Journal of Food Engineering 67 (2005) 157–165

www.elsevier.com/locate/jfoodeng

Effects of carbohydrate crystallization on stability of

dehydrated foods and ingredient formulations
Pilar Buera *,1, Carolina Schebor 1, Beatriz Elizalde
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina

Received 10 October 2003; accepted 1 May 2004

Abstract

Sugars are important components of the amorphous regions of dehydrated foods, and their structure may define product per-
formance. The objective of present work was to analyze the impact of sugar crystallization on product quality, and to discuss some
strategies to avoid sugar crystallization. Chemical changes, (non-enzymatic browning, lipid oxidation and enzymatic activity) were
accelerated upon crystallization of the sugar matrix. Oxidation of encapsulated pigments was not prevented in the glassy state but it
was delayed by crystallization inhibition of the encapsulating sugar. At a kinetic level, sugars promote the formation of glassy matri-
ces delaying deteriorative reactions. At a specific-interaction level, dehydroprotectants interact with proteins and membranes and
stabilize them by hydrogen bonding. Both aspects are related, since specific interactions cannot be manifested if the sugar is in
the crystalline state and vitrification is the property which ensures inhibition of crystallization. The study of delay/inhibition of sugar
crystallization in supercooled liquids may increase the range of applications of sugars for specific purposes.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Amorphous sugars; Dehydration; Crystallization; Dehydroprotectants; Structure

1. Introduction organisms under extreme conditions. Among them,

The preservation of biomolecules in the dry state has
acquired new interest in technological, clinical, biological
and pharmaceutical fields. During drying, a certain degree
of deterioration may occur, and the stabilization of bio-
materials by their incorporation into carbohydrate and/
or polymer solutions before drying is a known preserva-
tion procedure (Colaço, Sen, Thangavelu, Pinder, &
Roser, 1992; Crowe, Crowe, Carpenter, & Winstrom,
1987; Leslie, Israeli, Ligthart, Crowe, & Crowe, 1995;
Rossi, Buera, Moreno, & Chirife, 1997). Many sugars
are involved in preservation mechanisms of living
*
Corresponding author. Tel.: +54 11 4576 3397; fax: +54 11 4576
3366.
E-mail address: pilar@di.fcen.uba.ar (P. Buera).
1
Member of Consejo Nacional de Investigaciones Cientı́ficas y
Técnicas de la República Argentina.
the non-reducing sugar trehalose is found at particular
high concentrations in anhydrobiotic organisms, and res-
urrection plants (Salahas, Peslis, Georgiou, & Gavalas,
1997). Several seeds accumulate sucrose during develop-
ment and b-fructofuranosyl oligosaccharides (raffinose,
stachyose or verbascose) play a key role in the acquisition
of seed desiccation tolerance (Koster & Leopold, 1988;
Obendorf, 1997). These solutes have proved also to pro-
vide stabilization of dried or frozen labile biomaterials
in vitro, which may be required for technological or re-
search applications in different fields (Arakawa & Tima-
sheff, 1982; Colaço et al., 1992; Crowe, Carpenter, &
Crowe, 1998; Duddu & Dal Monte, 1997). Several
mechanisms have been proposed to explain this protective
effect. In the water replacement hypotheses, the stabiliza-
tion was attributed to the formation of hydrogen bonds
between sensitive components and disaccharide mole-
cules when water is removed. The structural integrity of

