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LDRT #1141781, VOL 0, ISS 0
Spray and freeze drying of human milk on the retention of immunoglobulins
(IgA, IgG, IgM)
Jorge Castro-Albarrán, Blanca Rosa Aguilar-Uscanga, Frédéric Calon, Isabelle St-Amour, Josué Solís-Pacheco,
Linda Saucier, and Cristina Ratti
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Spray and freeze drying of human milk on the retention of immunoglobulins (IgA, IgG, IgM)
Jorge Castro-Albarrán, Blanca Rosa Aguilar-Uscanga, Frédéric Calon, Isabelle St-Amour, Josué Solís-Pacheco, Linda Saucier, and Cristina Ratti
DRYING TECHNOLOGY
http://dx.doi.org/10.1080/07373937.2016.1141781
5Spray and freeze drying of human milk on the retention of immunoglobulins
(IgA, IgG, IgM)
Jorge Castro-Albarrána, Blanca Rosa Aguilar-Uscangaa, Frédéric Calonb,c, Isabelle St-Amourc, Josué Solís-Pachecoa,
Linda Saucierb, and Cristina Rattib
aLaboratorio de Microbiología Industrial, Centro Universitario de Ciencias Exactas e Ingeniería, Universidad de Guadalajara, Guadalajara,
10 Jalisco, México; bInstitut sur la nutrition et les aliments fonctionnels (INAF), Université Laval, Québec, Québec, Canada; cCentre de Recherche
du CHU de Québec (CHUL), Axe Neurosciences, Québec, Québec, Canada
ABSTRACT
Several freeze-drying and spray-drying methods were investigated in relation to the retention of
immunoglobulins (Ig) A, IgG, and IgM. Spray drying produced human milk powders with 2%
humidity and a good retention of IgG (>88%) and IgM (∼70%). However, only 38% of IgA remained
after spray drying. For freeze drying, only the highest heating plate temperature used in this study
(40°C) brought IgA content down to 55% in powder with 1.75% residual humidity, whereas milk
samples undergoing lower temperatures had higher preservation rates (75% for IgA and 80% for
IgG and IgM) and higher residual moisture contents. From these results, it can be concluded that
IgA is the most sensitive Ig lost during drying processing of human milk. The best method to
generate human milk powders without a significant loss of Ig was thus freeze drying at 30°C
heating plate temperature, which accelerated the process compared to lower processing
temperatures, but still had good overall Ig retention.
KEYWORDS
Freeze drying; human milk;
immunological properties;
spray drying
30
Introduction
Mother’s milk contains compounds that greatly
contribute to the development of immune and digestive
systems, as well as the general growth and immuno-
35 logical support of infants.
[1]
A wide array of important
compounds, such as vitamins and fatty acids, immuno-
globulins (Ig) A, IgG, IgM, and IgD, are all found in
human milk. Of these, IgA, which appears to be both
synthesized and stored in the breast,
[2]
plays a crucial
40 role. Indeed, IgA sheets the intestinal epithelium,
thereby protecting the mucosal surfaces against
entry of pathogenic bacteria and enteroviruses. It
bestows protection against Escherichia coli, Salmonellae,
Shigellae, Streptococci, Staphylococci, Pneumococci,
45 poliovirus, and the rotaviruses.
[3]
When the mother’s own milk is unavailable, the
American Academics of Pediatrics
[4]
recommends using
donor milk. For this purpose, human milk banks have
been created. To reduce the risk of microbial contami-
50 nation that can occur during collection and handling
of human milk, it has to be pasteurized to reduce the
number of viable pathogens.
[5]
The guideline for human
milk banks in the USA and in Spain is to use low-
temperature long-time pasteurization,
[6]
usually 62.5°C
55for 30 min. A study done by Evans et al.
[7]
on pasteur-
ization of human milk for 30 min and temperatures
ranging from 60 to 70°C showed that IgA was preserved
with relative little loss until 70°C (33% loss), while IgG
and lactoferrin were much more labile, displaying losses
60of 77.2 and 85%, respectively, at 65°C. Following
heating for 30 min at 62.5°C, the initial contents of
IgG, lactoferrin, and lysozyme were reduced by 34, 57,
and, 23%, respectively, while IgA content remained
stable.
[7]
However, Permanyer et al.
