The Journal of Maternal-Fetal & Neonatal Medicine

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The human milk oligosaccharides are not affected

by pasteurization and freeze-drying

Won-ho Hahn, Jaehan Kim, Seunghyun Song, Suyeon Park & Nam Mi Kang

To cite this article: Won-ho Hahn, Jaehan Kim, Seunghyun Song, Suyeon Park & Nam Mi Kang
(2017): The human milk oligosaccharides are not affected by pasteurization and freeze-drying, The
Journal of Maternal-Fetal & Neonatal Medicine, DOI: 10.1080/14767058.2017.1397122

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Published online: 06 Nov 2017.

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Download by: [University of New England] Date: 08 November 2017, At: 07:18
 THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE, 2017
[Link]

ORIGINAL ARTICLE

The human milk oligosaccharides are not affected by pasteurization and

freeze-drying
Won-ho Hahna, Jaehan Kimb, Seunghyun Songc, Suyeon Parkd and Nam Mi Kange
a
Department of Pediatrics, Seoul Hospital, School of Medicine, Soon Chun Hyang University, Seoul, Republic of Korea; bDepartment of
Food and Nutrition, College of Human Ecology, Chungnam National University, Daejeon, Republic of Korea; cDepartment of Political
Sciences, Faculty of Social Sciences, University of Antwerp, Antwerp, Belgium; dDepartment of Biostatistics, Soon Chun Hyang Medical
Center, Seoul, Republic of Korea; eDepartment of Nursing, Konkuk University, Chungju, Republic of Korea

ABSTRACT ARTICLE HISTORY

Objectives: Human milk oligosaccharides (HMOs) are known as important factors in neurologic Received 11 July 2017
and immunologic development of neonates. Moreover, freeze-drying seems to be a promising Revised 19 October 2017
Downloaded by [University of New England] at 07:18 08 November 2017

storage method to improve the processes of human milk banks. However, the effects of pasteur- Accepted 23 October 2017
ization and freeze-drying on HMOs were not evaluated yet. The purpose of this study is to ana-
lyze and compare the HMOs profiles of human milk collected before and after the pasteurization KEYWORDS
and freeze-drying. Freeze-drying; human milk;
Methods: Totally nine fresh human milk samples were collected from three healthy mothers at mass spectrometry;
the first, second, and third week after delivery. The samples were treated with Holder pasteuriza- oligosaccharides;
tion and freeze-drying. HMOs profiles were analyzed by matrix-assisted laser desorption/ioniza- pasteurization
tion (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry and compared between
samples collected before and after the treatments.
Results: Human milk samples showed significantly different HMO patterns between mothers.
However, HMOs were not affected by lactation periods within 3 weeks after delivery
(r2 ¼ 0.972–0.999, p < .001). Moreover, both of pasteurization and freeze-drying were found not
to affect HMO patterns in a correlation analysis (r2 ¼ 0.989–0.999, p < .001).
Conclusion: HMO patterns were found not to be affected by pasteurization and freeze-drying of
donor milks. We hope that introducing freeze-drying to the human milk banks would be encour-
aged by the present study. However, the storage length without composition changes of HMOs
after freeze-drying needs to be evaluated in the further studies.

Introduction vulnerable neonates is recommended as a next best

option [10]. The main processes in the human milk
Human milk oligosaccharides (HMOs) were suggested
banks include Holder pasteurization and freezing for
to play important roles in the development of immune
and nervous systems in neonates [1–5]. HMOs were the safety of donated human milks. Even though the
found to affect the composition of intestinal probiotic macronutrients are generally known not to be
bacteria leading to well-balanced gut microflora envir- affected, some biological, immunologic, and anti-
onment that is important for the development of infective factors are found to be destructed during the
infantile immune system [2,3,6]. The antibacterial prop- processes [10].
erties of HMOs were also suggested by mimicking the Interestingly, there were some attempts to evaluate
natural ligands of various bacteria inhibiting their the effectiveness of freeze-drying of human milks to
adhesion [3,7]. Moreover, the sialic acid containing minimize the loss of nutrients and immunologic fac-
HMOs were found to be important for neurodevelop- tors with fewer resources for longer storages and eas-
ment possibly by increasing synaptogenesis [8,9]. ier transports [11–14]. According to the studies, it was
Human milk feeding is well-known gold standard to suggested as one of the most promising storage
provide optimal nutrition and immunological protec- methods for human milk banking in the future.
tion for the infants. However, when own mother’s milk However, no study investigated the effects of pas-
is not available, providing the donated human milk to teurization and freeze-drying on the HMOs. Thus, we