0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.05.052
158 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165
membranes and proteins is thus maintained (Crowe et al.,
1987; Womersley & Smith, 1981). A second hypothesis is
related to the ability of carbohydrates and polymers to
form a glassy structure during drying under suitable con-
ditions (Crowe, Crowe, & Carpenter, 1993; Franks, 1993;
Slade & Levine, 1991) where the sensitive components are
embedded. Amorphous glassy solids are meta-stable
materials in a non-equilibrium state, since it is located
below the saturation curve in the temperature-composi-
tion state diagram (Fig. 1), where the stable form is the
crystal. However, crystallization of the amorphous solid
is kinetically delayed if the sample remains in the glassy
state, below the glass transition temperature (Tg). The
Tg of a material is a function of the relative proportion
of its glass-forming components and of the water content.
In the glassy state, most structural changes occur very
slowly and only small motions of molecules, mainly rota-
tional motions of side chains and vibrations, may occur
(Levine & Slade, 1986; Slade & Levine, 1991). Molecular
mobility in the amorphous region of the solid is important
in determining its physicochemical stability (Hancock &
Zografi, 1997). Crystallization of sugars may occur as
the material undergoes the glass transition and above
the transition with a rate dependent on the temperature
difference, T–Tg (Roos & Karel, 1991). Most physical
changes in amorphous materials (including stickiness
and structural collapse, or shrinkage) result from the
sharp decrease of viscosity which occurs above their Tg
(Levine & Slade, 1986; Roos & Karel, 1991; Slade &
Levine, 1991). There is evidence, however, that the main-
tenance of a glassy structure is not the only factor
controlling enzyme stability, or chemical reactions like
non-enzymatic browning and lipid oxidation (Cardona,
Schebor, Buera, Karel, & Chirife, 1997; Crowe et al.,
1993; Crowe et al., 1998; Mazzobre, Buera, & Chirife,
1997).
The action of cryo and dehydroprotectants can thus
be ascribed to both kinetic and specific effects. At the ki-
netic level, cryo and dehydroprotectants promote the
formation of amorphous, glassy systems, inhibit
crystallization and influence the kinetics of deteriorative
Fig. 1. Phase diagram indicating the equilibrium solubility curve, the
glass transition curve, and the time-dependent crystallization zone for
sugars.
reactions upon storage. At the specific-interaction level,
protectants are believed to interact with biological struc-
tures and stabilize them during both, freezing or drying,
although by different mechanisms. Crowe et al. (1998)
reported that vitrification of the structure is necessary
to improve enzyme and liposome stability, but specific
hydrogen-bond interactions between sugars and the bio-
material are also needed.
The objective of the present work was to analyze the
relationship between several specific properties of amor-
phous sugars and the impact of their crystallization on
the maintenance of product quality, and to discuss sev-
eral strategies to avoid sugar crystallization.
2. Modification of physico-chemical aspects due to sugar
crystallization
The stabilizing effect of sugars in the amorphous state
can be related to decreasing the rates of: (a) chemical
reactions (non-enzymic browning, enzymic activity, acid
hydrolysis of sucrose), (b) protein denaturation or
enzyme inactivation, (c) volatiles release, and (d) mem-
brane disruption. The mechanisms by which amorphous
sugars manifest these different protective effects are,
however, of very different nature on each case. Amor-
phous sugars are highly hygroscopic and they may sorb
large amounts of water from the surroundings, resulting
in crystallization during storage above a critical, temper-
ature-dependent relative humidity (Levine & Slade,
1986). Once crystallization is initiated, sugar molecules
become tightly packed, and the amount of water that
can be held therefore decreases, and, in closed contain-
ers, water remains in the amorphous phase and causes
a significant depression of the Tg. Therefore, crystalliza-
tion may also increase the rate of diffusion-controlled
reactions (Roos, Jouppila, & Zielasko, 1996). Sugars
that form anhydrous crystals (sucrose, lactose) may
crystallize at room temperature after adsorbing the
amount of water needed to decrease their Tg to below
room temperature (water contents about 3–4% for
sucrose and 5% for lactose, in dry basis). Sugars that
form hydrated crystals (trehalose dihydrate and raffi-
nose tri, tetra or penta hydrates), retain higher amounts
of water (about 10–12% in dry basis) without crystalliza-
tion, since they need not only the water content neces-
sary to get a Tg value below room temperature, but
they also need to have enough water to form the
hydrated crystals (Crowe, Reid, & Crowe, 1996;
Iglesias, Buera, & Chirife, 1997; Iglesias, Schebor,
Buera, & Chirife, 2000; Kajiwara & Franks, 1997;
Saleki-Gerhardt, Stowell, Byrn, & Zografi, 1995).
Table 1 lists the results of several experiments which
showed that crystallization of the amorphous sugar
matrices markedly accelerated some deteriorative reac-
tions in foods, biological and model systems.
 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165 159

