Effect of composition and drying method on glass transition temperature, water sorption characteristics and surface morphology of newly designed β-lactoglobulin/retinyl palmitate/disaccharides systems
Effect of composition and drying method on glass transition temperature, water sorption characteristics and surface morphology of newly designed b-lactoglobulin/retinyl palmitate/ disaccharides systems
Agata Go´ rska 0 1
Karolina Szulc 0 1
Ewa Ostrowska-Lige˛za 0 1
Joanna Brys´ 0 1
Magdalena Wirkowska-Wojdyła 0 1
0 Department of Food Engineering and Process Management, Faculty of Food Sciences, Warsaw University of Life Sciences , 166 Nowoursynowska Street, 02-787 Warsaw , Poland
1 Department of Chemistry, Faculty of Food Sciences, Warsaw University of Life Sciences , 166 Nowoursynowska Street, 02-787 Warsaw , Poland
In the study, MDSC method was used to evaluate thermal properties of b-lactoglobulin-retinyl palmitate products enriched in disaccharides. Additionally, sorption behavior of powders was determined to define their stability. The obtained powders were also examined and visualized with scanning electron microscopy to find the relationship between food processing conditions and morphological changes in products. For the samples tested, a single glass transition was observed. The results have shown the impact of the composition of studied samples on the glass transition temperatures. The carbohydrate- blactoglobulin systems were characterized by higher Tg values than disaccharides alone. At low water activity, all powdered complexes showed typical sorption behavior of food systems with the sigmoidal shape of the curve. The present study has shown that spray-dried and freeze-dried powdered products with lactose or trehalose have different time-dependent crystallization behavior. Trehalose as a carbohydrate component of samples, in comparison with lactose, delayed the occurrence of crystallization. The obtained images presented the dependence of the shape of the particles on the drying method used. Spray-dried powders were characterized by a spherical or close to spherical structure with the surface of larger particles often deformed. In the case of freeze-dried particles, irregular structures were observed.
b-Lactoglobulin; Retinyl palmitate; MDSC; Thermal properties; Sorption isotherms; Scanning electron microscopy
The fortification of food in vitamin A is usually carried
out with some forms of fat. Nowadays, low-fat products
become more popular, and consequently, the reduction of
vitamin A in the diet rendered a nutritional concern.
Searching for new possibilities to enrich products in
vitamin A is a big challenge for scientists.
b-lactoglobulin—a major whey protein—is of direct interest to the
food industry since its properties can be advantageous in
dairy products and processing. It is worth mentioning that
biochemical oxygen demand of whey possesses a
worldwide disposal and pollution problem for the dairy
industry. It is urgently needed to search for new applications
for whey proteins. The binding properties of
b-lactoglobulin make it a potential ingredient to deliver retinyl
palmitate in a form of spray- or freeze-dried powders to
fat-free food systems [1–4]. Drying is the most commonly
used method in production of stable food ingredients and
food products. Powdered products are preferred by food
industry because they are easier to transport, storage and
handling. Freeze-drying is designed for unstable or heat
labile foods and allows the production of high-quality
dried products. High production costs, high energy
consumptions and low throughputs limit often its application
. Spray drying is the dehydration process preferred for
liquid food products with high initial moisture contents.
The wide availability of the equipment, a large variety of
carriers and good final product stability make the method
one of the mostly used drying processes in the food
industry [6, 7].
It is worth mentioning that rapid cooling or removal of
solvent water during drying can result in the production of
amorphous materials. Powdered food products, containing
amorphous materials, such as disaccharides are prone to
sticking, caking, collapse and crystallization, which are
undesirable physical changes that can impact on quality
and functionality of the final product . Physical changes
in food powders are related to the glass transition
phenomenon. The most important change in the amorphous
state occurs over the glass transition temperature (Tg),
which is the temperature at which polymeric materials
change from an amorphous solid (glass) to an amorphous
rubber . The glass transition temperature affects the
texture of foods as well as storage stability of dried foods
. Understanding the glass transition phenomenon
provides an insight into the causes of the cohesiveness of
many important powders influencing the wettability or
solubility of the powder, which is important for new
product development. Food products are expected to be fairly
stable below the Tg, but when the temperature rises above
Tg, a solid structure is transformed to a supercooled liquid
state with time-dependent flow. The glass transition
behavior of disaccharides (lactose, trehalose) is well
known, but higher Tg may be observed for carbohydrate–
protein systems than for carbohydrates alone [11, 12].
