STATE TRANSITIONS AND ENZYMATIC ACTIVITY IN...
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STATE TRANSITIONS AND ENZYMATIC ACTIVITY IN LOW MOISTURE CARBOHYDRATE FOOD SYSTEMS
Gilles Kouame Kouassi
ACADEMIC DISSERTATION
To be presented, with permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in lecture hall B2, Viikki, Helsinki, on
January 31st, 2003, at 12 o'clock noon.
Helsingin yliopisto Soveltavan kemian ja mikrobiologian laitos
Elintarviketeknologian laitos
University of Helsinki Department of Applied Chemistry and Microbiology
Department of Food Technology
EKT-sarja 1278 EKT series 1278
Custos
Professor Vieno PiironenDepartment of Applied Chemistry and MicrobiologyUniversity of Helsinki
Supervisors
Professor Yrjö H. RoosDepartment of Food and Nutritional SciencesUniversity College, CorkCork, Ireland
and
Professor Kirsi JouppilaDepartment of Food TechnologyUniversity of Helsinki
Reviewers
Dr Pilar BueraDepartment de IndustriasFacultad de Ciencias y Naturales1428 Buenos Aires, Argentina
and
Dr Roger ParkerInstitute of Food ResearchFood Molecular Biochemistry DepartmentNorwich Research Park, Colney LaneNorwich NR7 3UA, UK
Opponent
Professor Elisabeth DumoulinDepartment of Food Process EngineeringENSIA, France
ISBN 952-10-0888-1 (paperback)ISBN 952-10-0893-8 (HTML)ISBN 952-10-0889-x (pdf)ISSN 0355-1180
Helsinki University PressHelsinki, 2003
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Kouassi, K.G 2003. State transitions and enzymatic activity in low moisture carbohydrate food systems. EKT series 1278. University of Helsinki, Department of Food Technology, ABSTRACT Effects of thermal transitions (glass transition, crystallization), water, and glycerol on invertase
activity in freeze-dried carbohydrate systems were investigated. Efficacy of κ-carrageenan to
delay sugar crystallization in freeze-dried carbohydrate systems was also studied.
Water sorption properties and glass transition temperatures, Tg, were determined for freeze-dried
carbohydrate-sucrose systems embedded with invertase, and stored under various relative
humidity (RH) conditions, at 24°C. Invertase activity was determined by measuring glucose,
fructose, and sucrose contents using HPLC or glucose content using an enzymatic method as a
function of storage time. The extent of crystallization in lactose-sucrose-invertase (LSI) and
lactose-sucrose-invertase-carrageenan (LSI-carrageenan) was determined by powder X-ray
diffraction and reflected on changes in water sorption behavior. DSC thermograms of samples
stored at various RH conditions were obtained by scanning samples at a heating rate of 5°C/min
from at least 35°C below the glass transition temperature ranges. Relationships between RH,
water content, Tg, and rates of sucrose hydrolysis were analyzed.
Crystallization in lactose-sucrose-invertase systems was observed at RH as low as 44.4% and it
became substantial above RH 53.8% concomitantly with sucrose hydrolysis. In non-crystallizing
systems, sucrose hydrolysis was effective at RH 66.2 % and above, but was not directly related to
Tg. In systems composed of maltodextrin matrix, crystallization was not manifested in changes of
sorption properties. Maltodextrin could be used in preparation of food materials to delay
crystallization. Water sorption study and X-ray diffraction results demonstrated that κ-
carrageenan was effective in delaying crystallization. This result is of great importance for
industrial processes where crystallization needs to be controlled. Glycerol enhanced the rate of
sucrose hydrolysis. Plasticizers, such as glycerol, can affect the enzyme activity even at low RH.
Water is the most important plasticizer but also a key factor controlling the activity of the enzyme
and possibly plasticization of the enzyme itself.
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PREFACE The present study was carried out at the Department of Food Technology, University of Helsinki during the years 1998-2002. During that period, I spent seven months at the Division of Food Science, University of Nottingham, UK. Professor Yrjö Roos supervised my studies and introduced the field of “Food Material Science” to me. Through numerous cordial and interesting discussions, he provided me with the necessary ground for understanding state and phase transitions and their relationships with rates of chemical reactions in foods. I highly appreciate his guidance and the excellent research environment he provided me with. His insightful advice and support in experimental work and reporting was priceless. I wish to express my sincerest thanks and deepest gratitude for his help and support of all kinds during these years. I am deeply indebted to supervision given by Professor Kirsi Jouppila and her constant supportive and friendly attitude to me. I am grateful for the very fruitful comments and suggestions in experiments as well as in writing and reporting. She was eager to find solutions to most of my problems. I wish to thank her from my heart for her attention. I wish to thank Professor Vieno Piironen for her comments and suggestions on my thesis. My thanks go also to Professor Lea Hyvönen for giving me the opportunity to carry out my research in the Department of Food Technology. I am grateful to Professor John Mitchell for giving me the opportunity to conduct part of my study in the Division of Food Science of the University of Nottingham. I wish to thank Dr Bill MacNaugtan for his guidance in Solid State NMR, and all my nice friends in Sutton Bonington. I am grateful to the reviewers of my thesis, Dr Pilar Buera and Dr Roger Parker for their enriching and constructive comments and suggestions. The present study was financially supported by the Commission of the European Communities, Agriculture and Fisheries (FAIR) Specific RTD program, CT 96-1085, Enhancement of the Quality of Food and Related Systems by Control of Molecular Mobility (Molecular Mobility in Foods). It does not necessarily reflect its views and in no way anticipate the Commission's future policy in the area. Financial support from the Academy of Finland, project number 163246, is also appreciated. I want to thank my colleagues and friends for their help and valuable comments during these years, and also for sharing with me wonderful moments. Finally, I would like to thank my family and relatives for their help and encouragement. I owe special thank to my mother for her constant believe in me. I would not forget my dear Marie Claire for her support and attention, and Joel Alexis (Jojo) my wonderful son. Helsinki, December 2002
Gilles Kouame Kouassi
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CONTENTS ABSTRACT 2 PREFACE 3 LIST OF ORIGINAL PAPERS 6 ABBREVIATIONS 7 1 INTRODUCTION 8 2 JUSTIFICATION OF THE PRESENT STUDY 10 3 LITERATURE REVIEW 11
3.1 State and reactions of low-moisture food systems 11 3.1.1 Amorphous state and state transitions 11 3.1.2 Water sorption and plasticization 12 3.1.3 Glass transition and crystallization 17 3.1.4 Composition, dynamics, and phase separation in amorphous systems 19
3.2 Enzyme activity in low-moisture food systems 21 3.2.1 Hydrolytic enzyme and observed changes 21 3.2.2 Water activity control/dependence 23 3.2.3 Effect of substrate 25 3.2.4 pH in low moisture systems 27 3.2.5 Effect of temperature 28 3.3.6 Other rate controlling factors 29
4 MATERIALS AND METHODS 30 4.1 Preparation of amorphous carbohydrate food systems 30
4.1.1 Lactose-sucrose-invertase (LSI) and lactose-sucrose-invertase 31 -carrageenan (LSI-carrageenan) systems
4.1.2 Maltodextrin-sucrose-invertase (MDSI) and maltodextrin-lactose- sucrose-invertase (MDLSI) systems 31
4.1.3 Pullulan-sucrose (PS), pullulan-sucrose-invertase (PSI) and pullulan- sucrose-invertase-glycerol (PSIG) systems 31
4.2 Characterization of food systems 32 4.2.1 Water sorption isotherms 32 4.2.2 Glass transition 32
4.3 Evaluation of crystallization 33 4.3.1 Gravimetric method 33 4.3.2 X-ray diffraction 35
4.4 Determination of sucrose hydrolysis 35 4.4.1 Determination of glucose content by spectrophotometry: enzymatic 36
method 4.4.2 Determination of sugar contents by HPLC 38
5 RESULTS AND DISCUSSION 39 5.1 Water sorption isotherms 39
5.1.1 Lactose-sucrose-invertase and lactose-sucrose-invertase-carrageenan systems 39
5.1.2 Maltodextrin-sucrose-invertase systems with and without lactose 42 5.1.3 Pullulan-sucrose-systems: effects of glycerol 42
5.2 Glass transition 43 5.2.1 Lactose-sucrose-invertase and lactose-sucrose-carrageenan systems 43 5.2.2 Maltodextrin–sucrose-invertase systems with and without lactose 44 5.2.3 Pullulan-sucrose-systems: effects of glycerol 45
5.3 Sucrose hydrolysis 47
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5.3.1 Effect of glass transition 47 5.3.2 Effect of water 49 5.3.3 Effect of nonaqueous plasticizers, additives, and composition change 50 5.3.4 Crystallization and sucrose hydrolysis 52
6 CONCLUSIONS 53 7 REFERENCES 56
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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original articles, which are referred by their Roman
numbers I-IV.