Table 1
Reactions that were observed to be accelerated upon crystallization of sugar matrices
Reaction Reference
Non-enzymatic browning Karmas and Karel (1994) and Burin et al. (2000)
Enzymatic activity Kouassi and Roos (2000, 2001)
Acid hydrolysis of sucrose Schebor et al. (1994)
Enzyme activity loss Cardona et al. (1997), Mazzobre et al. (1997), Suzuki et al. (1997),
and Miller et al. (1998)
Damage of vesicles, biological membranes and liposomes Crowe et al. (1987, 1996, 1998), Sun et al. (1996),
Sun and Leopold (1997)
Dry liposome rupture Sun et al. (1996)
Release and oxidation of encapsulated lipids Labrousse et al. (1992)
Diacetyl loss Senoussi et al. (1995)

2.1. Chemical reactions

The increased rates of chemical reactions upon sugar

crystallization may be explained through the scheme
depicted in Fig. 2a. When the matrix is amorphous,
chemical reactions are delayed because the reactants (a) Amorphous matrix Crystalline matrix
remain diluted in a highly viscous medium. When sugar
crystallizes, they are concentrated in the matrix, excluded Reactants
from the crystals, and the reaction rate increases. It is Amorphous sugar
well recognized that non-enzymatic browning reaction
Crystalline sugar
is a major deteriorative factor in the storage of dehy-
drated food products (Labuza & Saltmarch, 1981; Roos
Protein protected Protein denatured
et al., 1996). Karmas and Karel (1994) showed how in an amorphous in a crystalline
browning development was delayed by the presence of (b) matrix matrix
maltodextrin in a trehalose matrix containing browning
reactants, in comparison with a matrix of trehalose
alone. At the water activity at which trehalose crystal-
lizes at 25 °C (aw = 0.43), browning reactions are accel-
erated, and this was not observed in the maltodextrin
system, in which trehalose crystallization was delayed. Membrane Membrane
protected in an disrupted in a
Kouassi and Roos (2000, 2001) observed that in a amorphous sugar crystalline matrix
lactose matrix the enzymatic activity of b-fructofuranos- (c) matrix
idase was detected concomitantly with sugar crystalliza-
tion, and that the inhibition of crystallization by the Solute encapsulated
incorporation of maltodextrin retarded the enzyme in a crystalline sugar
matrix
activity by about one order of magnitude. In another
kind of chemical reaction, acid hydrolysis of sucrose
occurred faster in crystallizing lactose matrices than in
systems in which lactose crystallization was delayed by Solute released from
the crystalline sugar
the presence of carboxymethyl cellulose and trehalose matrix
Solute encapsulated
(Schebor, Mazzobre, Burin, Buera, & Chirife, 1994). in an amorphous
(d) sugar matrix

