4, 8, 24, 48
and 72 h was investigated. The degree of proteolysis of both skim
milk and fat globule associated proteins, thiol oxidation, protein
carbonylation, and reduction in lipase activity were measured.
2. Materials and methods
2.1. Chemicals
Unless otherwise stated, all chemicals and reagents were
obtained from SigmaeAldrich (NSW, Australia).
2.2. Subjects and milk collection
Milk samples were obtained from healthy lactating mothers
through the Breastfeeding Centre at King Edward Memorial
Hospital, Subiaco, Western Australia. Mothers were at different
stages of lactation (between 3 and 21 weeks postpartum), and their
nipples were cleaned prior to expression. For the analysis of skim
milk proteins, 1 mL samples were collected by manual expression
from a single breast at the end of a feed. For the analysis of fat
globule proteins, milk was expressed using an electric pump,
mixed, and a 10 mL aliquot collected from the total expression
volume. For the purposes of analyzing protein oxidation, control
milk samples were collected directly from the surface of the nipple
using a 1.0 mL plastic syringe, excluding air, and the oxidative state
was trapped by adding one volume of milk to four volumes of 20%
(w/v) trichloroacetic acid (TCA)/acetone. Milk samples were
transported to the laboratory within 30 min of expression and the
time of arrival designated t ¼ 0 h.
All mothers gave written consent to participate in the research,
and the study was approved by The University of Western Australia,
Human Research Ethics Committee.
2.3. Storage protocol
Milk samples from 8 mothers were used for the analysis of
skim milk proteins. One aliquot from each milk sample was
immediately frozen at 80
C. The remainder of each milk sample
was divided into two treatment groups. A mammalian protease-
inhibitor cocktail containing 4-(2-aminoethyl)benzenesulfonyl
fluoride, E-64, bestatin, leupeptin, aprotinin, sodium EDTA was
added to samples in Group 1, and Group 2 samples were left
untreated. Both treatment groups were incubated at 25
C and
after 2, 4, 8, 24, 48, and 72 h, they were mixed, and aliquots
immediately frozen at 80
C.
For the analysis of milk fat globule proteins, milk samples from
5 different mothers were used. The mammalian protease-inhibitor
cocktail was added to two samples and three were left untreated.
Immediately, aliquots were removed from each sample and frozen
at 20
C for 7 d. The remaining milk from each of the five samples
was incubated at 25
C and aliquots removed after 0, 8, 24, and
48 h. The fat globule membrane proteins were isolated from each
aliquot using the double extraction protocol described by
Quaranta et al. (2001). All containers used to store milk were
made of polypropylene.
287
2.4. Protein concentration
Protein concentrations were determined using a Bicinchoninic
acid kit (Sigma-Aldrich, NSW, Australia). Human milk standards
were used to calibrate the measurements as previously described
(Mitoulas et al., 2002). The recovery of a known amount of protein
added to the milk samples was 99(SEM 1.03)% (n ¼ 12), the inter-
assay CV was 5.0%, and the detection limit was 0.015 g L1.
2.5. SDS-PAGE and image analysis
A fluorescently labeled human serum albumin standard was
prepared by incubating human serum albumin with BODIPYÒFL
N-(2-aminoethyl)maleimide (Invitrogen, Carlsbad, CA, USA), and
precipitating the labeled protein with ethanol. The fluorescently
labeled protein was washed twice in ethanol, and resuspended
in 8 M urea, 4% 3-((3-cholamidopropyl)dimethylammonio)-1-
propanesulfonic acid (CHAPS).
Milk samples were thawed and after mixing, spun at 10,000 g
for 10 min and the fat discarded. Equal volumes of the samples
within each time course (corresponding to 30 mg of the sample
collected at t ¼ 0), were each mixed with 2 ng of the fluorescently
labeled serum albumin standard and analyzed using SDS-PAGE.
This amount of fluorescently labeled standard was too low to be
visible with Coomassie staining. Both the skim milk proteins and fat
globule associated proteins were analyzed in this way.
SDS-PAGE analysis was conducted as described by Laemmli
(1970) using the HOEFER gel apparatus (HOEFER Scientific Instru-
ments, San Francisco, CA, USA). Fifteen micrograms of the protein
mixture containing the fluorescent standard were loaded into each
lane of a 12.5% acrylamide gel. Samples were run in duplicate and 2
samples collected at t ¼ 0 were included on each gel. Gels were run
at a constant current of 15 mA for 16 h at 4
C and then immediately
scanned using a Typhoon Trio variable mode imager (GE Healthcare
Biosciences, Pittsburgh, PA, USA) with an excitation wavelength of
488 nm.
The gels were fixed for 2 h in a solution containing 50% (v/v)
methanol and 10% (v/v) TCA, washed using double deionised water,
stained using Coomassie Brilliant Blue R-250 overnight, and scan-
ned using a flatbed light scanner (Epson, Nagano, Japan). Gel
images were analyzed using the open access software package
ImageJ 1.410 (Abramoff, Magelhaes, & Ram, 2004). Seven clearly
defined protein bands were selected for analysis. The intensity of
each band and the internal fluorescent standard in each lane were
measured. The variation in sample loading between wells was
accounted for by calculating a corrected intensity:
Corrected intensity ¼ intensity of protein band/
intensity of internal standard (1)
To enable comparisons between gels, the intensity of each
protein band in the t ¼ 0 samples were set at 100%, and all corre-
sponding bands on the same gel were expressed relative to this
value.
Relative intensity ¼
(corrected intensity of protein band)/
(corrected intensity of protein band in sample t ¼ 0)  100 (2)
All gels were run in duplicate, and contained duplicates of each
sample. Measurements of the intensity of each band using ImageJ
were also made in duplicate.
The average coefficient of variance (CV) between repeat intensity
measurements of the same protein band was 4.5% (n ¼ 2016). The
within-gel variability, given by the average CV of the fluorescent
288 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293