CONTACT Nam Mi Kang nmkang03@[Link] 268 Chungwondaero, Chungcheongbuk-do, College of Nursing, Konkuk University, Chungju-si,
Republic of Korea
Supplemental data for this article can be accessed here.
! 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 W.-H. HAHN ET AL.

conducted this study to evaluate the changes of the at 13,000 rpm for 30 min at 4 # C. Carefully extract the
HMOs after these treatments. middle layer into a 15 mL centrifuge tube and add
four volumes of chloroform/methanol (2:1, v/v). The
mixture was centrifuged at 4000 % g for 30 min at 4 # C.
Methods
The aqueous layer was recovered and subjected to an
Milk samples ethanol precipitation for removal of protein. Proteins
Human donor milk was obtained from the mothers who were precipitated at "40 # C for 1 h. After 30 min of
agreed to participate in the present study in April 2016. centrifugation at 4000 % g at 4 # C, the upper liquid
This study was approved by the ethics review commit- fraction was collected and dried to completion using a
tee of the Medical Research Institute, Konkuk University speed vacuum.
hospital (KUH-7001355-201511-HR-093). Inclusion crite-
ria were the milks donated from nonsmoking healthy Reduction and purification of HMOs
mothers who delivered their baby vaginally without
Dried samples dissolved in 0.5 mL of deionized water.
positive results in the serologic tests for hepatitis B virus,
Fifty microliters of aqueous HMOs were reduced to an
syphilis and human immunodeficiency virus, and who
alditol form by adding 1.0 M NaBH4 and incubating at
Downloaded by [University of New England] at 07:18 08 November 2017

followed the nutritional instructions for breastfeeding

65 # C heating block for 1 h followed by solid-phase
mothers [15]. Totally nine fresh human milks were col-
extraction (SPE) using a PGC cartridge to remove bor-
lected from three mothers at the first, second, and third
ate salts and fractionate the mixtures. Prior to use, the
week after delivery (periods 1, 2, and 3, respectively).
porous graphitized carbon-solid-phase extraction (PGC-
The mean age of mothers was 30.0 ± 4.4 years. Each col-
SPE) cartridge was washed with 80% acetonitrile in
lected sample was divided into four plastic tubes con-
0.1% trifluoroacetic acid (TFA) (v/v) followed by deion-
taining 20 mL of human milks; (1) non-pasteurized/not
ized water. After loading of the milk oligosaccharide
freeze-dried (liquid), (2) pasteurized/not freeze-dried
mixture onto a cartridge, salts were removed by wash-
(liquid), (3) non-pasteurized/freeze-dried (powder), and
ing with several cartridge volumes of deionized water.
(4) pasteurized/freeze-dried (powder). Totally 12 sam-
The milk oligosaccharides were fractionated using 20%
ples were sent for the HMO analysis.
acetonitrile in water (v/v) and 40% acetonitrile in
The samples were frozen and stored at recom-
0.05% TFA (v/v) as eluting solvents. Each fraction was
mended temperature of "80 # C after each treatment.
collected and evaporated in vacuo prior to mass spec-
Pasteurization of donor milk was performed at the rec-
trometry (MS) analysis.
ommended temperature of 62.5 # C for a period of
30 min prior to its use (Holder pasteurization method).
Freeze-drying was performed for 24 h with 5 mL of Mass spectrometry analysis of HMOs using
milk samples at $ "70 # C and $1.33 Pa, using Freeze- MALDI-TOF/TOF MS
dryer (Freezone 4.5 Liter Benchtop Freeze dry systemV,
R