2.2. Protein denaturation
The effect of crystallization on protein denaturation is
schematically represented in Fig. 2b. In this case, the
hydroxyl groups of sugars have to interact with the
hydrophilic sites of proteins in dehydrated systems in or-
der to avoid protein denaturation. When sugar crystal-
lizes, this effect cannot be performed anymore, and the
protein is excluded from the sugar crystals, where, be-
sides lacking the stabilizing effect of hydroxyl groups
Fig. 2. Schematic interpretation of the effect of sugar crystallization
on: the acceleration of chemical reactions (a), protein denaturation (b),
membrane integrity (c) and release of encapsulated compounds (d).
of the sugar, the changes in pH, concentration of reac-
tive groups and ionic strength may also negatively affect
their stability. This behavior was observed for b-galac-
tosidase and b-fructofuranosidase in dehydrated sugar
matrices. In a crystallizing matrix of trehalose the degree
160 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165
of enzyme inactivation sharply increased in the aw re-
gion at which trehalose crystallizes (Cardona et al.,
1997; Mazzobre et al., 1997).
2.3. Membrane and cell damage
Certain solutes added to protect cells during drying
and rehydration may act on membrane integrity and/
or on protein structure of the cells. The hydrogen bond-
ing capacity of compounds added as protective agents of
phospholipid head groups and proteins of membranes
could be the critical factor which determines the survival
of cells submitted to different treatments (Sun, Leopold,
Crowe, & Crowe, 1996; Sun & Leopold, 1997). In the
case of membrane stabilization, sugars may act to pre-
vent the fusion of phospholipids heads. Fig. 2c schema-
tizes the action of sugars preventing damage to
membranes, and how this effect is lost when the sugar
crystallizes.
Crowe et al. (1987) found that maltose and trehalose
gave the best stability to freeze-dried unilamellar vesicles
and biological membranes, compared with other mono,
di or tri saccharides. They also showed that some poly-
mers (such as dextran and hydroxyethyl starch) did not
have direct interactions with the head group phosphates,
nor lowered the melting transition phase of the lipids
and did not protect liposomes during drying (Crowe
et al., 1996). The combination of trehalose to improve
membrane and protein thermal resistance and a high
molecular weight compound, such as maltodextrin, to
improve physical stability (delaying sugar crystalliza-
tion) was particularly analyzed in dehydrated yeast cells
(Lodato, Huergo, & Buera, 1999). The analysis of these
systems illustrated both the lack of correlation between
the physical stability of the external matrix and the
remaining stability of yeasts, and the importance of tre-
halose concentration in the external matrix. Mazzobre,
Hough, and Buera (2003) made a distinction between
the different requirements to protect yeast cells or en-
zymes in sugar matrices: while incorporation of 50%
maltodextrin was detrimental for yeast stability, it im-
proved enzyme stability, avoiding sugar crystallization.
2.4. Release of encapsulated volatiles and lipids
For encapsulated compounds there are even more
complex interactions than for soluble components. The
effect of amorphous sugars on aroma retention during
storage has been extensively investigated (Chirife, Karel,
& Flink, 1973; Flink & Karel, 1972). Senoussi, Dumou-
lin, and Berk (1995) showed that diacetyl retention
decreased sharply in a lactose matrix, when about 60%
of lactose crystallized. In dehydrated skim milk, how-
ever, lactose crystallization was delayed (probably by
the presence of proteins and salts), and the sharp in-
crease of diacetyl loss observed in the lactose matrix
did not occur. In systems containing encapsulated lipids,
the protective action of the solid matrix is lost when
crystallization occurs. In non-crystalline sugar systems,
the stability of encapsulated lipids may be increased
upon collapse of the samples above the glass transition
temperature (Labrousse, Karel, & Roos, 1992). A com-
promise between structural collapse (favorable for
retention of encapsulated compounds) and matrix crys-
tallinity (promoting the release of encapsulated com-
pounds) was always observed. It is interesting to note
that the loss of b-carotene was related to the crystalliza-
tion of the sugar matrix, but the moisture content of the
amorphous phase was determinant of the extent of car-
otene remaining encapsulated (Elizalde, Herrera, &
Buera, 2002; Prado, Elizalde, & Buera, 2002; Sutter,
Elizalde, & Buera, 2002).
The effect of crystallization on the release of volatiles
and lipids is schematically represented in Fig. 2d. Sugar
crystals can exclude the solute and it is released. How-
ever, depending on crystallization conditions the crystals
can encapsulate the solutes.
3. Modifying the kinetics of sugar crystallization
On the basis of previous studies, several strategies can
be proposed to avoid sugar crystallization in a formu-
lated system:
3.1. Vitrification
As previously discussed, regarding the phase diagram
shown in Fig. 1, when a solid is maintained below Tg, in
the glassy state, crystallization is virtually nonexistent.
Vitrification can then be considered as an important
strategy to avoid sugar crystallization. However, Crowe
et al. (1998) proposed that vitrification was necessary
but not the only requirement for stabilization of lipo-
somes at low moisture contents. The good glass-former
polymers maltodextrin and PVP, were not effective in
protecting the yeasts during heat treatment (Lodato
et al., 1999). On the contrary, maltose and trehalose
matrices protected cell viability very well even in the liq-
uid-supercooled state, but a critical concentration of
disaccharides was necessary for the retention of yeast
viability during heating. The protection provided by tre-
halose and maltose on yeast cells could be due to a spe-
cific effect, not directly associated with their capacity to
form an external glassy matrix. Suzuki, Imamura,
Yamamoto, Satoh, and Okazaki (1997) enhanced the
importance of the non-crystalline nature of the sugar
matrix on the stability of enzymes and Rossi et al.
(1997) confirmed that the stabilization of a restriction
enzyme could be achieved in liquid supercooled matri-
ces. Tan et al. (1995) demonstrated that not only the
physical stability (as determined by Tg values), but also
 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165 161

the hydrogen bonding capacity of the saccharides was

important to determine the number of germinating mold
spores after storage at 30 °C.
It can thus be generalized that if the adequate poli-
hydroxylic compounds (namely sugars) are maintained
vitrified; their protective action will be preserved. How-
ever, if sugar crystallization is inhibited or conveniently
delayed, the protective action may be extended to the
supercooled-liquid state.