standards on each gel, was 8.0% (n ¼ 16 gels). The between-gel
variability, given by the average CV of the relative intensities of
duplicate samples run on different gels, was 9.7% (n ¼ 56).
2.6. Two-dimensional gel electrophoresis (2DE)
Prior to analysis, protein samples were reduced with 6 mM Tris
(2-carboxyethyl)phosphine (TCEP) for 210 min and alkylated with
50 mM iodoacetamide for 30 min. Protein samples were then
precipitated using ethanol, and resuspended in an isoelectric
focusing (IEF) rehydration buffer containing 8 M urea, 4% CHAPS, 2%
IPG buffer pH 3e10, and 3% tributyl phosphine (TBP). Two-DE was
carried out according to the method of Lui, Lipscombe, and Arthur
(2009). The gels were fixed, stained and scanned as in Section 2.5.
2.7. Mass spectrometry and protein identification
Bands and spots of interest were cut from the gel, destained and
digested in-gel with trypsin (Roche Diagnostics, Castle Hill, NSW,
Australia) as described by Shevchenko, Tomas, Havlis, Olsen, and
Mann (2007). For mass spectrometric analysis, 1 mL of each
peptide mixture was mixed with 1 mL of matrix solution (1%, w/v, a-
cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile (ACN),
0.1% trifluoroacetic acid (TFA)) and applied to a target plate. Mass
spectra were obtained using an UltraFlex III MALDI-TOF/TOF
instrument (Bruker Daltonics, Bremen, Germany) in the mass range
800e4000 Da. A laser intensity of 50% was used, with 400 shots
fired at each spot. Up to 20 of the most intense peaks in each
spectrum meeting the criteria of intensity >300, S/N > 6 were
selected for subsequent MS/MS analysis. For MS/MS analysis, a laser
intensity of 70% was used, with 250 shots fired at each spot.
MS/MS data was imported into the database search engine
(Mascot, Version 2.3.01, www.matrixscience.com). Mascot
searches were conducted using the SwissProt Mammalia database
(49,887 sequences) with the following settings: number of missed
cleavages permitted ¼ 1; no fixed modifications; variable
modifications ¼ methionine oxidation, cysteine carbamidome-
thylation; peptide tolerance ¼ 1.2 Da, MS/MS tolerance ¼ 0.6 Da;
enzyme ¼ trypsin; and the peptide charge ¼ þ1. A Mascot score
greater than 50 with a minimum of two peptide matches was
considered to be a significant identification.
7.2 for 2 h at 37
C. Western blot analysis was also performed, as
described by Shacter, Williams, Lim, and Levine (1994).
2.10. Statistical analysis
Statistical analysis was carried out using the statistics package R
(R Development Core Team, 2008). The packages nlme (Pinheiro,
Bates, DebRoy, Sarkar, & R Development Core Team, 2009) and
multcomp (Hothorn, Bretz, & Westfall, 2008), were used for linear
mixed effects modeling and Tukey’s contrast analysis, respectively.
Univariate analysis for variables collected at multiple time points
was performed using a linear mixed models approach. This method
is considered appropriate, given the variation between different
milk samples and the repeated measures in the data. All models
included the individual milk samples as the grouping. For the
proteolysis analysis, analysis of variance of the fitted model objects
found that a grouping variable of the duplicate gels within the data
from each milk sample did not improve the models.
Values are presented as mean (SEM) unless otherwise specified.
p-Values < 0.05 were considered statistically significant and values
smaller than 0.001 have been reported as p < 0.001.
3. Results
3.1. SDS-PAGE analysis
The most abundant protein bands from both the skim milk
fraction (Fig. 1) and the fat globule protein fraction (Fig. 2) were
selected for analysis. In the skim milk and fat globule protein
fractions, the intensity of these bands represented 85(5)% and 33