I Kouassi, K. and Roos, Y.H. 2000. Glass transition and water effects on sucrose
inversion by invertase in lactose-sucrose system. J. Agric. Food Chem. 48: 2461-
2466.
II Kouassi, K. and Roos, Y.H. 2001. Glass transition and water effects on sucrose
inversion by invertase in non-crystalline carbohydrate food systems. Food Res.
Int. 34: 895-901.
III Kouassi, K., Jouppila, K. and Roos, Y.H. 2002. Effects of κ-carrageenan on
crystallization and sucrose inversion in lactose-sucrose-invertase systems J. Food
Sci. 67 (6): 2190-2195.
IV Kouassi, K. and Roos, Y.H. 2002. Glass transition, water, and glycerol effects on
sucrose inversion in pullulan-sucrose systems. J. Food Sci. 67 (9): 3402-3407.
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ABBREVIATIONS
Abl absorbance of blank
Asple absorbance of sample
aw water activity
BET Brunauer-Emmett-Teller (sorption isotherm model)
DSC differential scanning calorimetry
E enzyme
ES enzyme-substrate complex
ESR electron spin resonance GAB Guggenheim-Anderson-de Boer (sorption isotherm model)
GC glucose content
LSI lactose-sucrose-invertase (food system)
LSI-carr Lactose-sucrose-Invertase-carrageenan (food system)
MDSI maltodextrin-sucrose-invertase (food system)
MDLSI maltodextrin-sucrose-lactose-invertase (food system)
NMR nuclear magnetic resonance
P product
PS pullulan-sucrose (food system)
PSI pullulan-sucrose-invertase (food system)
PSIG pullulan-sucrose-invertase-glycerol (food system)
PVP polyvinylpyrrolidone
RH relative humidity
Std standard
S substrate
Tg glass transition temperature
Tm melting temperature
T-Tg temperature difference between storage temperature and glass transition
temperature
WL loss water
WT amount of water measured at a time T
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1 INTRODUCTION
In low moisture food systems, numerous changes can take place depending on physical
state, physicochemical properties of food materials and the environment. During
processing, distribution, and storage of food materials, enzymatic reactions may
contribute to a significant extent to these transformations. Enzymatic reactions may be
desirable in some cases but are often deleterious. Control of such reactions is necessary
for technological improvement (Drapon, 1985) as well as preservation of quality and
shelf life of foods.
Water with carbohydrates, lipids, and proteins, is one of the main components of foods. It
is used in processing of foods to improve texture, mixing, and flowing properties, and
functionality. Water is also involved in a number of interactions with other components
of foods. These interactions may contribute to the molecularly disordered, amorphous
state, e.g., in low moisture foods (Roos, 1995a). In amorphous food systems, the glass
transition is the characteristic temperature range over which a stiff material softens and
begins to behave in a leathery manner (Sperling, 1992). This change is a temperature-,
time- (or frequency) and composition-dependent, material specific change in physical
state, from a “glassy” mechanical solid to a “rubbery” viscous fluid.
The glass transition has been recognized as a possible factor affecting kinetics of
enzymatic changes in low-moisture and frozen foods (Lim and Reid, 1991; Slade and
Levine, 1991; Roos and Karel, 1991; Roos, 1998). Furthermore, relationship between the
glass transition and thermal stability of invertase and lactase in amorphous dried PVP and
maltodextrin matrices has been reported (Schebor et al., 1996; Mazzobre et al., 1997a).
The glassy state is accompanied by a reduction in translational and rotational motions of
component molecules, supporting the assumption that chemical and biological reactions
have reduced rates in glassy systems (Le Meste, 1995; Cardona et al., 1997). Rates of
changes occurring in amorphous materials increase dramatically within and above the
glass transition temperature range as a result of decreasing viscosity (Roos, 1995b).
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Yet, combined effects of glass transition, water content, concentration, pH, and
temperature on the kinetics of various chemical reactions and deterioration are not well
established (White and Cakebread, 1966; Slade and Levine, 1991; Nelson and Labuza,
1994; Champion et al., 2000). However, it is often assumed that physical stability is
maintained in the glassy state over a practical time frame. Also, that various changes may
occur above Tg with more significant rates determined by the temperature difference
between Tg and storage temperature, T-Tg (Levine and Slade, 1986, Slade and Levine
1991, Jouppila et al., 1998). An example of such changes is sugar crystallization (Roos
and Karel, 1991; Roos, 1995b; Andronis and Zografi, 1998) and it has been reported that
crystallization in amorphous materials may occur above the glass transition.
Crystallization of sugars is important factor affecting quality of food products as
observed in many processes where the extent of crystallinity is critical to acceptance of
the final product (Kedward et al., 1998). Crystallization of lactose in dairy powders is
often detrimental and known to result in increased rates of nonenzymatic browning and
loss of lysine (Saltmarch et al., 1981). This points out the importance of inhibiting or
controlling sugar crystallization and sugar crystal size and growth of crystals, to meet
desirability and longevity of the food materials.
Water plasticizes amorphous materials and enhances crystallization (Andronis et al.,
1997; Jouppila and Roos, 1998; paper I). The plasticizing effect of water gives rise to an
increase in the molecular mobility that facilitates the arrangement of molecules and
possibly enhances enzymatic reactions. The availability of water has also been
recognized as a factor affecting rates of enzymatic reactions in amorphous food systems
(Schwimmer, 1980; Silver and Karel, 1981; Chen et al., 1999). Food materials are
significantly plasticized by water. Plasticization is observed from a depression of Tg
(Roos and Karel, 1991; Slade and Levine, 1991; Biliaderis et al., 1999; Tolstoguzov,
1999). At increasing water contents, the materials also have higher water activities (aw).
Several reactions in low-moisture food systems have been shown to exhibit higher rates
above specific water contents and water activities (Silver and Karel 1981; Chen et al.
1999; Bell and Hageman, 1994). Other low molecular weight compounds, e.g., polyols
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(Biliaderis et al., 1999; Tolstoguzov, 1999), are also reported to plasticize food materials.
Plasticizers are used to improve flexibility and workability of polymers as well as reduce
viscosity (Griffin and Lynch, 1972). For instance, glycerol and water depressed the Tg
and glycerol interacted with starch reducing crystallization of amylopectin (Forssell et al,
1997). Thus, their possible effects on molecular mobility or diffusion-controlled related
phenomena such as enzymatic reactions are of concern. Rates of deteriorative changes
and rates of chemical reactions including enzymatic reactions have been related to Tg
(Slade and Levine, 1995; Hancock and Zografi, 1997).