3.2. Combination of sugars and polymers

Several authors have indicated that the presence of a

polymer promoted a delay in the crystallization process
of sugars: sucrose crystallization was inhibited by
starch (Roos & Karel, 1991) and by corn syrup polymers
(Gabarra & Hartel, 1998) and trehalose crystallization
was delayed by maltodextrins (Mazzobre et al., 1997).
In skim milk powder (Jouppila & Roos, 1994; Senoussi
et al., 1995) and modified-whey powders (Burin, Joup-
pila, Roos, Kansikas, & Buera, 2004) the presence of
proteins caused a delay of lactose crystallization, in
comparison with pure lactose systems. Fig. 3 shows
thermograms of mixtures of sugars and proteins. It
can be seen that the presence of gelatin inhibits the crys-
tallization of the sugar raffinose (Fig. 3a) and that in the Fig. 4. Delay of sugar crystallization by the incorportaion of a second
sugar. (a) Dynamic DSC thermograms showing sucrose (S) crystalli-
presence of BSA crystallization of both sucrose and
zation temperature shift to higher temperature values when raffinose
raffinose is inhibited (Fig. 3b), even at very high relative (R) is added. (b) Isothermal DSC thermograms showing the time
humidities (84% RH) (Schebor, Moreno, Chirife, & crystallization shift when raffinose is added to sucrose (Mazzobre
Buera, 2002). et al., 2003).

3.3. Combination of various sugars
In the case of seeds, embryos and plant tissues several
oligosaccharides occur, this fact was related to seed
longevity due to protection of proteins and mem-
branes, and to the prevention of sucrose crystallization
(Obendorf, 1997). The effect of the mixture of several
sugars was analyzed in freeze-dried model systems. In
trehalose–lactose systems the time to crystallization of
lactose increased when trehalose was added (Mazzobre,
Soto, Aguilera, & Buera, 2001). The addition of raffi-
nose had also a retarding effect on sucrose crystalliza-
tion (Fig. 4a and b) (Mazzobre et al., 2001). It can be
observed that in the dynamics runs (Fig. 4a) the onset
temperature for sugar crystallization increased when
raffinose was added, and was even avoided during the
run if enough amount raffinose was added. In the iso-
thermal run (Fig. 4b), both the crystallization time and
the rate of sucrose crystallization increased in the sys-
tems containing raffinose.