2.8. Lipase activity assay

Lipase content of human milk was measured using a Lipase

Assay Kit (QuantichromÔ Lipase Assay, Bioassay Systems, Hayward
CA, USA), according to the manufacturers instructions. The CV of all
duplicate measurements was less than 10%, and the inter-assay CV
was 4.4%.

2.9. Protein oxidation assays

The reduced thiol content of human milk was measured using

Ellman’s assay (Riener, Kada, & Gruber, 2003) in a 96-well format.
To measure the total thiol content, TCEP (4 mM) was added to
protein samples and incubated at room temperature for 60 min to
reduce the disulfide bonds. The TCEP was then removed by
precipitation with ethanol, and the thiol content measured as
above. The recovery of the addition of known amount of reduced
cysteine to the milk protein samples was 99(SEM 2.0)% (n ¼ 8). The
detection limit of this assay was 3.9 mM (n ¼ 8).
Protein carbonylation was determined using the spectrophoto-
Fig. 1. SDS-PAGE electrophoretograms of skim milk protein in aliquots removed at
metric method described by Hawkins, Morgan, and Davies (2009). regular intervals over 72 h (0, 2, 4, 8, 24, 48 and 72 h) from fresh milk incubated at
For use as a positive control, 1 mg of skim milk protein was incu- 25
C. Seven protein bands were identified using MALDI-TOFeTOF MS. The gel image is
bated with 200 mM H2O2, 25 mM ascorbate, and 100 mM FeSO4 at pH from a single representative milk sample.
 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293 289