[6]
found that
65pasteurization of human milk, using the same heating
conditions, induced a 30% decrease in IgA. Other
reports indicate that IgA is stable up to 56°C but
heat-labile at 62.5°C
[8]
and that temperature but not
process time is a critical parameter in determining
70IgA stability.
[9]
Studies by Friend et al.
[10]
reported
reductions of 47, 55, 39, and 73% of lactoperoxidase,
lipase, lysozyme, and protease, respectively, after pas-
teurization of human milk at 62.5°C for 30 min. Hence,
even if human milk is safe for consumption from
75a microbiological point of view after low-temperature/
long-time pasteurization, the potential detrimental
effect of this traditional type of preservation method
on bioactive compounds within human milk should also
be taken into account.
CONTACT Cristina Ratti cristina.ratti@fsaa.ulaval.ca Institut sur la nutrition et les aliments fonctionnels, Université Laval, SGA–FSAA,
2425 rue de l’agriculture, Québec (QC), G1V 0A6, Canada.
© 2016 Taylor & Francis
3b2 Version Number : 11.0.3184/W Unicode (Apr 10 2014)
File path : {1TFJATS}LDRT/v0n0/LDRT1141781/LDRT_A_1141781_J.3d
Date and Time : 16/9/16 and 17:31
80 Storage of human milk was studied after
pasteurization,
[7,10]
cooling or freezing,
[7,11]
and freeze
drying.
[7,10,12]
Recently, Lozano et al.
[12]
suggested that
the stability of human milk, in terms of vitamins, fatty
acids, and antioxidant levels, was higher in freeze-dried
85 milk powders than the reported values for frozen or
fresh milk after the same length of storage.
Milk can be converted into shelf-stable powders by
spray-drying or freeze-drying methods. Spray drying is
a dehydration method where a liquid/slurry is sprayed
90 in fine droplets in contact with air at elevated tempera-
tures. This method is commonly used to dry milk, whey,
yeast, and other high-value products in industry due to
the good final quality of spray-dried powders. Feed flow
rate, atomizer rotation speed, and inlet air temperature
95 have been identified as key parameters affecting powder
quality during spray-drying dairy emulsions such
as whole milk.
[13]
Energy consumption is, however,
a restriction in the widespread use of this drying
method. In addition, the oxygen present in the large
100 volumes of air mixed with the droplets as well as the
high operating temperatures during spray drying could
have a negative impact not only on fat-soluble vitamins
and in CLA contents in milk due to oxidation but also
on other heat-labile compounds such as IgG and IgA.
105 To the best of our knowledge, no studies have been
reported on spray drying of human milk.
Freeze drying is an alternative dehydration method
based on sublimation of the ice contained in a frozen
material and is recognized for producing final products
110 of the highest quality.
[14]
This method is an expensive
process due to increased energy consumption during
the long processing times under vacuum and thus its
application to the food industry has been limited. Freeze
drying of human milk (previously frozen at 80°C)
115 during 24 h in a benchtop unit at 10
3
mBar pressure
and 46°C condenser temperature was proposed
as a good alternative to preserve human milk.
[12]
When
compared to frozen milk at 20°C, the concentrations
of vitamins C and E as well as antioxidant capacity
120 are better retained in freeze-dried human milk. In terms
of Ig, a previous study done by Evans et al.
[7]
suggests
that deep freezing at 20°C is a satisfactory procedure
compared to the more expensive freeze drying (no
operating conditions specified), which showed no
125 additional benefit in this study.
Most of the previous studies on preservation
of human milk have used pasteurization or heat treat-
ments, followed by cool storage or freezing, providing
recommendations mainly focused on bacterial content.
130 However, the combination of both processes (heat pro-
cessing and storage conditions) can lead to a decrease in
bioactive compounds, such as Ig. Other preservation
methods like freeze drying have been studied from the
standpoint of feasibility and final quality of the product,
135but little has been analyzed on the determination of
optimal process conditions in order to decrease costs.
Also, no previous studies have addressed the effect
of human milk dehydration processes on bioactive
immunological components. Furthermore, the use of
140a dehydration method to preserve mother’s milk could
be more beneficial than pasteurization by producing
a powder that can be stored at cold temperatures for
long times without loss in bioactive properties. Thus,
the aim of this work was to study freeze-drying and
145spray-drying methods with different operating con-
ditions for the conservation of human milk with specific
focus on Ig preservation. Determinations of sorption
isotherms and glass transition temperature (T
g
) of
the final powders complete this work in order to have
150indicators about their specific storage conditions.