Fig. 3. DSC thermograms of sugar/protein samples showing the crystallization of the sugar and/or the denaturation of the protein. Raffinose/gelatin
samples (a), BSA/sucrose and BSA/raffinose (b) (Schebor et al., 2002).
162 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165
In the food industry the addition of a polymeric
material to avoid crystallization, as previously discussed
cannot always be performed, due to quality and senso-
rial considerations. It would be thus necessary to use a
material of similar characteristics to the one it is meant
to stabilize, and the employment of a second sugar may
serve to this purpose (Mazzobre et al., 2001).
3.4. Combination of sugars and salts
Evidence exists on the modification of some physical
properties of sugars by salts (Miller, de Pablo, & Corti,
1997; Miller, Anderson, & de Pablo, 1998). The effect of
electrolytes is of special interest because of their univer-
sal presence in biological systems, their major influence
on water structure and their possible interactions with
biomolecules. The synergistic effects of sugars and diva-
lent cations on protein stabilization have been reported
(Carpenter, Crowe, & Arakawa, 1986) but they are yet
to be explained. Several physical methods have provided
evidence for the existence of sugar–metal complexes in
solution and many complexes of sugars and sugar deriv-
atives with inorganic salts have been isolated in solid,
and often in crystalline form (Angyal, 1973; Morel-
Desrosier, Lhermet, & Morel, 1991; Rongere, Morel-
Desrosiers, & Morel, 1995). Mazzobre and Buera
(1999) reported that magnesium chloride could be a
synergistic compound to extend the range of trehalose
on the protection of the enzyme b-galactosidase. Fig. 5
shows that the inhibition/delay of trehalose crystalliza-
tion was afforded by the presence of salts, and especially
MgCl2 and KCl, with parallel retention of the enzymatic
activity. It is interesting to note that the Tg values of the
mixtures trehalose-salts had the same values than the
pure sugar systems (Mazzobre & Buera, 1999). Recent
experiments employing sucrose confirmed that the
presence of salts affected the kinetics of sucrose crys-
tallization, besides modifying the sorption behavior
Mg
K
75
% Remaining activity
Na
50
Cs
25
0 1994). Water crystallization occurs at higher moisture
0 10 20 30 40 50
Fig. 5. Effect of salts on the degree of trehalose crystallization, and
parallel b-galactosidase activity retention in trehalose or trehalose-salt
matrices of mass fraction of water w = 0.1, after storage at 45 °C.
(increasing the water uptake) and the kinetics of ice crys-
tallization in solutions of higher water content. The de-
gree at which the salts modified the properties of the
sugar systems was in the order Mg > K > Na > Cs,
which is the same as the charge/radius ratios of those
cations. It is proposed that the salt effect on sugar crys-
tallization occurs at a molecular level, but in such a dy-
namic way that the cooperative relaxations leading to
glass transitions are not affected (Longinotti, Mazzobre,
Buera, & Corti, 2002). It is interesting to note that by
the addition of salts the protective effects of the sugars
can be extended to a liquid-supercooled region of the
phase diagram, at which sugar crystallization occurs fast
in the absence of salts.
4. Evidences of delayed sugar crystallization in biological
and food systems
The several examples analyzed in the previous sec-
tions indicated that the quality of the product may be
maintained when crystallization is delayed/inhibited.
As mentioned before, in those matrices formed mainly
by sugars, crystallization has a direct impact on quality
loss. Since biological and food materials are frequently
very complex systems, it can be expected that sugar crys-
tallization is naturally delayed in them, in comparison
with that of pure sugars, by the presence of polymers,
other sugars and/or salts. Indeed, Table 2 lists some lit-
erature references which show that sugar crystallization
is naturally delayed in complex foods and biological sys-
tems. Fig. 6 shows thermograms of yeast components
mixed with trehalose. It can be seen that trehalose crys-
tallization was not possible in the presence of yeast cyto-
plasmatic solutes. However, in the presence of yeast
proteins trehalose crystallization was detected, which
indicates that other solutes present in the cytoplasm
are responsible for the delay/inhibition of sugar crystal-
lization (Espinosa, Schebor, Buera, Moreno, & Chirife,
2002).
Crystallization in the cytoplasm was proposed as a
cause of viability loss in living organisms, however, no
studies exist in which sugar crystallization was detected
in vivo (Buitink, Hoekstra, & Hemminga, 2000a; Bui-
tink, van den Dries, Hoekstra, Alberda, & Hemminga,
2000b; Schebor, Galvagno, Buera, & Chirife, 2000;
Sun & Leopold, 1997). The only components with
detectable crystallization in seeds and embryos (with
No salt deleterious consequences) are water and lipids, although
at subzero temperatures (Vertucci, 1989; Williams,
60 70 levels than those of dehydrated media (the moisture con-
tents need to observe water crystallization were from
25% (in dry basis) for yeast, recalcitrant seeds, cereal
leaves and strawberries to about 50% (in dry basis),
for silver maple seed) (Vertucci, 1989).
 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165 163