HMOs were analyzed by Bruker ultrafleXtreme matrix-

Labconco, Kansas City, MO). assisted laser desorption/ionization (MALDI) time-of-
flight/time-of-flight (TOF/TOF) mass spectrometry (MS)
Reagents (Bruker Daltonics, Bremen, Germany) in positive and
negative ion modes. DHB was used as a matrix (5 mg/
Porous graphitized carbon (PGC) cartridges for desalt- 100 lL in 50% acetonitrile) for both positive and nega-
ing were purchased from Thermo Fisher Scientific (San
tive modes. NaCl (0.01 M in water) was used as a cat-
Jose, CA). Sodium borohydride (NaBH4, 98%), 2, 5-dihy-
ion dopant for the positive mode. Each fraction was
droxybenzoic acid (DHB), and sodium chloride (NaCl)
spotted onto a stainless steel target plate along with a
were obtained from Sigma Aldrich (St. Louis, MO).
DHB; rapidly dried under vacuum to make more
Solvents were of HPLC grade. All other reagents were
homogeneous crystal; and loaded into a MS system.
of analytical grade or higher.
The HMOs in the 20% acetonitrile (v/v) fraction were
detected primarily as [M þ Na]þ ions in the positive
Sample preparation ion mode while the HMOs in the 40% acetonitrile in
0.05% TFA (v/v) fraction were analyzed in the negative
Extraction of HMOs
mode as [M " H]". Mass spectra were externally cali-
All milks were stored at "80 # C until thawed for imme- brated using malto-oligosaccharides allowing mass
diate use. An initial volume of 0.5 mL of milk was accuracy of 10 ppm or better. MALDI-TOF mass spectra
diluted with 0.5 mL of deionized water and centrifuged were acquired in the positive mode over the m/z
 THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE 3

range from 400 to 3000 and in the negative mode Impact of the pasteurization on the HMOs
over the m/z range from 900 to 3000 for a total of
Table 1 demonstrates the HMO patterns in the pas-
2400 laser shots using a 1 kHz laser. Raw MS and MS/
teurized milks from three different mothers. In the
MS data were produced with flexAnalysis software
Wilcoxon signed rank test, no significant difference of
(version 3.3, Bruker Daltonics, Bremen, Germany).
HMO composition was found between milks collected
before and after the pasteurization (p ¼ .899). Similarly,
Statistical analysis there was strong correlation between the amounts of
HMOs of the milks collected before and after the pas-
The mean percentage of peak intensities of each HMO teurization, which indicated there was no significant
was compared between paired data sets (pre- and post- change of HMOs patterns after the pasteurization
pasteurized milks, and between pre- and post-freeze (r ¼ 0.925, p < .001, Figure 2(A)).
dried milks) were calculated by Wilcoxon signed rank
test using SPSSV version 18.0 (SPSS Inc, Chicago, IL) to
R

find out the effects of each treatment on the HMO com- Impact of the freeze-drying on HMOs
position. Spearman’s correlation test was performed The HMO levels before and after the freeze-drying is
using MedCalcV version 12.3 (MedCalc, Mariakerke,
R
Downloaded by [University of New England] at 07:18 08 November 2017