Differential scanning calorimetry (DSC) is the most widely
used thermal analysis technique to measure the temperature
and heat flows associated with phase transitions in
materials, as a function of time and temperature. Such
measurements can provide both quantitative information and
qualitative information concerning physical and chemical
changes that involve endothermic and exothermic
processes, or changes in heat capacity. DSC is particularly
suitable for analysis of food systems because they are often
subject to heating or cooling during processing. The
calorimetric information from DSC can be directly used to
understand the thermal transitions that the food system
undergoes during processing or storage [13–17].
Modulated differential scanning calorimetry (MDSC) which is an
enhancement to conventional DSC and involves the
application of a sinusoidal heating or cooling signal to a
sample and the subsequent measurement of the reversing
and non-reversing components of the heat flow response is
widely used technique for studying the glass transition
Disaccharides such as trehalose and lactose are
commonly used to protect biomolecules from drying-related
stresses . They were found to limit the conformational
changes and stabilize whey proteins during spray drying
. The saccharides are able to form a glassy state of a
very high viscosity and low mobility and restrict the
mobility of protein as well as its unfolding . Additives
such as lactose and trehalose remain in the amorphous
phase with the protein and bind to the protein in the place
of water during drying that prevent the stability problems.
Crystallization is a phase transition occurring in
freezedried and spray-dried amorphous lactose, trehalose or other
crystallizing carbohydrates. Crystallization occurs time
dependently when water content or temperature exceeds a
critical value . A number of researchers have
investigated lactose [26, 27] or trehalose  crystallization
occurring in powdered mixtures during water sorption
Water vapor sorption behavior and glass–rubber
transition data provide information that is useful in selecting
processing conditions for dried powders, and that influence
physical characteristics (hygroscopicity, stickiness and
caking behavior) and stability (storing and handling) of the
final product. Water vapor sorption isotherms show the
quantity of water absorbed by powders and the relative
humidity at which carbohydrate crystallizes in powders
The aim of the present study was to determine the effect
of disaccharides incorporation and the parameters used in
the drying process on thermal and sorption properties of
blactoglobulin–retinyl palmitate powdered complexes. The
knowledge of Tg profiles together with isotherms provides
important information about the stability of powdered
products enriched in disaccharides. Additionally, the
morphology of particles was discussed.
Materials and methods
BioPURE b-lactoglobulin containing 95% b-lactoglobulin
was provided as powder by Davisco Foods International,
Inc. (Le Sueur, Minnesota). Retinyl palmitate and a-lactose
monohydrate were purchased from Sigma Chemical Co.
(St. Louis, Missouri) and were of the highest analytical
quality. Trehalose was purchased from Hortimex Ltd Co.
400 mL of b-lactoglobulin (M = 18 400 g mol-1) solution
was prepared by gently adding distilled water into 8.6 g
(0.47 mmol) of the protein while stirring slowly to avoid
heavy foaming. The mixture was kept at room temperature
until a homogenous clear solution was formed. Then,
0.50 g (0.94 mmol) of retinyl palmitate (vitamin A)
dissolved in 800 lL absolute ethanol was added into the
solution to obtain 2:1 molar ratio of vitamin A to protein.