the intensity of the LF band was reduced (p ¼ 0.03), however the

Fig. 2. SDS-PAGE electrophoretograms of milk fat globule membrane proteins, in
aliquots removed at regular intervals from fresh milk incubated at 25
C for 48 h (0, 8,
24, and 48 h), and after freezing (Fr) for 7 days at 20
C. Seven protein bands were
identified using MALDI-TOFeTOF MS. The gel image is from a single representative
milk sample.
(6)% of the total protein intensity, respectively. These were unam-
biguously identified by mass spectrometry (Table 1).
3.2. Skim milk protein analysis
In the fresh milk samples, there was no evidence for proteolysis
of lactoferrin (LF), serum albumin (SA) (Fig. 3B, D), secretory IgA
(sIgA) or a-lactalbumin (LA) (data not shown) after 24 h. At 48 h,
loss was slight, with 89(3)% remaining. Even at 72 h, 87(6)% of the
intact LF remained. Similarly, for SA, there was a reduction in
intensity seen only at 72 h (p ¼ 0.04), with 90(6)% of the intact
protein remaining. For sIgA and LA, there was no significant
reduction in protein intensity even after 72 h incubation. The
intensity of the bile-salt stimulated lipase (BSSL) band was reduced
(p ¼ 0.004) after 4 h with 85(4)% remaining. At 72 h, 72(5)% of the
intact BSSL remained (Fig. 3A).
The b-casein band at 30 kDa was rapidly hydrolyzed during
incubation at 25
C, with a reduction in intensity (p < 0.001)
observed after 4 h, and only 38(5)% remaining after 72 h (Fig. 3E).
Transient low molecular mass b-casein bands were observed in the
10e30 kDa molecular mass region between 4 and 72 h (Fig. 1). The
intensity of the major hydrolysis product (16 kDa), already present
at 0 h, increased until 24 h, and then returned to its original level by
48 h (Fig. 3C).
A qualitative examination of the 2DE gels provided a more
detailed image of the pattern of casein hydrolysis. There was an
abundance of protein spots in the 10e30 kDa region after 72 h of
storage at 25
C, which were not visible in the control sample at 0 h
(Fig. 4A, B). All of the additional spots in this region that could be
identified by MALDI-TOF-TOF MS, were b-casein hydrolysis prod-
ucts. The same two b-casein peptides were detected in each protein
spot, SPTIPFFDPQIPK (120e132, MW 1486.794) and VLPIPQQVV-
PYPQR (176e189, MW 1633.942), and therefore it was not possible
to determine any structural differences between the b-casein spots.
The addition of the protease inhibitor cocktail was effective at
preventing proteolysis from occurring. In the SDS-PAGE analysis, no
proteolysis was observed for any of the protein bands when
protease inhibitors were added (Fig. 3). Using 2DE, a small number
of additional protein spots in the 10e30 kDa region in the sample
containing protease inhibitor were visible after 72 h relative to the
control sample (Fig. 4A, C); however there were far fewer spots
than were present in the corresponding untreated milk sample
(Fig. 4B).
Different rates of hydrolysis were observed in each of the milk
samples. This was most apparent for the 30 kDa b-casein band.
After 8 h of storage at 25
C, the percentage of b-casein remaining
varied between 60 and 90% for the different milk samples, and,
after 72 h, this range increased to between 0 and 60% (Fig. 3F).
In summary, it was found that proteolysis does occur in milk
samples during storage at 25
C. b-Casein is rapidly hydrolyzed into
multiple different products. BSSL, LF and SA also undergo hydrolysis,

Table 1
Identification of proteins separated on electrophoresis gels.

Figure Protein identification Mass from Uniprot Mascot score Sequence Peptides
gel (kDa) number coverage
Fig. 1 Bile-salt stimulated lipase 120 P19835 90.61 2% 2
Lactoferrin 85 P02788 185.72 8% 5
Serum albumin 65 P02768 415.32 18% 10
Ig alpha-1 chain C region 60 P01876 86.21 6% 2
b-Casein 30 P05814 160.78 11% 2
b-Casein 16 P05814 140.04 11% 2
a-Lactalbumin 14 P00709 105.04 12% 2
Fig. 2 Xanthine dehydrogenase/oxidase 148 P47989 147.52 5% 6
Butyrophilin 63 Q13410 210.33 8% 4
Butyrophilin 60 Q13410 54.88 3% 2
Adipophilin 48 Q99541 103.06 15% 6
Lactadherin EGF-8 43 Q08431 218.98 16% 5
b-Casein 30 P05814 152.52 11% 2
Fatty-acid-binding protein 14 P05413 98.10 18% 2
Fig. 4 b-Casein 10e30 P05814 63e180 11% 2
a-Lactalbumin 14 P00709 85e110 12% 2
as1-Casein 24e26 P44710 72e110 12e15% 3e4
290 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293

Fig. 3. Changes in skim milk protein intensity over 72 h incubation at 25
C. Panels AeE are, respectively, the 120 kDa bile-salt stimulated lipase band, the 65 kDa serum albumin
band, the 16 kDa b-casein peptide band, the 85 kDa lactoferrin band, and the 30 kDa b-casein band. Closed squares (-) are fresh milk samples, open circles (B) are fresh milk
samples treated with protease inhibitor. All values given are the mean  SEM (n ¼ 8); values in the fresh milk samples that are significantly different from the intensity present at
0 h, are denoted by *(p < 0.05), and **(p < 0.001). Panel F shows the hydrolysis of the 30 kDa b-casein band in milk samples from 8 different mothers. Each line represents
a different milk sample.