Materials and methods
Ethical considerations
This study was approved by the Ethical Research
Committee of Université Laval (Québec, Canada) in
155April 2014 (# 2014-034/14-04-2014). Volunteer donors
provided a written agreement about the donation of
excess human milk for this study.
Human milk samples
Surplus human milk was obtained from healthy
160mothers during lactation between 4 and 8 months after
giving birth. Milk was collected in their homes using
an electric extraction pump (Medela
1
), placing the
collected milk in “Pump and Save Bags” (Medela
1
),
and storing afterwards in a freezer at 18°C until
165further processing.
Human milk preparation
All human milk samples (for spray or freeze drying)
were thawed in their storage bags in a water bath
at 30°C for 20–30 min. Several samples (n ¼10) were
170randomized and pooled for each repetition. The pooled
milk was subjected to one cycle of homogenization
at 5,000 psi in an Emulsiflex C50 (Avestin
1
, Mannheim,
Germany).
Freeze drying of human milk
175Thirty (30) mL of pooled milk was poured in Petri
dishes of 9-cm external diameter; the liquid samples
2 J. CASTRO-ALBARRÁN ET AL.
have a thickness of approximately 8 mm. The Petri
dishes were covered and placed inside a Sanyo medical
freezer (MDF 235, Gunma, Japan) at 40°C for a mini-
180 mum of 9 h. Then, the samples were placed inside the
drying chamber of a Virtis freeze dryer (Unitop 4001,
Gardinier, NY, USA), working under vacuum of less
than 30 mTorr, at 85°C condenser temperature (con-
denser separated from the drying chamber), and at 20,
185 30, 4°C heating plate temperatures. In order to establish
freeze-drying kinetics and thus to estimate the final
freeze-drying time at each heating plate temperature,
drying curves were obtained by periodically weighing
the milk samples for up to 10 h. The temperature at
190 the center of the milk sample was followed throughout
the freeze-drying process with a T thermocouple
(TMQSS-040G-18, Omega, Stamford, CT, USA), which
was inserted in the sample center prior to freezing. Final
humidity was determined as described later.
195 Simplified mathematical models were used to
quantify drying kinetics of various food products.
[15]
In this study, experimental data were fitted to the Page’s
equation:
[16]
XXe
X0Xe¼exp k tn
ð Þ ð1Þ
200 where X, X
o,
and X
e
are moisture content in dry basis,
initial, and equilibrium moisture content, respectively,
k is the drying constant (h
n
), n is the Page’s model
parameter, and t is the process time (h). Several
previous works have suggested that X
e
can be neglected
205 in Eq. (1) since it is significantly lower than moisture
content for most of the drying process.
[17]
Spray drying of human milk
Homogenized human milk was kept at 30°C until
the spray-drying process. Spray drying was done in
210 a Niro-Atomizer pilot unit with conical base (Model
209/S, Soeborg, Denmark) fed with a Watson Marlow
1
(Model 503U) peristaltic pump and a nozzle atomizer
(three bar pressure). In order to maximize the yield,
preliminary tests based on overall performance and final
215 powder humidity were done, from which the following
spray-drying operation variables were selected: 180 and
160°C inlet air temperature with feeding rates of 5 and
4 mL/min, respectively. Outlet air temperature was
recorded and yield could be estimated from gravimetric
220 measurements. Final humidity was determined
as described later.
In both spray-drying and freeze-drying processes, the
final product was kept in dark conditions, in desiccators
at 5°C with the presence of Drierite
R
, until further
225analysis.
Humidity and dry mass determination
Total solids and humidity of the dried samples were
determined with a halogen balance HR73-P (Mettler
Toledo
1
, Greifensee, Switzerland). Humidity was
230determined on a wet basis in duplicate measurements.
Immunoglobulin determination
Human milk powders (1 g) were rehydrated to
their initial moisture content by dissolution in distilled
water (approximately 7.15 mL) at room temperature,
235followed by agitation homogenization and centrifuga-
tion at 1500 rpm for 5 min in order to obtain the Ig
in the whey, which was separated and analyzed by
ELISA.
ELISA quantification was performed as previously
240described
[18]
to determine the concentrations of Ig.