The solution was incubated at 40 C for 2 h according to
the method described by Kontopidis et al. . Then,
lactose or trehalose was added to the b-lactoglobulin–retinyl
palmitate solution in a mass ratio 5:1. The formulations
were mixed until disaccharides were dissolved and
The aqueous b-LG–vitamin A–carbohydrates solutions
were spray-dried in a laboratory spray-dryer Lab S1
(Anhydro, Denmark) by a peristaltic pump and atomized to
small droplets by a spray disk speed of 39.000 rpm. The
operational conditions of the spray drying were: air inlet
temperature: 120 and 160 C, and raw material flux rate:
51.4 and 64.2 mL min-1. The powders were kept in plastic
bags and stored in vacuum desiccators over CaCl2 at room
temperature, in a dry and dark place until further analysis.
Before freeze-drying, the aqueous solutions were frozen at
-40 C for 4 h. Frozen samples were placed on the shelves
of a Gamma 1-16 LDC freeze dryer (Christ Company,
Germany). The freeze-drying process was conducted for
24 h under a constant pressure of 63 Pa at a constant shelf
temperature of 30 C. The powders were kept in plastic
bags and stored in vacuum desiccators over CaCl2 at room
temperature, in a dry and dark place until further analysis.
Modulated DSC experiments were performed on a TA
Instrument Q200 differential scanning calorimeter (New
Castle, USA). Modulated differential scanning calorimetry
(MDSC) was used to determine the glass transition
temperature of b-lactoglobulin–retinyl palmitate powders with
lactose or trehalose addition at water activity about 0. The
cell was purged with 50 mL min-1 dry nitrogen and
calibrated for baseline on an empty oven and for temperature
using standard pure indium. An empty sealed aluminum
pan was used as a reference in each test. Powders
(10–13 mg) were non-hermetically sealed in aluminum
pans (volume 30 lL) and cooled from room temperature to
10 C at heating rate 5 C per min and equilibrated for
5 min. In the case of MDSC, samples were scanned from
10 to 170 C at a constant cooling rate of 2 C per min
with an amplitude of ±1 C and 60 s period of modulation.
Curves were analyzed with respect to the total, reversible
and non-reversible heat flow [31, 32]. Glass transition was
reported with parameters indicating its onset, midpoint and
endpoint of a vertical shift in the reversing transition curve.
TA Instruments Universal Analysis software was used to
analyze the glass transition temperature. The measurements
were carried out in three replicates for each sample.
Water vapor sorption isotherms were determined using the
static gravimetric method. The products were stored at a
stable relative humidity, which ranged from 0.0 to 0.92, for
3 months [23, 24]. The products to be analyzed were
placed in desiccators and were kept at a constant
temperature of 25 C. Saturated salt solutions were prepared as
hygroscopic factors. The salts used were: CaCl2, LiCl,
CH3COOK, MgCl2, K2CO3, Mg(NO3)2, NaNO2, NaCl,
(NH4)2SO4 and NH4H2PO4 with corresponding water
activities of 0, 0.11, 0.23, 0.33, 0.44, 0.53, 0.65, 0.75, 0.81
and 0.92, respectively .
Table 1 Composition and drying method for products A1–A10
Method of drying
The measurement of water vapor sorption kinetics was
conducted using a stand which ensured continuous measurement
of mass increase in the conditions of constant temperature and
relative humidity . Adsorption kinetics was determined at
a temperature of 25 C within 24 h at three levels of relative
humidity of the environment (0.33, 0.65 and 0.92) obtained
using saturated MgCl2, NaNO2 and NH4H2PO4 solutions. The
investigated samples mass increase was registered using the
measurement for DOS software.
Analysis of the sample structure was carried out on the
basis of the obtained images. The morphology of studied
powders was examined using scanning electron microscope
(SEM), FEI QUANTA 200 with EDS microanalyzer and
digital video recording. Photographs were taken at a
voltage prevailing in the generator of 30 kV, the distance of the
head from a sample of 10 mm and a pressure of 1 Torr.
FLD detector was used. Analysis of images was performed
using the program MultiScan v 13.1.
Each measurement was triplicate. The data were reported
as the mean ± standard deviation. Two-way ANOVA was
conducted using Statgraphics Plus for Windows program,
version 4.1 (Statistical Graphics Corporation, Warrenton,
VA, USA). Differences were considered to be significant at
a p value of 0.05, according to Tukey’s multiple range test.