In amorphous systems, mobility of components could be dependent on the composition
and structure of the system. Sperling (1986) and Contreras-Lopez et al. (2000) have
shown that polymers are able to form entanglements in which small molecules can be
entrapped. The use of plant polymeric materials to improve physical and structural
properties of food systems has become popular and may contribute to modify the
reactivity (Kasapis et al., 1999) and kinetics of deteriorative changes in foods (Contreras-
Lopez et al., 2000). Since crystallization and enzymatic reactions in food are mobility-
related, and to some extent, glass transition-related, control of these phenomena in
amorphous food materials could be achieved by regulating mobility of the component
molecules through incorporations of additives such as polymers or plasticizers.
2 JUSTIFICATION OF THE STUDY
Enzymatic reactions are often responsible for deleterious changes in low moisture foods.
The rates of these changes may be related to changes in the physical state such as the
glass transition. Water is the most important plasticizer of food materials. Water and
other plasticizers also affect rates of enzymatic reactions. Food systems including
carbohydrates, such as sugars, are very susceptible to crystallization even at reduced
moisture level. Upon crystallization, the sorbed water may be expelled to the food
materials changing the moisture level of the food systems and possibly affect rate of
enzymatic reactions. The hypothesis of the present study was that plasticizers including
water and polyols and, the glass transition affect the rates of enzymatic reactions in low
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moisture foods and are important factors on maintaining quality and shelf life of low
moisture food systems.
The aims of the study were:
1. To examine enzyme activity in low water content systems undergoing physical
changes.
2. To examine the effects of water sorption properties, glass transition temperature of
freeze-dried carbohydrate systems and plasticizers embedded with invertase on invertase
activity.
3. To investigate the effects of a network forming polysaccharide (κ–carrageenan) on
sugar crystallization and sucrose inversion in systems undergoing changes in water
plasticization and physical state.
3 LITERATURE REVIEW
3.1 Physical state and properties of low-moisture food systems
Low-moisture food systems refer to foods with a low water activity, aw. In general, they
are characterized by high viscosity, and they may contain amorphous and crystalline
components. The physical state of their component compounds defines their
microstructure (Roos, 1995a). The physical state of food systems will depend on the
amount of water and other plasticizers, and the types of molecular interactions that
involve all the components.
3.1.1 Amorphous state and state transitions
Amorphous materials are characterized by the random disposition of molecules in the
matrix, in opposition to crystalline structures. A presumption was made that amorphous
sugars in foods are stable below Tg where they exist as solid “glasses”. Above Tg, they
exist in a liquid "syrup-like" state (Roos, 1993). A material in the glassy state is described
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as a brittle substance with a viscosity in the order of 1012 Pa (Ferry, 1980; Noel et al.,
1990), with very limited molecular mobility and diffusion (Karel, 1985; Drapon, 1985;
Levine and Slade, 1990). The constraint in molecular mobility and diffusion has been
regarded as a possible factor affecting rates of deteriorative changes including enzymatic
reactions (Slade and Levine, 1991; Roos, 1998). As the temperature increases through
and above the glass transition, the glassy material enters in the supercooled liquid state,
softens, and becomes rubbery or leathery or behaves like syrup as a result of decreased
viscosity and increased transitional mobility. Subsequently, it can be inferred that the
increase in mobility is associated with increased rates of physical, chemical, and
biological changes, since the temperature difference between Tg and the storage
temperature T-Tg, was assumed to control the rate of physical, chemical and biological
changes (Champion et al., 2000). Changes in the physical state often include stickiness,
collapse and crystallization (Roos, 1987; Roos and Karel, 1991). These changes occur in
the rubbery state above Tg as a result of the transformation of the solid material to the
supercooled liquid state (Roos, 1998). Understanding the glass transition and its
relationships with physicochemical changes is very important for predicting the state and
the behavior of food during processing, distribution, and storage.
3.1.2 Water sorption and plasticization
Water is present in foods either as a constituent of food materials or added during food
processing. The water in foods influences the physical and textural characteristics of the
product as well as its chemical stability (Bell and Labuza, 2000). The control of water in
foods is of primary importance for understanding the physicochemical relationship of
water to the various components of foods. This relationship depends on structure,
appearance, and stability, and can enable improvement in processing and storage of
foods. Since ancient time dehydration that consists of removing water from foods has
been one of the most efficient ways to decrease perishability. However, it has been
observed that various types of food with the same water content differ significantly in
perishability (Fennema, 1996; Rahman and Labuza, 1999). Thus, water content alone
does not fully describe the kinetics of dynamic changes. The notion of aw, a temperature-
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dependent thermodynamic property was developed. This notion is based on the
assumption that the activity of a given molecular species in the gas is equal to its partial
vapor pressure divided by the pressure the pure species would have at the same
temperature if the air space was completely saturated with that molecular species (Chang,
1981). Thus, at steady state, aw is defined as the vapor pressure of water in food (p)
divided by the vapor pressure of pure water (p0) or the equilibrium relative humidity
(ERH) of the air surrounding the system at the same temperature (1).
aw = 1000
ERHpp = (1)
However, the combination of water content and aw is widely used in the literature as well
as in practical applications for material characterization. It is possible to obtain
simultaneous information on water contents and aw using water sorption isotherms. The
water sorption isotherm describes the dependence of water content on water activity of a
material at constant temperature (Rahman and Labuza, 1999). Numerous techniques are
used for measuring water sorption properties of food materials including gravimetric,
hygrometric, and manometric techniques. The gravimetric technique is the most
commonly used. It consists of determining the weight changes of samples placed in
desiccators or chambers at RH obtained using saturated salt solutions with or without
vacuum. Brunauer et al. (1938) described five basic forms of sorption. The types I, II, and
III of those isotherms (Fig. 1) are the most common to water sorption in foods. The type I
isotherm is typical of a non-swelling porous or capillary solids and anticaking agents
resulting in the holding of large amounts of water at low aw. The type II isotherm has a
sigmoid shape and it is characteristic of most foods, such as cereals. The sigmoid curve
result from the combination of colligative effects, capillary effects, and surface-water
interactions (Rahman and Labuza, 1999). The type III curve is indicative of the
adsorption isotherm of a pure crystalline substance (e.g., sucrose) and shows very little
moisture gain until the aw reaches the point where water begins to dissolve crystals at
their surface (Bell and Labuza, 2000).
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Figure 1. Type I, II, and III of water sorption isotherms adapted from Roos (1995a).
Mathematical models are often used to fit to experimental data to calculate the sorption
isotherm. The most popular models in food applications are the Brunauer-Emmett-Teller
(BET) model (Brunauer et al., 1938) and the Guggenheim-Anderson-de Boer (GAB)
model (van den Berg and Bruin, 1981). The BET model is described by equation (2)
( ) ( )[ ]ww
w
m aKaa
mm
111 −+−= (2)
This equation can be written in a linearized form as equation (3)
( ) wmmw
w aKm
KKmam
a 111
−+=−
(3)
where m is the moisture content of the sample, mm is the monolayer value, the moisture
content at which each polar and ionic group is bound to a water molecule, and K is a
constant.
The GAB isotherm model is described by the equation (4).
( ) ( )[ ]ww
w
m CaKCaCaK
mm
111 −′+−′
= (4)
The GAB model can also be written to the form of the second-order polynomial
0.0 0.2 0.4 0.6 0.80
5
10
15
20
25
30
II
III
IW
ATE
R C
ON
TEN
T (g
/100
g so
lids)
WATER ACTIVITY
15
( ) γβα ++= www aa
ma 2 (5)
from which the constants C, K’ and mm can be calculated by the following equations (6),
(7), and (8) (Roos, 1995a):
C = ( )
γβ
2/1
−− mm (6)
K’=γCmm
1 (7)
241
βαγ −−=mm (8)
Water is the most important solvent, dispersion medium, and plasticizer in biological and
food systems (Matveev et al., 2000). Plasticization and its modulating effect on
temperature location of the glass transition is a key technological aspect of synthetic
polymer technology where a plasticizer is defined as a material incorporated in a polymer
to increase the material’s workability, flexibility, or extensibility (Sears and Darby,
1982). The plasticizing effect is usually described by the dependence of the glass
transition temperature on either the weight, the volume, or molar fraction of water
(Matveev et al., 2000). Water plasticization can be observed from the decrease in the
glass transition temperature with increasing water content which may also improve the
detectability of the transition (Roos et al., 1996). Slade and Levine (1994) found that the
Tg of an undiluted polymer was much higher than that of a typical low molecular weight
glass forming diluent. An increase in the diluent concentration results in a monotone
decrease in the Tg. Since the average molecular weight of the homogeneous polymer-
plasticizer mixture decreases, its free volume (the volume of the mixture that is not
occupied by molecules) may increase, and the local viscosity decreases with a
concomitant increase in mobility (Ferry, 1980). It is well known that water, acting as a
plasticizer, affects the Tg of completely amorphous polymers and both the Tg and the
melting temperature (Tm) of partially crystalline polymers (Levine and Slade, 1990).