Table 2
Evidences of delayed sugar crystallization in biological and food systems
System Involved sugars Reference
Vegetables Glucose/fructose/sucrose Roos (1987) and Karmas et al. (1992)
Milk Lactose Jouppila and Roos (1994) and Senoussi et al. (1995)
Yeasts Trehalose Schebor et al. (2000) and Espinosa et al. (2002)
Seeds Sucrose, raffinose Sun and Leopold (1997), Williams (1994), and Buitink et al. (2000a)
Embryos Sucrose, raffinose Koster (1991) and Sun and Leopold (1997)
Several proteins Lactose Haque and Roos (in press)

glass transition temperature of the systems is one of

Fig. 6. DSC thermograms showing the effect of yeast components on
trehalose crystallization in yeast extract-trehalose and yeast protein-
trehalose mixtures.
5. Concluding remarks
Table 3 summarizes some of the explanations for the
stabilizing properties of the amorphous sugars, and the
additional requirements, besides their amorphous condi-
tion, that are needed to perform the protective effect.
Several discussions have arisen during the last decade
about the real impact of the glass transition temperature
on food stability. The conclusion of the above observa-
tions, may establish that in matrices composed mainly
by sugars, vitrification is the property which ensures
the amorphous state in the matrix, and avoiding sugar
crystallization is the first step to define product stability.
This is the case of some pharmaceutical preparations,
ingredients and model systems. In these systems the first
limiting factor of their stability is the crystallization of
the amorphous matrix. The glass transition of the sys-
tems is indirectly involved, since it is the factor that al-
lows crystallization to be observed. Therefore, the
the main factors to take into account to predict stability.
However, chemical reactions are not completely inhib-
ited in them, since below the macroscopic Tg there
may be local heterogeneities that allow diffusion and
reaction. If the reaction starts in local zones below the
macroscopic Tg (as determined by DSC), the water con-
tent around the reaction site may increase, allowing fur-
ther reaction to occur through further plasticization.
Deteriorative reactions were also related to the physical
structure of the matrix, either porous or mechanically
compressed (Buera & Karel, 1995; Burin, Jouppila,
Roos, Kansikas, & Buera, 2000).
In complex natural biological systems and foods, in
which the presence of polymers, other sugars and salts
naturally inhibit or delay sugar crystallization, the sta-
bility of the system will be limited by the degree at which
physicochemical reactions (mainly non-enzymatic
browning, lipid oxidation or protein denaturation) can
occur. In fact, amino-carbonyl condensations (or Mail-
lard reaction), decreased enzyme activities and lipid oxi-
dation are known as important factors limiting the
viability of seeds, pollen and dehydrated yeasts. These
reactions are on turn affected by several factors, such
as temperature, water activity, Tg. The heterogeneous
nature of biological materials makes it difficult to estab-
lish a clear relationship between an overall physical
property (such as glass transition temperature) and the
kinetics of a chemical reaction. Local viscosity (at a
molecular level) instead of bulk viscosity (related to
the glass/supercooled liquid) seems to determine the
mobility of small molecules (Chinachotti, 1997). Supra-
molecular relaxations, such as those occurring at Tg,
do not necessarily have a direct impact on changes

Table 3
Stabilizing properties of amorphous sugars, mode of action, and additional structural requirements to perform the protection
Stabilizing property
Inhibition of browning
and enzymatic reactions
Protective effect on proteins
Protective effect on membranes
Lipid encapsulation
Explained by
Formation of high viscosity media
Interaction of sugar hydroxyls with
hydrophilic protein sites
Interaction with phospholipid heads preventing
melting; decreasing membrane phase transitions;
protection to protein membranes
Surface properties
Additional structural requirements
Non-reducing stable sugars in glassy state
is the best combination, avoiding collapse or compression
Not crystalline (either glassy or supercooled)
Not crystalline (either glassy or supercooled)
Not crystalline, collapsed, compressed
164 P. Buera et al. / Journal of Food Engineering 67 (2005) 157–165
occurring at a molecular level, such as those above-men-
tioned reactions. On the basis of previous work of other
researchers and of our group it could be hypothesized
that kinetics and specific interactions of sugars and bio-
molecules are important and are somehow related, since
the specific interactions leading to biomolecule protec-
tion can only be manifested if the sugar is in the amor-
phous state (Suzuki et al., 1997), and vitrification is the
property which ensures inhibition of crystallization.
Acknowledgments
Financial support from Universidad de Buenos Aires
(EX107), CONICET, ANPCyT (PICTS 06-5066 and
09 = 6251) and from the International Foundation for
Science (Sweden) is acknowledged. This work is part
of CYTED Project XI.13.
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