but to a much lesser degree. Protease-inhibitor treatment was
effective at preventing most of the proteolytic activity.
3.3. Milk fat globule membrane protein analysis
No difference was found between the fresh milk samples and
those treated with protease inhibitor, for any of the protein bands
measured across the 5 time points (p ¼ 0.21). Therefore, all milk
samples were grouped together for statistical analysis. The band
intensity of butyrophilin, adipophilin, lactadherin EGF-8 and
b-casein did not change over either the 48 h incubation or with
freezing (Fig. 2). The xanthine dehydrogenase/oxidase (XDH) band
at 148 kDa was reduced after 48 h, with 80(1)% remaining
(p ¼ 0.01) (Fig. 5A). No difference was observed for XDH with
freezing.
The fatty-acid-binding protein (FABP) band at 14 kDa was also
reduced during storage at 25
C (p ¼ 0.02). By 48 h of incubation,
the intensity of this band was reduced to 51(10)% of its original level
(Fig. 5B). By freezing the sample, this effect was more pronounced,
with only 13(5)% of the FABP remaining.
The intensity of the b-casein band at 30 kDa was the most
variable of all the bands measured, showing no consistent trend in
intensity over the storage period in any of the milk samples.
3.4. Determination of lipase activity
The activity of lipase in the 0 h samples was 53(11) U mL1
(n ¼ 5). After 2 and 4 h, 79(5)% (p ¼ 0.027) and 65(10)% (p < 0.001),
respectively, of the lipase activity remained. After 4 h, the lipase
activity remained stable up to 72 h (p > 0.31).
3.5. Protein oxidation
No oxidation of protein thiols was observed over the 72 h incu-
bation period. In addition, the thiol groups in human milk proteins
were almost exclusively in the oxidized disulfide form upon
expression from the breast. The milk samples collected anaerobically
and analyzed immediately had a negligible percentage of their total
thiols in the reduced form, i.e., 1.4(1.5)%. There was no difference in
the level of either reduced or total protein thiols seen over time. With
the exception of the oxidized control sample, carbonylation of protein
was not detected in any of the samples analyzed, using either the
spectrophotometric assay or the more sensitive Western blot
analysis.
4. Discussion
Previous studies investigating the stability of human milk
proteins during storage have either used a global approach,
measuring changes in the concentration of total protein nitrogen in
milk (Hamosh et al., 1996), or a more focused approach, considering
only a small number of specific proteins (Elmlinger et al., 1999;
Ferranti et al., 2004). Using the gel-based method in the present
study, we were able to measure the effect of proteolysis on each of
the major proteins in human milk, accounting for more than 85% of
the total proteins in skim milk and 33% of the total proteins asso-
ciated with the milk fat globule. Therefore, the present study
represents the most robust and comprehensive analysis to date of
how human milk proteins are affected by proteolysis during storage
at 25
C.
It was found that the major immunological proteins LF and sIgA
were resistant to proteolytic breakdown during 24 h of storage at
25
C, with sIgA also resisting breakdown after 72 h. These results
support previous studies that have found that the heavy glycosylation
 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293
Fig. 4. Two-dimensional electrophoresis gels of skim milk proteins 10e30 kDa (A) 0 h
control sample, (B) fresh milk sample after 72 h incubation at 25
C, (C) protease-
inhibitor-treated fresh milk sample after 72 h incubation at 25
C. All aliquots analyzed
were from the same milk sample. Significant protein identifications are denoted by (1)
b-casein P05814 (2) a-lactalbumin P00709 (3) as1-casein P47710.
of these proteins confers protection against enzymatic breakdown
(Arnold, Wormald, Sim, Rudd, & Dwek, 2007). Indeed, human milk LF
and sIgA have been found in their intact form in infant stools and
urine (Brines & Brock, 1983; Goldman, Garza, Schanler, & Goldblum,
1990). Brines and Brock (1983) suggested that this resistance to
digestion may be an evolutionary development designed to preserve
the activity of immunological proteins throughout the infant gut.
BSSL did undergo proteolysis during storage, with a 20%
decrease in intensity seen after 4 h. Due to the importance of lipase
for the digestion and absorption of triacylglycerols by the infant
(Hernell, Blackberg, & Bernback, 1989), and its known sensitivity to