Results and discussion
In the study, powdered products (A1–A10) diverse in terms
of composition and method of drying were obtained
It has been proved that glass transition temperature can
be an effective indicator for food quality changes during
storage . In the present study, DSC curves of reversing
heat flow versus temperature were recorded to determine
glass transitions’ temperatures of products. The example of
such DSC curve is shown in Fig. 1.
The glass transitions presented in Table 2 are reported
with parameters indicating their onset, midpoint and
endpoint, so the width of the transitions is clear. In the case of
b-lactoglobulin–retinyl palmitate powders enriched in
disaccharides, a single glass transition was observed. In the
case of lactose incorporation, the values of glass transition
temperatures were lower than in the case of trehalose
presence in the product. In the samples containing lactose,
the onset Tg ranged from 111.45 ± 1.35 to
113.07 C ± 1.98, the midpoint Tg from 113.05 ± 1.37 to
115.60 C ± 1.15 and the endpoint Tg from 115.16 ± 1.58
Tg midpoint/ C
Tg endpoint/ C
Table 2 Experimental glass transition temperatures of
b-lactoglobulin–vitamin A complexes with the addition of lactose (A1–A4, A9)
or trehalose (A5–A8, A10)
Values represent means ± standard deviations
Different letters indicate that the samples are considered significantly
different at the 5% level (p \ 0.05)
to 117.14 C ± 0.85. In the case of
b-lactoglobulin–vitamin A–trehalose powders, the onset Tg ranged from
118.45 ± 1.00 to 118.98 C ± 1.98, the midpoint Tg from
120.05 ± 1.37 to 121.88 C ± 1.90 and the endpoint Tg
from 121.81 ± 1.18 to 123.14 C ± 0.85. Similar results
were observed in the case of
b-lactoglobulin–cholecalciferol–lactose powdered products. The onset glass transition
temperature of the samples in the study ranged from
112.93 ± 1.08 to 112.99 C ± 0.96, the midpoint Tg from
118.42 ± 1.27 to 119.20 C ± 1.18 and the endpoint Tg
from 122.07 ± 1.01 to 125.08 C ± 1.92 . The
obtained results show that glass transition temperatures
vary depending on the composition of studied complexes.
The effect of disaccharides incorporation on the glass
transition temperatures was observed. It is worth
mentioning that carbohydrate–protein systems were
characterized by higher Tg values than disaccharides (lactose,
trehalose) alone. The obtained results are in agreement
with researches presented by Haque and Roos, who studied
water sorption and plasticization behavior of spray-dried
lactose/protein mixtures . The higher Tg of
carbohydrate–protein mixtures may increase the stability of the
final food products. The method and parameters of drying
did not affect the values of Tg. The results confirmed the
statement that the glass transition temperatures of foods are
mainly dependent on the moisture content and chemical
composition of the material [28, 31, 33].
Figure 2 shows the sorption isotherm curves obtained
for powdered complexes at the temperature of 25 C. For
all powders, sigmoidal shape typical for the most foods,
independently of drying methods and process parameters,
was observed. Sorption isotherms of powdered complexes
with lactose as a main component showed a decrease in
mass at water activity of 0.44–0.65 (Fig. 2a). The loss of
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Fig. 2 Water vapor sorption isotherms of powdered complexes: a
blactoglobulin–vitamin A–lactose and b b-lactoglobulin–vitamin A–
sorbed water was due to the process of lactose
crystallization. Sugars in some processed foods, such as
spraydried or freeze-dried, are usually present in the amorphous
state. All amorphous sugars are hygroscopic and have a
strong tendency to absorb the surrounding water
[30, 34, 35]. The amount of water sorbed by powdered
blactoglobulin–vitamin A–trehalose products (A5–A6, A10)
increased within full range of water activity (Fig. 2b).
Crystallization has not been observed for these complexes.