16
The addition of low molecular weight plasticizers to an amorphous matrix has the same
effect as an increase in temperature on molecular mobility (Biliaderis et al., 1999).
Indeed, the plasticizing effect of polyols such as glycerol (Gontard et al., 1993; Forssell et
al., 1997; Arvanitoyannis and Biliaderis, 1998; Biliaderis et al., 1999; Moates et al.,
2001) has been demonstrated. In addition to glycerol, glycol and urea have been shown to
plasticize corn starch (Shogren, 1992). These substances are important in food to which
they are incorporated to lower their water activity (Sloan and Labuza, 1975). The
relationship between Tg, aw, and water content was first established by Roos (1987). He
found a decrease in glass transition temperature of freeze-dried strawberries with
increasing RH and aw. Fig. 2 shows a representation of a modified state diagram
indicating depression of the glass transition temperature, Tg, with increasing aw.
Prediction of Tg depression as a result of the water plasticization is useful in evaluating
effects of food composition on Tg, since glass transition-related changes may affect shelf
life and quality (Roos et al., 1996). There is a very significant literature describing
equations for predicting the plasticizing effect of water. The Gordon-Taylor equation
(Gordon and Taylor, 1952) given in equation (9) is probably the most popular equation
used in modeling of water plasticization of amorphous carbohydrates.
21
2211gT
kwwTkwTw gg
++
= (9)
where w1 and w2 are the respective weight fractions of the anhydrous amorphous
carbohydrate and water, Tg 1 is the Tg of the anhydrous carbohydrate, Tg 2 is the Tg of
amorphous water, −135°C (Johari et al., 1987), and k a constant. Water plasticization can
also be modeled using the Couchman-Karasz equation (10) (Couchman and Karasz,
1978).
2211
222111gT
pp
gpgp
CwCwTCwTCw
∆+∆∆+∆
= (10)
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Figure 2. Glass transition temperature, Tg, as function of water sorption isotherm at 24°C
for maltodextrin-sucrose-invertase (MDSI) system. Tg decreased with increasing water
content. The monolayer value was 5.8g.
Equation (10) is a thermodynamic form of the Gordon-Taylor equation with ∆Cp1 and
∆Cp2 being the change in heat capacity of anhydrous solids and water, respectively,
giving 1
2
p
p
CCk ∆
∆= . A ∆Cp2 of 1.94J g-1°C is often used for amorphous water and gives the
best fit (Kalichevsky and Blanshard, 1993) compared to many other (Hallbrucker et al.,
1989) and significantly lower values.
3.1.3 Glass transition and crystallization
In the condition of increased relative humidity, for example when amorphous sugars are
stored at high RH, the water sorption results in crystallization. Upon crystallization, the
sorbed water might be released with a substantial weight loss (Saleki-Gerhardt and
Zografi, 1994; Buckton and Darcy, 1995; Roos et al., 1996). Water plasticization and
depression of Tg to below ambient temperature are responsible for crystallization of
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
-120
-80
-40
0
40
80
Tg
GAB SORPTION ISOTHERM
5.8g
TEM
PER
ATU
RE
(°C
)
WATER ACTIVITY
0
4
8
12
16
20
24
WA
TER
CO
NTE
NT
(g/1
00/o
f Sol
ids)
18
amorphous sugars in foods, as a result of increased free volume and molecular mobility,
decreased viscosity, and enhanced diffusion (Roos and Karel, 1990; Roos, 1995b).
Crystallization of sugars for instance is important in food products, and the extent of
crystallization is critical to acceptance of the final product (Kedward et al., 1998). The
role of crystallization is significant in confectionery where it is necessary to control
growth of sugar crystals and crystal size and to achieve products with better desirability.
However, crystallization may be detrimental and has been reported to increase rate of
nonenzymatic browning and loss of lysine (Saltmarch et al., 1981) in dairy powders and
present an industrial problems in tabletting and storage of micronized powders (Schmitt
et al., 1999). Crystallization of amorphous sugars in foods may enhance both physical
and chemical deterioration (Saltmarch et al., 1981; Shimada et al., 1991; Hartel and
Shastry, 1991). Crystallization is universally described as a three-step mechanism
involving the following sequential steps: (1) nucleation – formation of crystal nuclei, (2)
propagation and growth of crystals from nuclei by intermolecular association, and (3)
maturation − crystal perfection (by annealing of metastable microcrystallites) (Slade and
Levine, 1987). According to Roos (1995b), crystallization may occur at temperatures
above the Tg but below the equilibrium melting point. Later, Shneidman and Uhlmann
(1998) demonstrated that the maximum nucleation rate will either correspond to Tg, or
will be located at a somewhat higher temperature depending on rate of heating. The
extent of crystallization often depends on factors such as time, temperature, structure of
the crystallizing compounds, and the effects of different substances present in the food
matrix. Addition of a second polymer, such as maltodextrin (Roos and Karel, 1991) or
corn syrup and their saccharides (Tjuradi and Hartel, 1995; Gabarra and Hartel, 1998) to
an amorphous matrix, has been found to delay crystallization of sucrose by reducing the
crystal growth rate. Similarly, studies have demonstrated the significant effects of three
sugars (trehalose, lactose, and raffinose) on the crystallization of amorphous sucrose
when each of these sugars was colyophilized with sucrose and exposed to high
temperatures and relative humidities (Saleki-Gerhardt and Zografi, 1994). Lactose crystal
growth was found to increase from 15 to 41% by addition of tiny amounts of LiCl. This
salt was used because of its ability to reorganize the structure of water due to its high
charge density (Bhargava and Jelen, 1996). Indeed sugars, such as lactose and sucrose
19
have higher tendency to crystallization compared to long chain polymers. The addition of
different substances to control crystallization in amorphous food systems is a common
way to quality improvement.
3.1.4 Composition, dynamics, and phase separation in amorphous foods
Amorphous carbohydrate food systems are blends of multi-component structures from
low to high molecular weight. They may be made of their aqueous solutions that are then
freeze-dried. The preparation of those systems involves a number of events, e.g. mixing,
and drying. For each of those events, factors such as the temperature, moisture, and time
that defined the concept of equilibrium versus non-equilibrium conditions are of major
significance (Flink, 1983). The physical state of these systems and their properties could
be determined with the particular values of these factors and the sequence of events
associated with their formation (Flink, 1983). In addition to these events, the structure
and reactivity of the systems might be related to the composition of the systems and the
interactions of their components. This relation can be successfully explained and often
predicted by associating the structure aspects of food with functional aspects, depending
on mobility and described in terms of water dynamics or glass dynamics. Slade and
Levine (1994) provided an appropriate kinetics description of the non-equilibrium
thermomechanical behavior of foods in which water acts as plasticizer, in terms of state
diagrams. Glass dynamics focuses on 1) the glass-forming solids in an aqueous food
system, 2) the Tg of the resulting supercooled matrix that can be produced by cooling to
T<Tg, and 3) the effect of the glass transition on processing and process control (Slade
and Levine, 1991). The concept of glass dynamics emphasized the absence of molecular
mobility (translational), the stability, and unreactive situation that can be obtained during
product storage at temperature and moisture content corresponding to the glassy solid
state at T<Tg. It can also be used to describe time-dependent phenomena, translational
diffusion-limited deterioration processes (physical, chemical, or enzymatic) that can
occur in amorphous food materials and products during storage in the rubbery liquid state
at T>Tg. Slade and Levine (1994) used the dynamic map, a state diagram describing the
mobility transformations in non-equilibrium metastable and rubbery food systems to
20
elucidate the physicochemical mechanisms and kinetics of structure/mechanical changes
involved in various melting, annealing, and crystallization processes.