shown in Table 2. In the Wilcoxon signed rank test, no

Belgium) to calculate correlations between the HMO significant difference of HMO composition was found
composition of milks from different mothers and lacta- between the milks collected before and after the
tion periods, and samples with different treatments. freeze-drying (p ¼ .838). The correlation analysis
revealed strong correlation between the milks col-
lected before and after the freeze-drying (r ¼ 0.927,
Results p < .001, Figure 2(B)).
HMOs in mother milks
Totally, 33 different oligosaccharides were detected in Correlation analysis between HMOs of overall
human milk samples. As shown in the correlation ana- samples
lysis, HMO amounts of three different lactation stages Figure, Supplemental Digital Content 1 demonstrates
(periods) in each mother were significantly correlated; correlation between HMOs of the overall samples. It
r ¼ 0.817–0.970, p < .001 (Figure 1). However, there shows that both of the pasteurization and the freeze-
was no correlation between the samples from different drying did not affect the levels of HMOs, but there
mothers. Moreover, most abundant HMOs were differ- were significant differences of HMO levels between
ent between the milk samples from different mothers milks from different mothers. It indicates that each
(data not shown). In addition, acidic glycan containing mother might have unique HMO patterns in their
sialic acid was not found in the sample B. milks.

Figure 1. Quantitative correlation of the human milk oligosaccharides between different lactation periods and different three
mothers. Notice that the samples from individual mothers are strongly grouped and there is strong correlation between the differ-
ent periods in the samples from same mother.
 4 W.-H. HAHN ET AL.

Table 1. List and mean percent of peak intensities of human milk oligosaccharides (HMOs) found in the pre-
and postpasteurized milks from different three mothers.
Pre-pasteurization Post-pasteurization
a
Code m/z Mother A Mother B Mother C Mother A Mother B Mother C
2_0_1_0 513.164 0.0 ± 0.0 55.6 ± 3.7 5.4 ± 0.8 0.0 ± 0.0 61.2 ± 11.3 7.1 ± 1.1
2_0_2_0 659.236 0.0 ± 0.0 6.2 ± 0.6 0.3 ± 0.1 0.0 ± 0.0 6.1 ± 0.9 0.5 ± 0.3
3_1_0_0 732.254 67.7 ± 2.6 4.5 ± 0.6 37.9 ± 3.3 66.8 ± 3.6 4.6 ± 0.3 34.6 ± 3.5
3_1_1_0 878.311 1.2 ± 0.5 18.1 ± 1.4 28.4 ± 0.7 1.0 ± 0.2 16.9 ± 1.1 27.7 ± 1.3
4_1_0_0 894.27 0.4 ± 0.1 0.0 ± 0.0 0.1 ± 0.1 0.4 ± 0.1 0.0 ± 0.0 0.0 ± 0.1
3_2_0_0 935.337 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0
3_1_2_0 1024.368 0.0 ± 0.0 12.8 ± 1.5 9.6 ± 1.0 0.0 ± 0.0 11.4 ± 1.6 10.7 ± 2.6
4_1_1_0 1040.362 0.0 ± 0.0 0.5 ± 0.2 0.1 ± 0.1 0.0 ± 0.0 0.6 ± 0.1 0.1 ± 0.1
4_2_0_0 1097.386 26.2 ± 2.4 0.0 ± 0.0 1.4 ± 0.2 27.1 ± 2.7 0.0 ± 0.0 1.2 ± 0.2
4_2_1_0 1243.440 1.1 ± 0.1 0.3 ± 0.2 10.2 ± 1.4 1.0 ± 0.2 0.5 ± 0.2 10.9 ± 1.8
4_2_2_0 1389.501 0.0 ± 0.0 2.0 ± 0.5 4.2 ± 0.8 0.0 ± 0.0 2.0 ± 0.4 4.6 ± 0.8
5_3_0_0 1462.520 2.9 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 2.9 ± 0.6 0.0 ± 0.0 0.0 ± 0.0
4_2_3_0 1535.530 0.0 ± 0.0 0.1 ± 0.2 0.6 ± 0.2 0.0 ± 0.0 0.1 ± 0.2 0.7 ± 0.2
5_3_1_0 1608.576 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.1
5_3_2_0 1754.628 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1
6_4_0_0 1827.650 0.5 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.3 0.0 ± 0.0 0.0 ± 0.0
5_3_3_0 1900.680 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.0
Downloaded by [University of New England] at 07:18 08 November 2017