The results are in agreement with researches conducted by
Sitaula and Bhowmick , in which crystallization in the
trehalose–PBS (phosphate-buffered saline) mixtures was
not observed. The amount of water sorbed by complexes
containing lactose (A1–A4, A9) was lower comparing to
those with trehalose (A5–A8, A10). The final water content
depends on the type of sugar crystalline structure and other
components. Sugars that form hydrated crystals (trehalose)
retain higher amounts of water than sugars that form
anhydrous crystals (lactose) [34, 35].
Adsorption kinetics of b-lactoglobulin–vitamin
A–carbohydrates complexes are given in Fig. 3. At low water
activity (0.33), all powdered complexes showed typical
(a) aW = 0.33
(d) aW = 0.65
(b) aW = 0.33
(e) aW = 0.92
(f) aW = 0.92
(c) aW = 0.65
Fig. 3 Water vapor sorption kinetics of powdered complexes: b-lactoglobulin–vitamin A–lactose (a, c, e) and b-lactoglobulin–vitamin A–
trehalose (b, d, f)
sorption behavior of food powders (Fig. 3a, b). Powdered
complexes with lactose or trehalose did not show loss of
sorbed water at relative humidity of 33%. After 24 h of
adsorption at water activity of 0.33, the lowest
hygroscopicity was observed for the powders obtained by spray
drying at inlet temperature of 120 C and flux rate
64.2 mL min-1, independently of carbohydrates used.
Lactose crystallization was observed in complexes at water
activity of 0.65, independently of process parameters of
spray drying or freeze-drying (Fig. 3c). Loss of sorbed
water in spray-dried and freeze-dried products with lactose
as the main component was observed after the same period
of time (\5 h). Similar results were obtained by Haque and
Roos , who described that powdered lactose/protein
mixtures crystallized when the material was stored at water
activity 0.65. Trehalose as a main carbohydrate component
of powdered samples A5–A8 and A10, in comparison
with lactose, reduced the occurrence of crystallization. In
powdered b-LG–vitamin A–trehalose complex (A10)
obtained by freeze-drying, crystallization was observed
after 14 h of water vapor adsorption (Fig. 3d). The loss of
adsorbed water at water activity of 0.65 in b-LG–vitamin
A–carbohydrate system is the result of carbohydrate
crystallization. Crystallization properties of lactose and
trehalose are quite different . It was observed that
crystallization in powders with trehalose was delayed in
comparison with those containing lactose. Carbohydrates
(lactose, trehalose) crystallization has occurred in some
processed foods (spray drying, freeze-drying),
independently on the operating conditions [22, 28]. Lactose or
trehalose crystallization was not observed in samples
stored at water activity 0.92 for both types of powders
(Fig. 3e, f). Freeze-dried complexes with lactose and
trehalose sorbed less amounts of water than spray-dried
powders at higher relative humidity (92%).
In recent years, the study of the microstructure of food is
of increasing significance since the structure of foods can
have a profound influence on its nutritional value, rheology
and textural attributes. Food processing, such as thermal
and nonthermal processes, can alter the natural structure
and the composition of food materials [37, 38]. The
technique that is widely used to study the influence of
processing conditions and ingredients on food structure is
scanning electron microscopy (SEM) [39–41]. It is a very
useful tool to visualize food structure because it combines
the best features of light microscopy (LM) and
transmission electron microscopy (TEM). The obtained powdered
products were examined and visualized with SEM. Images
are presented in Figs. 4–6. In the studied samples, visible
differences in the shapes of the particles obtained by
different drying methods can be observed (Fig. 4a, b). The
powders obtained by spray drying had a spherical or close
Fig. 4 SEM images (magnification 9400) illustrating the
morphology of b-lactoglobulin–vitamin A–lactose particles obtained in the
form of powders by a spray drying with an inlet air temperature of
120 C and b freeze-drying with the temperature of heating shelves in
freeze dryer 30 C
Fig. 5 SEM images (magnification 94000) illustrating the
morphology of b-lactoglobulin–vitamin A–lactose particles obtained in the
form of powders by spray drying with an inlet air temperature of
a 120 C and b 160 C
to spherical structure and a great diversity of size. The
surface of larger particles was often deformed (Fig. 5a, b).