Goat antibodies specific to the Fc fragment of human
IgG, IgM, or IgA were used for capture, and the
corresponding HRP-conjugated antibodies, for detec-
tion (all purchased from Jackson Immuno Research
245Laboratories Inc., West Grove, PA, USA). The standard
curves were performed using IgA from human
colostrum (1–10 ng/mL, Sigma-Aldrich, Saint Louis,
MO, USA), IgG from human serum (2.5–50 ng/mL,
Sigma-Aldrich), and IgM from human serum
250(2.5–50 ng/mL, Sigma-Aldrich). The results were
expressed in micrograms per milliliter (µg/mL) of
rehydrated milk.
Sorption isotherms
Lithium chloride (LiCl), sodium chloride (NaCl),
255sodium bromide (NaBr), magnesium chloride (MgCl
2
),
and potassium acetate (CH
3
COOK) saturated
salt solutions were prepared according to the method
described by Ratti et al.
[19]
Relative humidity of the
solutions was verified at ambient temperature
260(20°C) with an AquaLab (Series 3, Decagon Devices
Inc., Pullman, Washington, USA): 14.1% (LiCl), 75.6%
(NaCl), 57% (NaBr), 33% (MgCl
2
), and 25.3%
(CH
3
COOK). Freeze-dried and spray-dried human milk
powders (approximately 300 mg) were placed in alumi-
265num cups over the saturated salt solutions in desiccators
at constant temperature (20°C) until equilibrium was
reached in approximately 7 days.
The dry matter of the solids was determined at 60°C
in a vacuum oven using P
2
O
5
as desiccant. Sorption
270experiments were done in duplicate.
DRYING TECHNOLOGY 3
Experimental sorption data were fitted to the GAB
model
[20]
using the nonlinear regression function of
SigmaPlot 11.0:
[30]
Xe¼XmC K aw
1K aw
ð Þ 1K awþC K aw
ð Þ ð2Þ
275 where a
w
is the water activity and X
m
, C, and K are the
GAB model constants.
Glass transition temperature
Glass transition temperature (midpoint) was deter-
mined by differential scanning calorimeter (DSC) using
280 a thermal analysis system DSC Pyris 1 (Perkin Elmer)
connected to a PC for simultaneous data treatment
(Pyris Software for Windows version 3.52) and to a
refrigeration system with a compressor. The instrument
was calibrated for temperature and heat flow with
285 indium (T
m
¼156.6°C and ΔH ¼28.45 J/g, Perkin
Elmer standard) and checked with distilled water for
which T
m
¼0°C and ΔH ¼333 J/g.
[21]
A 30-mg sample
of each human milk powder was transferred into a high-
volume stainless steel pan (Product #03190029, Perkin
290 Elmer), where an O-ring was inserted. The capsule
was sealed with a cover and immediately weighed. A
similar empty capsule was used as a reference. Capsules
were cooled to 20°C. Scanning was performed by
heating at 5°C/min from 20 to 120°C. The glass tran-
295 sition appeared as an endothermic shift in the specific
heat capacity. Results were obtained in triplicate.
Statistical analysis
Due to limited quantity of collected mother’s milk
available for these studies, only duplicate experiments
300 (n ¼2) were done for each treatment. Results are
reported as the average value with associated standard
error. Nonparametric ANOVA was performed on the
data (Friedman and Kruskal–Wallis tests) using Dunn’s
multiple comparison and with initial concentration
305 as control. Differences were considered significant
at p <0.05.
Results and discussion
Spray drying
Initial solid content of fresh human milk was found to
310be 11.57 0.58%(w/w). Lawrence
[22]
reported a solid
content of 12.0% in mature human milk and 12.8% in
colostrum, whereas Picciano et al.
[23]
found values of
11.85 1.43%. Table 1 shows the parameters resulting
from spray drying of human milk at different operating
315variables. For both combinations of inlet temperature
and flow rate operating variables, the average of powder
humidity obtained after the process was 2.05 0.14%.
However, exit air temperature was 10° higher for a 20°
increase in inlet air temperature, even if the liquid flow
320rate was increased from 4 to 5 mL/min.
Freeze drying
Figure 1a shows the product temperature (dotted line)
during freeze drying of human milk samples at 20, 30,
and 40°C heating plate temperature (solid lines).
325The temperature curves obtained in this work were
similar to most curves available in the literature for pro-
duct temperature increase during the freeze-drying
process.