6_4_1_0 1973.700 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1
6_4_2_0 2119.739 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
3_1_0_1 999.352 41.3 ± 2.4 0.0 ± 0.0 62.3 ± 7.9 41.3 ± 2.3 0.0 ± 0.0 55.3 ± 7.8
3_1_0_2 1290.462 0.4 ± 0.2 0.0 ± 0.0 0.6 ± 0.2 0.3 ± 0.2 0.0 ± 0.0 0.6 ± 0.2
3_2_0_1 1202.376 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0
4_2_0_1 1364.484 53.8 ± 1.4 0.0 ± 0.0 9.6 ± 1.5 54.2 ± 2.8 0.0 ± 0.0 10.7 ± 2.1
4_2_0_2 1655.515 0.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0
4_2_1_1 1510.492 0.4 ± 0.5 0.0 ± 0.0 24.1 ± 6.0 0.3 ± 0.1 0.0 ± 0.0 29.9 ± 6.3
4_2_2_1 1656.535 0.0 ± 0.0 0.0 ± 0.0 1.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 0.3
5_3_0_1 1729.622 3.1 ± 1.6 0.0 ± 0.0 0.1 ± 0.1 3.1 ± 1.6 0.0 ± 0.0 0.0 ± 0.1
5_3_1_1 1875.604 0.3 ± 0.8 0.0 ± 0.0 1.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 0.3
5_3_2_1 2021.658 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.3
6_4_0_1 2094.793 0.7 ± 0.6 0.0 ± 0.0 0.0 ± 0.1 0.5 ± 0.6 0.0 ± 0.0 0.0 ± 0.1
6_4_1_1 2240.891 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.1
6_4_2_1 2386.924 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1
7_5_0_1 2459.808 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
a
Code is the glycan composition of HMOs designated by the number of monosaccharides; from left to right, hexose, N-acetylhexos-
amine, fucose and N-acetylneuraminic acid, respectively.

(A) 90 (B) 90

80 80

70 70
Post-pasteurization

Post-freeze drying

60 60

50 50

40 40

30 30

20 20

10 Spearman r = 0.925, 10 Spearman r = 0.927,

P < 0.0001 P < 0.0001
0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Pre-pasteurization Pre-freeze drying

Figure 2. Quantitative correlation of the human milk oligosaccharides between the milk samples collected pre- and post-pasteur-
ization (A), and between pre- and post-freeze drying (B).

Discussion
on the HMO patterns. In addition, the lactation stages
In the present study, the authors report the presence did not influence the HMO profiles.
of large differences in HMOs between mothers and no The pasteurization and freezing is the most
significant effects of pasteurization and freeze-drying essential processes of human milk banking [10].
 THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE 5