The functional behavior of plant polysaccharides and carbohydrates in food materials is
often influenced by their phase behavior and dynamics (Gunning et al., 1999). Phase
behavior involves polysaccharide interactions with water, solutes, and other polymers.
Phase separation and dissolution are controlled by three variables: temperature, pressure,
and concentration (Sperling, 1986). It is generally believed that phase separation takes
place when the total concentration of the higher molecular weight components exceeds a
critical value. This is described as a limited thermodynamic compatibility or limited
cosolubility of biopolymers (Tolstoguzov, 2000a; Tolstoguzov, 2000b). A
thermodynamic justification of phase separation (Kalichevsky et al., 1992; Taylor and
Zografi, 1998; Tolstoguzov, 2000b) is that for compatibility the Gibbs free energy of
mixing (∆Gm) must be negative. This implies that there must be a negative enthalpy of
mixing (∆Hm) or an increase in entropy (∆Sm) of mixing (since ∆Gm = ∆Hm − T∆Sm).
Keller and Cheng (1998) found a relation between phase separation and mobility. They
demonstrated how the phase separation processes could be halted by a change in mobility
of one component. Thereafter, the effect of glass transition on phase separation has been
investigated. Isayeva et al. (1998) and De Graaf et al. (1996) found that crystallization
and phase separation were arrested by addition of glassy materials. This is where the
addition of polymers such as maltodextrin (Roos et al., 1996; paper II), corn syrup and
their saccharides (Tjuradi and Hartel, 1995; Garbarra and Hartel, 1998) to control crystal
growth rate find its relevance. Similarly, De Graaf et al.(1996) reported from a study of
mixtures of polystyrene and metacrylate a physical arrest of phase separation as the
polystyrene rich phase underwent a glass transition. In food systems, such as pullulan-
sucrose (PS), pullulan-sucrose-invertase (PSI), and pullulan-sucrose-invertase-glucose
(PSIG), where pullulan, the biopolymer, represents a very significant part of the matrix,
phase separation might occur as result of thermodynamic incompatibility between
pullulan and sucrose molecules. Phase separated systems can exist either as liquid-liquid,
polymer-polymer, or polymer-liquid phases (Sperling, 1986; Tolstoguzov, 2000b). Phase
separated systems have been studied by a number of experimental tools. Differential
21
scanning calorimetry (DSC) was used to measure the change in heat capacity of the
systems (Biliaderis, 1999). Dynamic mechanical spectroscopy was used for measuring
the shift in the glass transition temperature, which provides a measure of the extent of
molecular mixing in partly mixed systems (Sperling, 1986). Kwei et al. (1974) and
Shimada et al. (1988) measured different relaxation times for segments of different
components in systems by nuclear magnetic resonance spectroscopy (NMR) and electron
spin resonance (ESR) while Donatelli and Sperling (1976) used electron microscopy to
determine domain size and shape. Small angle X-ray scattering was used for measuring a
thermodynamic interaction coefficient between two polymers (Isayeva et al., 1998).
Turbidimetric titration (Antonov and Gonçalves, 1999) was also one of the techniques
used for the assessment of phase separation. The existence of two Tg suggested phase
separation in the system, on the basis of single glass transition criterion (Olabisi et al.,
1979; Bosma et al., 1998). Assessment of phase separation by Shamblin et al. (1998) was
based on the deviation of Tg values from free volume and thermodynamic models.
In summary, there exist some relationships between the dynamics of food systems and
their physical structure such as phase separation. The basic principle of phase separation
is the thermodynamic incompatibility between constituents, which reflects the type of
interactions occurring between individual constituents.
3.2 Enzyme activity in low-moisture food systems
3.2.1 Hydrolytic enzymes and observed changes
Before examining the activity of invertase, it is necessary to get an insight of the group of
hydrolytic enzymes to which invertase belongs. Enzymes are classified according to the
report of a Nomenclature Committee appointed by the International Union of
Biochemistry. This Enzyme Commission assigned each enzyme a recommended name
and a 4-part distinguishing number. However, the traditional use of the ending "ase" is
still retained. The Enzyme Commission (EC) numbers divide enzymes into six main
22
groups according to the type of chemical reaction catalysed: (1) oxidoreductases, (2)
transferases, (3) hydrolases, (4) lyases, (5) isomerases, and (6) ligases (Parkin, 1984).
Hydrolases or hydrolytic enzymes are enzymes in which water participates in breakage of
covalent bonds of the substrate, with concurrent addition of the element of water to the
principles of those bonds (Whitaker, 1996). An equation can be described as transfer of
the components of the substrate to water (11). Hydrolases are a large group of enzymes
having in common the involvement of water as a co-substrate of the primary substrate in
formation of products (Whitaker, 1972).
Hydrolytic reactions by nature yield two products that result from the cleavage of the
parent substrate molecule. Whitaker (1972) has proposed more detailed information on
the mechanism of hydrolysis and the classification of hydrolytic enzymes.
Invertase (β-D fructofuranosidase, EC 3.2.1.26) is an enzyme discovered in yeast. Its
name refers to its ability to change the direction of optical rotation of sucrose solution as
a result of hydrolysis to glucose and fructose (Whitaker, 1972). Although invertase is
primarily known for the inversion of sucrose, it can be used to digest other disaccharides,
trisaccharides, and fructans to make syrups of invert sugars, meliobiose from raffinose,
gentiobiose from genanone, and fructose from inulin (Bigelis, 1993). The hydrolysis of
sucrose by invertase was used as a model by Michaelis and Menten (1913) and served as
a basis for most of theories of enzyme kinetics using sucrose and invertase. The
Michaelis and Menten theory given by equation (12) predicts the following two step
mechanism for enzyme catalysis:
where E is the enzyme, S the substrate, [ES] the enzyme-substrate complex, and P the
product. k1, k-1 and k2 are the rate constants corresponding to the enzyme–substrate
association, the enzyme−substrate dissociation, and the transformation into product,
respectively. The global reaction rate constant K can be calculated using equation (13)
Y + HOH HX + YOHX (11)
E + Sk1
k-1ES E + P
k2 (12)
23
[ ][ ]ESKV =0 (13)
where 0V , K, [S], and [E] are the initial velocity of the reaction determined
experimentaly, the rate constant, the substrate concentration, and the enzyme
concentration, respectively. The rate of hydrolysis is expressed as equation (14):
[ ][ ]SK
SVVm
m
+=0 (14)
where [S] is the substrate concentration; Km is the Michaelis constant which is a
combination of rate constant equal to [(k-1 + k2) / k1], and Vm is the maximum reaction
velocity. Nelson and Schubert (1928) considered two fundamental assumptions from the
Michaelis and Menten theory which were:
(1) sucrose combines with invertase according to the mass law and
(2) the velocity of inversion is proportional to the amount of invertase combined
with sucrose. However, invertase failed to obey the Michaelis and Menten theory at
sucrose concentration >5-6% (Nelson and Schubert, 1928; McLaren, 1963; Bowski et al.,
1971).
3.2.2 Water activity control/dependence
Even in low moisture foods, enzymatic changes can occur despite the low aw. The
occurrence of these reactions reduces the storage stability of products (Acker, 1969).