[24]
The initial temperature of both the heating
plate and product after freezing was 30°C. As the
330temperature of the heating plate was raised, product
temperature increased with a delay corresponding to
the sublimation time. The duration of the sublimation
step had a good correlation with the heating plate
temperature (1.3, 3.0, and 4.5 h for 40, 30, and 20°C,
335respectively). After all the ice was sublimated, the product
temperature increased gradually to reach the heating
plate temperature, the times when the product tempera-
ture reaches 20, 30, or 40°C being 5.2, 7.7, and 8.5 h,
respectively.
340Freeze-drying curves of human milk are presented in
Fig. 1b. Drying rates were fast during the first phase of
drying and slowed down at the end of the process as
expected from the increase of the dry-layer resistance
to heat. An important effect of heating plate tempera-
345ture on residual moisture content was observed. This
positive effect was amplified as the heating plate
Table 1. Operation variables during spray drying and freeze drying of human milk.
Spray drying
Initial milk
total solids (%) T
inlet
(°C) Pressure (bar)
Flow rate
(mL/min) T
exit
(°C)
Final
moisture (%)
11.57 0.58
160 3 4 77.5 2.5 2.09 0.09
180 3 5 87.5 2.5 2.00 0.13
Freeze drying
Initial milk
total solids (%)
Heating plate
temperature (°C)
Freeze-drying
time (h)
Final
moisture (%) k (h
1
) N
11.21 0.41 20 9 2.65 005 0.115 1.52
30 8 3.05 0.15 0.133 1.53
40 6 1.75 0.075 0.170 1.64
4 J. CASTRO-ALBARRÁN ET AL.
temperature increased from 30 to 40°C (Fig. 1b). Times
to complete freeze drying can be extrapolated from
kinetic curves as 9, 8, and 6 h for 20, 30, and 40°C,
350 respectively, which corresponds approximately with
the times when the product temperature reached the
heating plate temperature.
Experimental data of moisture content decrease
as a function of time were fitted to Page’s model,
355Eq. (1). In Fig. 1b, Page’s model predictions are shown
together with experimental data. As can be observed,
this model predicted the freeze-drying kinetics in
human milk samples. The Page’s model fittings para-
meters are presented in Table 1. The rate constant (k)
360in the Page’s model involves the moisture diffusion
coefficient, which is temperature-dependent. Therefore,
as temperature increases, k increases. From Table 1, it
can be seen the rate constant (k) increased slightly
between 20 and 30°C, but as observed previously from
365the kinetic data, the rate constant was markedly
increased when using a 40°C heating plate temperature.
Table 1 also shows the final moisture content of human
milk powders freeze-dried at different heating plate
temperatures, which have a mean value of 2.48 0.61%.
370Effect of dehydration methods on IgA, IgG, and
IgM Content
Immunoglobulins concentrations were measured before
and after spray drying or freeze drying of human milk
and are presented in Table 2. Initial Ig concentrations
375were 215.80–262.68 µg/mL (IgA), 13.92–19.59 µg/mL
(IgG), and 21.95–22.48 µg/mL (IgM). It has to be noted
that each experiment for freeze drying or spray drying
was done with a different pool of homogenized
human milk, explaining the different initial Ig content
380values presented in Table 2. Human milk Ig contents
have been reported to vary depending on different
parameters such as the length of breastfeeding,
the breastfeeding stage, the time of the day when the
milk is extracted, and the geographic origin of the
385mothers.
[25,26]
Permanyer et al.
[10]
Q1reported IgA content
in mature human milk from 247 to 488 µg/mL and an
average of 13.47 µg/mL for IgG, whereas an IgM
concentration of 22.9 µg/mL was observed in the work
of Contador et al.
[25]
Therefore, our results on Ig
390concentration of human milk are in close agreement
with previous studies.
Based on the data presented in Table 2, graphs on
Ig retention were constructed (Figs. 2a and 2b for
spray drying and freeze drying, respectively, at differ-
395ent operation conditions). Spray-drying produced
Figure 1. Freeze-drying of human milk, (a) temperature profile
during freeze drying at 20, 30, and 40°C heating plate tempera-
tures (dotted lines represent product temperature while solid
lines, heating plate temperature), and (b) freeze-drying kinetic
curves of human milk at varying heating plate temperatures
(●20, 30, and !40°C).