The fine particles were concentrated in the immediate
vicinity of large components. In the case of spray-dried
products, SEM images showed the presence of
semispherical microcapsules which showed dents and rough
surfaces but no evidence of fracture (Figs. 4a, 5a, b). From
the SEM images, it can be observed that lower drying
temperature led to relatively uniform size and shape with
smooth particle surface, whereas higher drying temperature
resulted in size variations and wrinkled particle surfaces
(Fig. 5a, b). Krishnan et al.  evaluated the blend of
carriers for encapsulation of cardamom oleoresin.
Microcapsules were found to be nearly spherical, but had many
dents on the surface. The morphology of powders which
were produced with blend of carriers was spherical and had
a smooth surface. Among the drying methods which are
used in food processing industries, freeze-drying is
considered one of the most advanced methods for drying high
value products sensitive to heat. Freeze-drying is carried
out in two stages: the product is first frozen, and then, the
ice is removed by sublimation directly from the solid to the
vapor phase. During freeze-drying, ice sublimation causes
significant changes in the shape and volume of the food
products. Depending on the process conditions, pores or
gaps with different characteristics are created by the ice
crystals which sublimated [43, 44]. The studied
freezedried particles were characterized by irregular structure
(Fig. 6). A denser structure with less pore sizes resulting in
harder texture was observed (Figs. 4b, 6). The origin of this
morphology is probably due to the freeze-drying
processing technique used in this work. Similar results were
obtained by Oikonomopoulou et al. . They used SEM
to visualize the microstructure of freeze-dried rice kernels
and observed particles of different shapes. The increase in
boiling time caused the increase in both porosity and
average pore size. Rhim et al.  also employed scanning
electron microscope to investigate the microstructure of
freeze-dried rice porridge samples. SEM results showed
Fig. 6 SEM images (magnification 94000) illustrating the
morphology of b-lactoglobulin–vitamin A–lactose particles obtained in the
form of powders by freeze-drying with the temperature of heating
shelves in freeze dryer 30 C
rigid and porous cube type rice porridges that were
obtained after freeze dehydration. No shrinkage was
observed with all samples.
The obtained results have shown the disaccharides impact on
thermal properties of the final products. The glass transition
temperatures were dependent on the composition of studied
samples. In the case of trehalose incorporation into products,
the temperatures were higher than in samples containing
lactose. The carbohydrate- b-lactoglobulin systems were
characterized by higher Tg values than disaccharides alone.
In food systems, a higher Tg can be assumed to improve
protection and stability of encapsulated compounds, such as
vitamin A. Sorption isotherm curves obtained for powdered
complexes showed the sigmoidal shape. The amount of
water sorbed by powdered b-lactoglobulin–vitamin
A–lactose complexes was lower comparing to those with trehalose,
independently of environmental water activity. The present
study has shown that spray-dried and freeze-dried powdered
complexes with lactose or trehalose have different
time-dependent crystallization behavior. In the case of
b-lactoglobulin–vitamin A–trehalose products, the carbohydrate
crystallization at water activity of 0.65 was delayed in
comparison with b-lactoglobulin–vitamin A–lactose
samples. The addition of trehalose to food systems could
improve the quality of the powders by delaying
crystallization. It is of great importance because the extent of
crystallization is critical to acceptance of the final product. The
study showed the impact of different drying methods on the
shapes of the particles. The powders obtained by spray
drying had a spherical or close to spherical structure and a
great diversity of size. The surface of larger particles was
often deformed. The studied freeze-dried particles were
characterized by irregular structure. The information on
microstructure changes is essential for finding the
relationship between food processing conditions and morphological
changes of the food components. It enables better process
control and improvement in the appearance of the sample by
optimizing pretreatment and drying parameters. As the
structure of foods can have a profound influence on its
nutritional value, the proper conditions can help to retain the
quality of the final product.
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