According to Drapon (1985), water can play several different roles in food systems:
(1) Water may act as second substrate. It is well known that the spatial structure of
protein, which governs their functional properties, is stabilized by several kinds of
interactions that include hydrogen bonds, between polar groups or between polar groups
and water, and hydrophobic bonds associated with the structure of water around the
protein molecule.
(2) As disrupter of hydrogen bond and consequently contributing to the alteration of
protein structure.
(3) As a solving medium facilitating the diffusion of reactants.
(4) As a reagent in the case of hydrolysis reaction.
24
It is, therefore, necessary to examine the condition in which water can be available in
assuming these functions. The study of Acker (1969) showed that hydrolysis of lecithin
by phospholipase was measurable within hours at a high aw. At low aw, hydrolysis did not
take place even when moisture level was sufficient for completing hydrolysis. However,
hydrolysis at each different level of aw proceeds towards a different final value. Also,
different samples investigated had the same enzyme and substrate concentration but
differed only in water content. At an increasing aw, the enzyme activity occurred and
hydrolysis approached the value obtained for a sample stored at the higher aw from the
very beginning. These results were in agreement with the results of Schwimmer (1980)
who suggested that enzyme activity should be measured on the basis of knowledge on aw
rather than moisture content. Including water sorption isotherm on the graph showing the
dependence of enzyme activity on water activity (Acker, 1969), it appears that below the
monolayer value, water behaves as described in assumption 1. At water contents above
the monolayer value, it could be expected that some of the water in the liquid state be in
the conditions described as (2), (3), and (4) by Drapon (1985). In solid systems, since the
size of water cavities is limited, it is likely that only part of the substrate available can be
attacked and split inside them. The extent of the liquid phase depends on the water
activity and this explains why different degrees of hydrolysis are reached at different
levels of aw. As a summary, enzyme activity depends on water-enzyme, water-substrate
and water-matrix interactions. Besides, matrix-substrate and matrix-enzyme interactions
may be involved.
The first study that really addressed the relationship of water content, aw, and invertase
activity was done by Silver and Karel (1981). The model systems used were
microcrystalline cellulose (Avicel)-sucrose-invertase, agar-sucrose-invertase, and starch-
sucrose-invertase. This study showed that measurable reactions occurred at aw as low as
0.580 for the Avicel system. At aw above 0.750, complete hydrolysis was observed.
However, for crystalline sucrose the onset of hydrolysis has been hypothesized to
coincide at the mobilization point of the substrate, the point at which a true solution is
formed (Karel, 1985). Drapon (1985) reported that hydrolysis of amylose by α- and β-
amylase in a freeze-dried starch, and at 30°C started within the 0.65-0.70 aw range. Chen
25
et al. (1999) studied aw and the glass transition effects on sucrose hydrolysis in
polyvinylpyrrolidone (PVP) systems. The requirement of aw > 0.62 was found for sucrose
hydrolysis with rate constant increasing concomitantly with aw. Furthermore, occurrence
of sucrose hydrolysis was limited to the rubbery state. The large molecular weight of PVP
molecules was also considered as a limiting factor for hydrolysis due to their ability to
block the diffusion of sucrose to active sites of invertase. The water activity at which
enzyme activity is effective can be modified with changes in the reaction medium. For
example, if the aw at which water acting as a solvent medium is modified by addition of a
suitable substance having normally no effect upon enzyme activity, the aw at which
enzymatic activity begins can correspondingly change. Addition of such a substance to
the reaction mixture reduced the water activity at which enzymatic activity began to a
value lower than that found for control mixtures with no additives (Drapon, 1985).
Finally, the occurrence of enzyme-catalyzed reactions in low moisture systems requires a
certain quantity of water in order to facilitate both mobility and diffusion of reactants.
This quantity may change according to the characteristics of the enzyme and the
solubility and molecular size of the substrate.
3.2.3 Effects of substrate
Rates of enzyme-catalyzed reactions are influenced by a number of experimental
parameters. Among these parameters are substrate concentration, enzyme concentration,
pH, temperature, presence of activators, inhibitors, dielectric constant and ionic strength
(Whitaker, 1972). Effects of substrate concentration, pH, and temperature will be
successively discussed. An enzymatic reaction involves two steps: a binding step in
which the substrate is attracted to and captured by the enzyme, followed by the actual
reaction step that forms the products. The effects of substrate concentration can be better
observed by keeping all other parameters constant, while the initial concentration of the
substrate is varied. The enzymes operate most efficiently when an excess of substrate is
available. If the substrate concentration is low compared to that of the enzyme, successful
collisions are few. When the substrate concentration is very high compared to the enzyme
26
concentration, successful collisions occur very rapidly, ensuring that most of the enzyme
is bound in an enzyme–substrate complex. Product is then produced at a maximum rate
(Mathewson, 1998). Once all enzyme molecules have combined with substrates, further
increases in substrate concentration will not increase the rate of reaction (Whitaker, 1972;
Mathewson, 1998). The velocity at this point is called maximum velocity, Vm, which is
used in equation (13).
Many investigators have studied the relationship between the rate of hydrolysis of
sucrose by invertase from yeast and the concentration of the sugar. They have observed
that the initial rate of reaction increased until about 5% sucrose concentration is reached.
Above this concentration, the initial reaction rate fell markedly (Nelson and Schubert,
1928; McLaren, 1963; Ritchi and McLaren, 1964). This observation deviated from the
Michaelis-Menten theory. Nelson and Schubert (1928) justified the fall in the velocity of
sucrose hydrolysis at sucrose concentration above 5 % by two means: (1) the increasing
formation of sucrose-invertase complex and (2) the decrease in water concentration as
sucrose concentration increased. The latter justification was based on their experiment
that consisted of varying the sucrose and water concentration independently of each other
using a constant quantity of alcohol in various solutions. Latter on, a peculiar decrease in
the rate of sucrose inversion was found to result from a transferring action of invertase.
This was based on a quantitative analysis of sucrose hydrolysis products that revealed
that the amount of fructose was lower than that of glucose, confirming that a part of the
fructose was converted to glucose by invertase (Andersen et al., 1969). They then
postulated that sucrose hydrolysis was a two step transfer in which the first step is the
formation of an active enzyme-fructose complex and the next step a liberation of glucose
and a simultaneous transfer of the fructosyl group to water or sucrose. This observation
was later confirmed by Bowski et al. (1971). However, since the velocity drops off
beyond 5% sucrose concentration, it has been customary to neglect the portion of the
curve corresponding to sucrose concentrations above 5% and to assume that, at the
experimental maximum, all the enzyme is combined with sucrose (Nelson and Schubert,
1928). Fig. 3 is a schematic presentation of the effects of sucrose concentration on
sucrose hydrolysis.
27
Figure 3. Schematic presentation of the effect of substrate concentration on rate of
enzymatic reaction.
3.2.4 pH in low moisture foods
It is well known that pH, affects the three-dimensional structure of proteins (Whitaker,
1972; Tipton and Dixon, 1983; Mathewson, 1998). Change in protein structure may result
in defect on its activity. The ability of the amino acids at the active sites of an enzyme to
interact with the substrate depends on their electrostatic state (Kang et al., 1994). That is
whether they are charged or uncharged as well as their spatial orientation. If the pH is not
appropriate, the charge on one or all of the required amino acids is such that the substrate
can neither bind nor react to produce a product (Mathewson, 1998). Most of enzymes are
active in rather narrow pH range of 6.0-8.0, although some exceptions exist. Silver and
Haskel (1990) observed a 10-fold increase in the activity of α-chymotrypsin in a
membrane, when the pH of the medium was lowered from 8.5 to 4. However, the
dependence of reaction rate on pH is often related to aw (Schwimmer, 1980; Bell and
Labuza, 1992, 1994). Schwimmer (1980) found that in a system composed of an enzyme,
Low reaction rateLow [substrate]
High reaction rateHigh [substrate]
ENZY
ME
AC
TIV
ITY
SUBSTRATE CONCENTRATION
28
a substrate and an organic solvent, at a low aw, enzyme activity was affected by a shift in
pH-profile of the medium upon removal of water from the reaction medium. Thus, in the
alkaline side of the shifted pH maximum, mushroom phenolase was found to be more
active at aw = 0.85 than at aw = 1, although at pH values lower than the optimum the
enzyme was inhibited from water rich to water-poor environments. These results
suggested that although enzyme activity is affected by pH, the effect of pH seems to be
related to water, probably because pH is a measure of the content of hydronium ions that
result from ionization of water. This statement is in agreement with Buera at al. (1995)
that found an effect of the pH change by change in water content in PVP matrices, which
affected acid-catalyzed sucrose hydrolysis.