Table 2. IgA, IgG, and IgM contents of human milk powder spray dried or freeze dried at different operation conditions.
Temperature (°C) IgA (µg/mL) IgG (µg/mL) IgM (µg/mL)
Spray drying Initial 215.80 6.84 13.92 0.80 21.95 5.15
160 (4 mL/min) 77.76 5.00 13.06 0.17 14.62 2.35
180 (5 mL/min) 83.20 1.22 12.26 0.55 16.10 3.39
Freeze drying Initial 262.68 56.40 19.59 0.17 22.48 5.84
20 199.48 33.91 15.36 0.33 19.57 6.28
30 200.62 4.75 16.00 0.70 18.84 5.12
40 144.72 7.00 15.38 0.20 18.40 8.40
Mean standard deviation (n ¼2).
DRYING TECHNOLOGY 5
human milk powders have good retention of IgG
(higher than 88%) and IgM (higher than 67%)
(Fig. 2a). However, only 38% of IgA could be pre-
served after spray dying. Please note from Table 1, that
400 both spray-dried powders have low residual moisture
contents (∼2%).
For freeze drying, only the highest heating plate
temperature used in this study (40°C) caused a low
retention of immunoglobulin IgA (55%). The other
405 conditions retained over 76% IgA and 80% IgG and
IgM. The reason why different Ig are retained
differently by the same process conditions is unknown
to us. It has been previously reported that IgA is stable
up to 56°C but heat-labile at 62.5°C
[8]
and that tempera-
410 ture rather than process time is a critical parameter in
keeping IgA content.
[9]
The lower retention of IgA at 40°C could be
explained by a longer exposure of the milk sample to
the heating plate temperature (Fig. 1a) as well as its
415 lower residual moisture content (Table 1). In freeze
drying, the lowest moisture content possible is not
necessarily the best optimal condition to preserve pro-
teins (i.e., Ig) since chemical and physical degradation
such as deamination, oxidation, and aggregation may
420 occur
[27]
and accelerate at very low water contents.
Nonparametric ANOVA performed on the Ig
concentrations data before and after freeze-drying/
spray-drying treatments failed to reveal evidence for
significant differences. However, Dunn’s multiple
425comparisons test suggested for spray drying at 160°C
a trend toward a significant reduction of IgA
(p ¼0.0651) and IgM (p ¼0.0911) as well as for IgG
at 180°C (p ¼0.0651), while for freeze drying such
a trend was observed for IgA concentrations between
430Initial vs 40°C treatment only (p ¼0.0605).
The present results indicated that IgA is the most
sensitive Ig during processing human milk by drying.
Reduced moisture contents obtained under specific
operating conditions as well as higher temperatures
435could be the causes of lower retention of IgA during
freeze drying and spray drying. Taking into account that
IgA is believed to be the most important Ig in human
milk,
[2]
the comparison of both dehydration methods
leads to the conclusion that freeze drying at 30°C is
440a particularly promising method to preserve human
milk. In addition, a heating plate of 30°C throughout the
whole sublimation process can accelerate the kinetics
(only 8 h processing time) with good overall immunoglob-
ulin retention (IgA 76.37 µg/mL 1.81%, IgG μg/mL
44581.65 3.61%, and IgM 83.80 µg/mL 22.75%).
Sorption isotherms
The sorption equilibrium isotherms of freeze-dried and
spray-dried human milk powders at 20°C are shown in
Fig. 3. Both curves showed a type-II sigmoid sorption
450form.
[28]
The sorption curves found in this work
showed a progressive increase in water content until
a
w
¼0.6, then the slope of the curve increases until
a
w
¼0.9. Soteras et al.
[29]
found similar behavior when
studying adsorption isotherms at 25°C of whole cow
Figure 2. Human milk IgA, IgG, and IgM retention after
(a) spray-drying at ( ) 160°C and ( ) 180°C and (b) freeze-drying
at heating plate temperatures of ( ) 20°C, ( ) 30°C, and ( )
40°C.
Figure 3. Sorption isotherms of human milk freeze-dried or
spray-dried powders.
6 J. CASTRO-ALBARRÁN ET AL.
455 milk samples, dried in an oven. In our study, differences
in moisture sorption by spray-dried or freeze-dried
human milk samples were not significant.