Table 2. List and mean percent of human milk oligosaccharides (HMOs) found in the pre- and post-freeze
dried milks from three mothers.
Pre-freeze drying Post-freeze drying
a
Code m/z Mother A Mother B Mother C Mother A Mother B Mother C
2_0_1_0 513.164 0.0 ± 0.0 56.5 ± 3.9 6.4 ± 1.2 0.0 ± 0.0 60.3 ± 11.7 6.1 ± 1.4
2_0_2_0 659.236 0.0 ± 0.0 6.0 ± 0.5 0.4 ± 0.2 0.0 ± 0.0 6.3 ± 0.9 0.4 ± 0.3
3_1_0_0 732.254 68.1 ± 3.1 4.6 ± 0.5 36.7 ± 3.9 66.5 ± 2.9 4.5 ± 0.3 35.8 ± 3.7
3_1_1_0 878.311 1.2 ± 0.4 17.4 ± 1.2 27.9 ± 1.3 1.0 ± 0.3 17.6 ± 1.6 28.1 ± 0.9
4_1_0_0 894.270 0.4 ± 0.1 0.0 ± 0.0 0.1 ± 0.1 0.4 ± 0.1 0.0 ± 0.0 0.1 ± 0.1
3_2_0_0 935.337 0.1 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.2 0.0 ± 0.0 0.0 ± 0.0
3_1_2_0 1024.368 0.0 ± 0.0 12.3 ± 1.8 10.1 ± 2.1 0.0 ± 0.0 11.9 ± 1.6 10.2 ± 2.0
4_1_1_0 1040.362 0.0 ± 0.0 0.6 ± 0.1 0.1 ± 0.1 0.0 ± 0.0 0.5 ± 0.2 0.1 ± 0.1
4_2_0_0 1097.386 25.7 ± 2.4 0.0 ± 0.0 1.3 ± 0.2 27.5 ± 2.4 0.0 ± 0.0 1.3 ± 0.3
4_2_1_0 1243.440 1.0 ± 0.1 0.5 ± 0.1 10.3 ± 1.8 1.1 ± 0.2 0.3 ± 0.3 10.9 ± 1.3
4_2_2_0 1389.501 0.0 ± 0.0 2.1 ± 0.4 4.3 ± 0.9 0.0 ± 0.0 1.9 ± 0.5 4.5 ± 0.7
5_3_0_0 1462.520 2.9 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 2.9 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
4_2_3_0 1535.530 0.0 ± 0.0 0.1 ± 0.2 0.6 ± 0.2 0.0 ± 0.0 0.1 ± 0.2 0.7 ± 0.2
5_3_1_0 1608.576 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.1
5_3_2_0 1754.628 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1
6_4_0_0 1827.650 0.6 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.1 0.0 ± 0.0 0.0 ± 0.0
5_3_3_0 1900.680 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.0
Downloaded by [University of New England] at 07:18 08 November 2017

6_4_1_0 1973.700 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1
6_4_2_0 2119.739 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
3_1_0_1 999.352 40.6 ± 2.6 0.0 ± 0.0 58.7 ± 8.7 42.0 ± 1.7 0.0 ± 0.0 58.9 ± 8.7
3_1_0_2 1290.462 0.3 ± 0.2 0.0 ± 0.0 0.7 ± 0.2 0.5 ± 0.1 0.0 ± 0.0 0.5 ± 0.1
3_2_0_1 1202.376 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0
4_2_0_1 1364.484 54.9 ± 2.0 0.0 ± 0.0 10.6 ± 1.6 53.1 ± 2.0 0.0 ± 0.0 9.8 ± 2.1
4_2_0_2 1655.515 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0
4_2_1_1 1510.492 0.3 ± 0.1 0.0 ± 0.0 27.0 ± 7.2 0.5 ± 0.4 0.0 ± 0.0 27.1 ± 6.7
4_2_2_1 1656.535 0.0 ± 0.0 0.0 ± 0.0 1.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 0.4
5_3_0_1 1729.622 3.1 ± 1.6 0.0 ± 0.0 0.1 ± 0.1 3.1 ± 1.5 0.0 ± 0.0 0.0 ± 0.1
5_3_1_1 1875.604 0.0 ± 0.0 0.0 ± 0.0 1.2 ± 0.1 0.3 ± 0.8 0.0 ± 0.0 1.3 ± 0.3
5_3_2_1 2021.658 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.1
6_4_0_1 2094.793 0.5 ± 0.5 0.0 ± 0.0 0.0 ± 0.1 0.8 ± 0.6 0.0 ± 0.0 0.0 ± 0.1
6_4_1_1 2240.891 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.1
6_4_2_1 2386.924 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1
7_5_0_1 2459.808 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
a
Code is the glycan composition of HMOs designated by the number of monosaccharides; from left to right, hexose, N-acetyl-
hexosamine, fucose and N-acetylneuraminic acid, respectively.