3.2.5 Effect of temperature
Like pH, temperature affects the three-dimensional structure of an enzyme (Whitaker,
1972; Mathewson, 1998). Supplying the system with more heat helps to overcome the
energy barrier, the energy necessary for ionization of groups in the active site of the ES
complex. This occurs because, the warmer the system is, the faster the molecules move.
Thus, the reactants collide more often with greater energy. Effect of temperature on
enzymes in amorphous food systems was extensively studied (Schebor et al., 1996;
Cardona et al., 1997; Mazzobre et al., 1997a, Mazzobre et al, 1997b; Burin et al., 2002).
However, these studies focused on the thermal resistance or stability of the enzyme as
related to Tg, rather than on enzyme activity. Drapon (1985) found that the effect of
temperature on enzyme activity was related to moisture content by demonstrating that at a
higher moisture content, inactivation and denaturation of a lipase occurred at a lower
temperature, while at low moisture content, a higher temperature was required for the
same processes. The effect of temperature on rate of enzyme-catalyzed reactions has also
been studied. Whitaker (1995) found that decreasing the temperature of a system to
below 0°C decreased the rate of enzyme-catalyzed reactions. Lund (1969) attributed this
decrease to the conversion of part of the bulk water in the system to ice, causing
reduction in mobility, and increased substrate inhibition as a result of increased substrate
concentration.
29
3.2.5 Other rate controlling factors
Enzymatic reactions involve the interaction of an enzyme with a substrate where often
water is associated either as a solvent or a second substrate. The hydrolysis of sucrose
required that invertase was in contact with the hydrolytic bond of sucrose. If the system is
dehydrated, the addition of water is necessary to restore the activity of the enzyme. There
is, therefore, a requirement of mobility of the components. Water has to diffuse through
the system, the enzyme may exert a certain mobility to reach the hydrolytic bond, or the
substrate needs to move toward the active site of the enzyme. The rate of enzymatic
reactions has to be dependent on the rate at which those motions take place, which
depends in turn on the structure of the matrix of the systems. The presence of
polysaccharides in viscoelastic liquid for example has been shown to cause entanglement
of the polysaccharide chain and restrict diffusion of water molecules (Goff et al., 1993).
McCurdy et al. (1994) demonstrated that dextrans underwent a conformational change
from random coil to a more compact coil when its concentration was raised from 4.7 to
19%. These authors suggested that the conformational change could modify the diffusion
rate of small molecules. Contreras-Lopez et al. (2000) demonstrated that pullulan at 10%
concentration induced a significant decrease in translational mobility measured using
fluorescein. This effect of pullulan was attributed to its ability to form physical
entanglements. Similar behavior was observed by Gillies et al. (1996) for gelatin gels.
When the concentration of gelatin reached 55%, translational motions were virtually
stopped (Contreras-Lopez et al., 2000). The role of polysaccharides in molecular motions
has also been investigated by Roozen et al. (1991) who suggested that small molecules
can freely rotate in cavities, "holes", created by polysaccharides. These results showed
that the structure and composition of the matrix could affect the motions of molecules,
affecting the probability of substrates and enzyme molecules to collide. Molecular
motions can subsequently influence the reaction rates. In water restricted systems, it
could be assumed that mobility would be limited. The activity of the enzyme would be
dependent on its closeness to the substrate. The enzyme should, therefore, be distributed
30
in such a way that it is available in the vicinity of the substrate. Poor miscibility could
also lead to reduced reaction rates since it may reduce interactions between molecules.
This review enlightens conditions under which enzymatic reactions occur in low moisture
food systems. Composition, structure, and environmental conditions including moisture
content, temperature, and pH, determine the physical state and the dynamics of the
systems.
4 MATERIALS AND METHODS
4.1 Preparation of amorphous food systems
The systems investigated in the present study were carbohydrate matrices in which
invertase was embedded. There were in total seven systems: lactose-sucrose-invertase
(LSI) (paper I and III), lactose-sucrose-invertase-carrageenan (LSI-carrageenan) (paper
III), maltodextrin-sucrose-invertase (MDSI) and maltodextrin-lactose-sucrose-invertase
(MDLSI) (paper II), and pullulan-sucrose (PS), pullulan-sucrose-invertase (PSI), and
pullulan-sucrose-invertase-glycerol (PSIG) (paper IV). PS was a control system without
invertase. The systems were prepared by freeze-drying solutions made of bulk
carbohydrates, sucrose, and invertase. The carbohydrate-sucrose solution was cooled at
+5°C for 20 min and invertase (β-D fructofuranosidase, EC 3.2.1.26, pH 4.5; V grade
from baker’s yeast; Sigma, USA) was dissolved into 5ml distilled water and added to the
solution in an ice bath (to minimize sucrose hydrolysis during mixing), as proposed by
Chen et al. (1999). The concentration of invertase was 94.4 units/g, 31.4 units/g, and 65.6
units/g in papers I, II and III, and IV, respectively. In all the systems the enzyme existed
in saturation. The solution was mixed with a magnetic stirrer for 30 seconds. Aliquots of
the solution (5 mL) were quickly prepared into 20ml vials and pre-frozen at −20°C for
2h. After pre-feezing, the aliquots were stored in a freezer at −80°C for about 24h, then
freeze-dried for 3-5 days at a pressure < 0.1 mbar using a Lyovac GT 2 (Amsco Finn-
Aqua GmbH) freeze-dryer. The freeze-dried samples were stored for 5 days in vacuum
desiccators over P2O5 for further dehydration. Two samples stored over P2O5 were daily
31
removed from desiccators and weighed. The samples were assumed as anhydrous when
their weights remained constant for at least three consecutive days.
4.1.1 Lactose-sucrose-invertase (LSI) systems (papers I and III) and lactose-sucrose-
invertase-carrageenan (LSI-carrageenan) system (paper III)
For the preparation of LSI, α-lactose monohydrate (66.6 % dry weight) and sucrose (33.3
%) from Sigma (St Louis, USA) were successively dissolved in distilled water in 4:1 ratio
the weight of solids, under mild heating and mixing to obtain a clear solution. For LSI-
carrageenan, κ-carrageenan obtained from Hercules (Lill-Skensved, Denmark) was
dialyzed using a dialysis tubing (molecular weight cut-off 12-14 kDa, Medicell
International, UK) and gently dissolved into distilled water. Lactose and sucrose in the
proportion (65:35) were added to the mixture and mixed. KCl (0.01M) from Merck
(Darmstadt, Germany) was added to the solution to facilitate network formation (Kasapis
et al., 1999)
4.1.2 Maltodextrin-sucrose-invertase (MDSI) and maltodextrin-lactose-sucrose-
invertase (MDLSI) systems (paper II)
MDSI was made of Maltrin M100 (Grain Product Corporation, Muscatine, USA) and
sucrose (2:1), and MDLSI contained Maltrin M100, α-lactose, and sucrose (1:1:1). The
concentration of sucrose was the same in both systems. The solid materials were
dissolved in distilled water in a ratio 1:5 under mild heating. The solution was cooled at
5°C and Invertase was added to it.