milk storage and treatment procedures (Berkow et al., 1984; Czank,
Simmer, & Hartmann, 2010), the lipase activity in the stored milk
291
Fig. 5. Changes in milk fat globule membrane protein intensity over 48 h incubation at
25
C, and with freezing for 7 days at 20
C: panel A, Xanthine dehydrogenase; panel
B, fatty-acid-binding protein. All values given are the mean  SEM (n ¼ 5). Values that
are significantly different to the intensity at 0 h are denoted by *(p < 0.05), and
**(p < 0.001).
samples was also measured, to assess how it was affected by the
observed proteolysis of BSSL. We found lipase activity to also be
reduced by milk storage at 25
C, although to a greater extent than
could be directly attributed to the proteolytic breakdown. This
suggests that while proteolysis of BSSL may be partly responsible
for the loss of lipase activity, it is unlikely to be the sole mechanism.
A greater loss of activity was found than was observed in a previous
study; however, it is possible that this discrepancy is due to the
different stages of lactation at which the milk was collected
(Hamosh, Henderson, Ellis, Mao, & Hamosh, 1997).
Ferranti et al. (2004) described the enzymatic pathways leading
to casein breakdown in human milk and, certainly in the present
study, the susceptibility of b-casein to proteolysis was evident. After
4 h storage at 25
C, the decrease in intensity of intact b-casein and
the concomitant increase in intensity of b-casein hydrolysis prod-
ucts at 16 kDa were clearly apparent. A significant proportion of the
b-casein remains in fragments greater than 10 kDa during the
incubation period, which may explain why studies using the levels
of total protein nitrogen to indicate protein breakdown did not find
any evidence of proteolysis (Garza, Johnson, Harrist, & Nichols,
1982; Hamosh et al., 1996).
The primary role of b-casein for the infant is nutritional, acting
in its micellar form as a source of amino acids, calcium and phos-
phate. Hydrolysis of b-casein prior to infant feeding does not alter
the delivery of these nutrients. Indeed, it has been suggested that
partially hydrolyzed casein in milk may facilitate digestion, and
represent an advantage for the immature digestive system of
newborn infants (Armaforte et al., 2010; Ferranti et al., 2004). In
addition to this nutritional role, b-casein also possesses bioactive
properties. A large number of small peptides that exhibit bioactivity
in vitro have been found in the b-casein sequence. These include
the opioid agonists b-casomorphin-7 and b-casomorphin-5 (Meisel
& FitzGerald, 2000), and immunomodulatory peptides (Parker
et al., 1984). Partial hydrolysis of b-casein during storage of milk
could potentially lead to an increase in the concentration of these
peptides in human milk. Further investigation into the action
of these peptides in vivo is required before the physiological
significance of hydrolysis of casein during milk storage can be
determined.
The hydrolysis of b-casein during storage is also an important
consideration when conducting human milk protein analyses in
a laboratory. Variability in the degree of casein hydrolysis has made
attempts to quantify the levels of casein in human milk problematic
(Chtourou et al., 1985; Ferranti et al., 2004), and rendered it difficult
to obtain an efficient separation of the whey and casein fractions
(Ferranti et al., 2004; Kunz & Lonnerdal, 1989, 1992). Hydrolysis of
292 C.E. Molinari et al. / International Dairy Journal 21 (2011) 286e293
casein also creates difficulties for proteomic studies of human milk.
The abundance of b-casein and its hydrolysis products between 10
and 30 kDa obscures the lesser abundant proteins within this same
molecular weight region (Fig. 4). Although not observed in this
study, it is likely that hydrolysis of as1-casein is also significant
(Armaforte et al., 2010), and may contribute to the difficulties
associated with casein quantification and identifications of lesser
abundant protein species. The present study indicates that the
addition of protease inhibitors to human milk upon expression can
prevent the majority of the casein hydrolysis over 72 h storage at
25
C (Fig. 3). Therefore, it recommended that, when conducting
proteomic analyses of human milk, the addition of protease
inhibitors become a routine part of sample treatment and prepa-
ration. The addition of protease inhibitors also merits consideration