However, the pasteurization is known to significantly polyphenol-related functionality, and oxidative integ-
reduce the content of lipid, energy, and/or carbohy- rity of human milk [11,12].
drates especially when it is combined with the freez- These evidences commonly imply that freeze-drying
ing process at both of "20 and "80 # C [16,17]. The is a promising option to improve the preservation of
pasteurization and/or freezing of raw human milk are human milk in banks. Small numbers of human milk
found to affect the vitamin D level, lactoferrin level banks are storing pasteurized human milk up to 12
and antioxidative capacity as well [18–22]. Even months after freeze-drying and transporting freeze-
though the gold standard method for human milk dried milks at room temperature [13,14]. However, the
banking is freezing milks at "70 or "80 # C, it requires effects of freeze-drying on the HMOs are not evaluated
lot of resources. Thus, there is an agreement on the yet. There is only one study that freeze-drying in the
freezing between "20 and "25 # C for milk storage stool samples does not make significant HMO changes
despite of less effective bacterial killing and nutrients in neonates [25].
preserving performance [16,23,24]. To the best of our knowledge, this is the first report
Recently, the freeze-drying is suggested as a better about the effects of pasteurization and freeze-drying
method to store longer maintaining nutrients and bac- on the HMO profiles. HMOs seem to be very stable to
teriostatic properties. Salcedo et al. [23] reported that the treatments and it would support the benefits of
freeze-drying showed similar bactericidal activity com- introducing freeze-drying in human milk bank for lon-
pared with storing at "80 # C and was superior to stor- ger, safer, and easier storage and transport of the
ing at "20 # C. Moreover, the freeze-drying could donor milks. However, there are several limitations in
achieve suitable protection of the milk properties the present study. First, the numbers of samples were
including protein, fatty acids, triglycerides, vitamins, not sufficient to emphasize the stability of HMOs
 6 W.-H. HAHN ET AL.

against the treatments. It could be one of the major [6] O’Hara AM, Shanahan F. The gut flora as a forgotten
limitations because the composition of human milk organ. EMBO Rep. 2006;7:688–693.
[7] Andreas NJ, Al-Khalidi A, Jaiteh M, et al. Role of
does vary between individuals [26–29]. However,
human milk oligosaccharides in group b streptococ-
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the samples were not stored for longer than months. development and cognition. Annu Rev Nutr.
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and glycoprotein sialic acid in breastfed compared
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milks of later stages need to be investigated [30,31]. Establishing an integrated human milk banking
In summary, the HMOs were found not to be approach to strengthen newborn care. J Perinatol.
2017;37:469–474.
affected by pasteurization and freeze-drying.
[11] Lozano B, Castellote AI, Montes R, et al. Vitamins, fatty
Moreover, there were large differences between the acids, and antioxidant capacity stability during storage
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mothers. As significant changes of HMOs were not of freeze-dried human milk. Int J Food Sci Nutr.
detected after pasteurization and freeze-drying, we 2014;65:703–707.
hope introducing freeze-drying in human milk banks [12] Cortez MV, Soria EA. The effect of freeze-drying on
the nutrient, polyphenol, and oxidant levels of breast
to be encouraged. However, the storage length with-
milk. Breastfeed Med. 2016;11:551–554.
out composition changes of HMOs after freeze-drying [13] Guti! errez D, de Almeida JA. Human milk banks in bra-
needs to be evaluated in the further studies. zil. J Hum Lact. 1998;14:333–335.
[14] Centre for Clinical Practice. National Institute for
Health and Clinical Excellence: guidance; Donor breast
Disclosure statement milk banks: the operation of donor milk bank services.
London: National Institute for Health and Clinical
The authors report no conflicts of interest. Excellence; 2010.
[15] Leung AK, Sauve RS. Breast is best for babies. J Natl
Med Assoc. 2005;97:1010–1019.
Funding [16] Garc!ıa-Lara NR, Vieco DE, De la Cruz-B! ertolo J, et al.
Effect of holder pasteurization and frozen storage
This work was supported by the National Research
on macronutrients and energy content of breast milk.
Foundation (NRF) of Korea grant funded by the Korean gov-
J Pediatr Gastroenterol Nutr. 2013;57:377–382.
ernment (MSIP) [No. 2015R1A2A1A15056046;
[17] Lev HM, Ovental A, Mandel D, et al. Major losses of
2017R1D1A1B03034270].
fat, carbohydrates and energy content of preterm
human milk frozen at "80 # C. J Perinatol. 2014;34:
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