Human milk freeze-dried at 30°C heating plate
temperature for 8 h presents 3.05% moisture content
460 in wet basis (i.e., 0.0315 kg water/kg dry solids in dry
basis) (Table 1). From Fig. 3, it can be observed that
at this moisture content, 0.2 is the corresponding
approximate water activity at ambient temperature.
Thus, storage of this powder at ambient temperature
465 would require a relative humidity lower than 20%,
or a moisture barrier packaging, to avoid rehydration
of the powder during storage.
The fitted constants of the GAB equation (Eq. (2))
for human milk are X
m
¼0.0596 kg water/kg dry solids,
470 C ¼4.0241 and K ¼0.7423. These nonlinear regression
constants have a standard error estimated to 0.0093.
[30]
Predictions of GAB equation with fitted parameters are
also presented in Fig. 3 together with the experimental
data. These results showed a good agreement between
475 experimental and predicted data. The X
m
parameter
is an important sorption value representing the water
molecular primary layer. Determining the moisture
content for the maximum shelf stability of a dehydrated
product involves the determination of the sorption
480 isotherm and the calculation of the value of X
m
in Eq. (2). The estimated X
m
parameter values from
our data are in agreement with literature values
determined by Garcia-Alvarado et al.
[31]
and Lim et al.,
[32]
ranging from 0.053 to 0.174 kg water/kg dry solids.
485 Glass transition temperature
Figure 4 shows a representative DSC thermograms
obtained for human milk powders processed by freeze
drying at different heating plates temperatures (Fig. 6a
Q2 )
and by spray drying at different air inlet temperatures
490 (Fig. 6bQ3 ). Since the intensity of the thermograms is
solely linked to the mass of the sample and the water
content, the comparison of these curves is done by
matching the temperatures where peaks and step change
in the heat flow appear. The thermograms for freeze-
495 dried and spray-dried human milk powder samples
were similar, presenting three main peaks at ∼5, 18,
and 33°C, which may correspond to the melting of the
main fatty acids in human milk. Also, glass transitions
are observed in a range of 65–75°C, which corresponds
500 to the glass transition of lactose, the main carbohydrate
present in human milk, at 2–3% water content.
[33]
From curves exampled in Fig. 4, glass transition
temperatures for human milk were estimated to be
63.90 7.57°C and 69.11 5.55°C for spray drying at
505 160 and 180°C inlet air temperature, respectively.
For freeze-dried human milk powders, T
g
values were
69.07 1.29°C, 72.09 5.29°C, and 73.12 0.45°C
at 20, 30, and 40°C heating plate temperatures, respect-
ively. Glass transition temperatures of human milk
510powders were above 60°C, which indicates a good
thermal stability at ambient temperature if the powder
is packaged with moisture-barrier materials. Similar
T
g
results of 61 and 62°C for whole and skim milk,
respectively, were found for cow milk powders.
[34–36]
515Conclusion
The results obtained from this study on dehydration
methods and Ig retention in human milk suggested that
IgA is particularly sensitive and specifically lost during
drying processing. Our data further support the use
520of freeze drying at 30°C heating plate temperature to
generate human milk powders, which can accelerate
the process compared to lower processing temperatures
and minimize the loss of Ig with good retention of
IgA (76.37 1.81%), IgG (81.65 3.61%), and IgM
525(83.80 22.75%). From sorption and glass transition
results, storage of this powder at ambient temperature
Figure 4. Heat flow curves as a function of scanning tempera-
ture (a) freeze-drying and (b) spray-drying. The glass transition is
marked with light circles.
DRYING TECHNOLOGY 7
of freeze-dried powders would be possible as long as the
milk powder is packaged in moisture-barrier materials.
Further research studies based on immunoglobulin
530 structure are recommended in order to explain the dif-
ferential impact of the same drying conditions on Ig
retention values.
Acknowledgments
535 We would like to acknowledge the fruitful discussions and
moral support to this project from nutritionists Marie-Ève
Paradis (INAF, Université Laval), and Julie Lauzière and
Huguette Turgeon-O’Brien (School of Nutrition, Université
Laval).
540 Funding
The research group in the present work would like to thank
the Ministère des Relations Internationales, de la Francopho-
nie et du Commerce Extérieur (MRIFCE), from Québec
(Canada), the Institute for Nutrition and Functional Foods
545 (INAF, Université Laval), and the Consejo Nacional de
Ciencia y Tecnología (CONACYT, México) for their financial
support to this project.
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