in all analyses of human milk for which the hydrolysis of casein may
be a confounding factor.
The fat globule associated proteins, butyrophilin, lactadherin,
XDH and adipophilin were all resistant to proteolysis during
storage at 25
C for 24 h and with freezing. However, the level of
FABP associated with the fat globule membrane was significantly
reduced with increased storage time (Fig. 5B). A proteolytic
mechanism is not supported, as no differences were observed with
the addition of the protease-inhibitor cocktail. However, it is
possible that the protease inhibitors used were unable to access the
hydrophobic fat globule membrane environment. Further investi-
gation is required to determine whether the loss of FABP from the
fat globule membrane is due to proteolysis, or whether it disasso-
ciates from the membrane and is released into skim milk during
storage. This change in location may be related to the affinity of
FABP to the increased levels of free fatty acids that are produced
during milk storage, and/or to structural changes in the milk fat
globule membrane. Certainly, both of these phenomena have been
shown to occur during milk storage, and to be accelerated by the
freezing and thawing process (Chappell, Clandinin, & McVey, 1985;
Wardell, Hill, & D’Souza, 1982).
A number of studies have identified skim milk proteins in
extracted milk fat globule protein (Caveletto, Giuffrida, & Conti,
1999; Quaranta et al., 2001; Schroten et al., 1999). This observa-
tion has been attributed to the presence of water-soluble proteins
in signets, which are small portions of cytoplasm that become
entrained between the outer membrane and the fat droplet during
milk fat globule formation (Patton & Huston, 1988). Alternate
explanations have also been given, with the presence of skim milk
proteins either described as an interaction between exposed
hydrophobic portions of the water-soluble proteins with the
hydrophobic membrane (Shimizu, Kamiya, & Yamauchi, 1981), or
described as an artifact of incomplete washing of the skim milk
components from the membrane fraction (Patton & Keenan, 1975).
The inconsistency in the levels of b-casein associated with the fat
globule membrane in the present study is consistent with the latter
explanation, and indicates that the efficiency of the washing steps
may not have been identical for each sample analyzed. However,
this does not preclude the possibility of small amounts of b-casein
and other skim milk proteins being constitutively associated with
the fat globule membrane.
Protein thiol oxidation was not observed during storage at 25
C,
as the protein thiols were already in the oxidized disulfide form
upon milk expression. Disulfide bond formation is a reversible
modification and it is possible that the oxidized disulfide form is
protective, as it prevents further thiol oxidation to sulfonic acid
from occurring (Mallis et al., 2005). If thiol groups were to be
irreversibly oxidized to the sulfonic acid form, they would not be
available for cellular metabolic processes, and their function may
be affected. Protein carbonylation, another irreversible protein
modification, was also not observed. Given that small antioxidant
molecules are readily oxidized and the total antioxidant capacity
reduced during storage at room temperature (Hanna et al., 2004;
Romeu-Nadal, Castellote, & Lupez-Sabater, 2008), evidence of
protein carbonylation would not have been surprising. The fact that
no carbonylation was observed again points toward the stability of
milk proteins.
5. Conclusions
The results of this study indicate that, with the exception of
b-casein, the major proteins in human milk are relatively resistant to
proteolysis and oxidation during storage at room temperature.
Nevertheless, it is apparent from the analysis of lipase, that the
activity of individual proteins can each be affected differently by milk
storage. Therefore, when studying particular proteins of biological
interest, storage conditions require careful consideration. Further
investigation is required to determine whether the observed effects
of short-term storage at 25
C upon milk proteins are of clinical
importance and their implications for short-term milk storage in
both home-based and hospital settings.
Acknowledgements
This study was supported financially by Medela AG
(Switzerland). The authors have no conflict of interest regarding the
content of this manuscript.
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