4.1.3 Pullulan-sucrose (PS), pullulan-sucrose-invertase (PSI) and pullulan-sucrose-
invertase-glycerol (PSIG) systems (paper IV)
Pullulan was a product of Hayashibara Biochem., Lab Inc. (Okayama, Japan) and
glycerol was from Sigma (St Louis, USA). The solution of the PS system was prepared as
follows: sucrose (33.3g) was dissolved into 500ml distilled water and pullulan (66.6g)
32
was added under mild heating and stirring until dissolution was completed. The same
procedure was followed in preparation of the solution of the PSI and PSIG systems
except that invertase (58.2mg) and glycerol (5g) were added, respectively. Glycerol was
added to distilled water before addition of other constituents, and invertase was added
after all other constituents were dissolved into distilled water. Dehydration of the sample
was achieved in the same way as described above. PSI and PSIG were used for water
sorption isotherm, differential scanning calorimetry (DSC) and kinetic studies while the
PS was used for physical characterization of the system using water sorption and DSC
studies.
4.2 Characterization of food systems
4.2.1 Water sorption isotherms
Water sorption isotherms for the different systems were determined by fitting GAB and
BET sorption models to experimental water contents obtained gravimetrically at 24°C.
Triplicate amorphous samples of 1g in the 20ml vials were removed from desiccators
containing P2O5. The samples were stored over saturated salt solutions until the sample
weight leveled off. The salts used were LiCl, CH3COOK, MgCl2, K2CO3, Mg(NO3)2, NaNO2, and NaCl. The corresponding RH were 11.3, 23.9, 33.0, 44.4, 53.8, 66.2, and
76.4% (Bell and Labuza, 2000). Sample weight was measured at time intervals during
storage. The GAB and the BET models were fitted to the experimental water sorption
data as reported by Roos (1993) (papers I, II, III, IV).
4.2.2 Glass transition
The glass transition temperature (Tg) of samples was measured using Differential
Scanning Calorimetry (DSC). Samples (5-20mg), triplicates at least, were prepared in
open aluminum pans (40µl; Mettler ME-27331 Al-crucibles) and stored in vacuum
desiccators over saturated salt solutions at 23.9, 33.0, 44.4, 53.8, 66.2, and 76.4% RH.
After 24 or 44h (depending on time for equilibrium), the pans were hermetically sealed
33
and scanned at 5°C/min from at least 35°C below the glass transition temperature range
using DSC (Mettler TA 4000 system with TC 15 TA processor, DSC 30 measuring cell,
and STARe Thermal analysis version 3.1 software; Mettler Toledo AG, Switzerland). An
empty pan was used as reference. Anhydrous samples of each system were prepared in
aluminium pans without being stored at RH and also scanned under the same conditions.
Each sample was immediately rescanned to remove endothermic baseline shift associated
in some cases with the glass transition. The Gordon-Taylor equation was fitted to the
glass transition data and used for predicting the plasticization of the systems at different
RH (papers III and IV).
4.3 Evaluation of crystallization
The occurrence and extent of crystallization was assessed by X-ray diffraction methods.
Gravimetric determination of water content was also related to the degree of crystallinity.
4.3.1 Gravimetric methods
The extent of crystallization was evaluated by monitoring the weight of samples under
various RH conditions. The change in sample weight resulting from loss of water can be
used as an estimation of the degree of crystallization. It is, therefore, a typical water
sorption study in which the sample is stored in a vacuum desiccator over a salt solution
and removed at time intervals, and weighed. The gravimetric measurement of
crystallinity from the loss of sorbed water for LSI and LSI-carrageenan systems is
presented in paper III. The decrease in the amount of sorbed water is an indication of
crystallization, and the extent of crystallization can be derived from water loss.
The percentage of loss water at any time Tx is calculated by the equation (15):
100t
Xt
WTWTWT%WL −= (15)
where WL is the amount of water loss, Wt the highest water content measured after a time
t, and WTx the moisture content at a given time. The extent of crystallization was referred
34
to the percentage of water loss. It was assumed that in case of total crystallinity the
sample would have 0 % water content.
The ratio of crystallization versus hydrolysis was calculated as water lost by
crystallization divided by water gained by hydrolysis 96h before and after the highest
water content or inflexion point at RH 76.4 %. That means that in the interval time 96-
196h, the water loss was referred to the extent of crystallization and in the interval
time196-296h, water gain was referred to the extent of hydrolysis (Fig. 4). This ratio (R)
describing the sort of competitiveness occurring between crystallization and sucrose
hydrolysis was calculated according to equation (16):
96196
196296
WTWTWTWTR
−−
= (16)
Where WT96, WT196, and WT296 were the water contents measured after 96, 196, and 296h,
respectively at RH 76.4%.
Figure 4. Water sorption at 76.4% RH used in the measurement of crystallization versus
hydrolysis in LSI and LSI-carrageenan.
0 50 100 150 200 250 3000
2
4
6
8
10
12
14
16
LSI LSI-arrageenan
WA
TER
CO
NTE
NT
(g/1
00g
solid
s)
TIME (h)
35
4.3.2 X-ray diffraction
X-ray diffractometer was used to monitor the extent of crystallization. Amorphous
samples were stored in evacuated desiccators under the same RH conditions used for
water sorption studies for 24h.Triplicate samples were quickly crushed into a fine powder
using a pestle and a mortar. A tiny amount of liquid nitrogen was added to the sample
during crushing in order to prevent sample stickiness. The sample was quickly filled into
a circular plastic sample holder, and the sample surface was leveled with a glass slide. All
the sampling operation was done using a glove box to prevent water condensation from
air. The sample was scanned using a D 5005 Siemens X-ray powder diffractometer, with
diffraction angle 2θ ranging from 4 to 38° with a step of 0.020°. The rotation speed was
15 rotations per minute. The X-ray generator was equipped with a copper tube at 40 kV
and 50mA producing CuKα radiation of 0.154nm wavelength. The spectra were analyzed
using a computer Diffract-Plus program. The baseline of the holder was extracted from
the recorded spectra and at each RH, the diffraction spectra were corrected for scattering
the amorphous area using the Diffract-Plus program. The increase in spectral resolution
was used to quantify the increased crystalline order. The crystallinity indices were
calculated using the methods described by Hermans and Weidinger, (1950) and Farhat et
al. (1996) based on the ratio of the relative area under the characteristic sharp peaks
versus the amorphous area.
4.4 Determination of sucrose hydrolysis
The freeze-dried materials in glass vials were ground and the amorphous powder was
transferred in Eppendorf polypropylene test tubes (Greiner, Germany). The samples
weight were between 80 and 100mg. The tubes were placed on supports made of
cardboard and stored desiccators under vacuum at 24°C over saturated salt solutions (RH
23.9-76.4%). The extent or the rate of sucrose hydrolysis in a sample after a given storage
period was determined by following the amount of fructose or glucose formed as well as
the amount of remaining sucrose as a function of time. The enzymatic glucose kit and
HPLC method were used to determine the contents of glucose, and glucose, fructose, and
36
sucrose, respectively. Glucose, fructose, and acetonitrile were products of Merck
(Darmstadt, Germany). The enzymatic kit was from Sigma (St Louis, USA)
In order to monitor the activity of invertase after the sample was exposed to a specific RH
for a certain time, it was essential to suppress its action so that the sugar concentration did
not change further. Folkes and Jordan (1996) suggested that acetonitrile deactivates any
enzyme. In this study, this was demonstrated for invertase. The task was to use
acetonitrile (99.7%, HPLC grade, Merck, Germany) at the concentration that can
deactivate the enzyme and also is suitable for the mobile phase used in the HPLC
method. A solution of 20 mg/ml of sucrose in distilled water was prepared. 1ml of freshly
prepared solution of invertase (0.5mg/ml) was added to 2ml of the sucrose solution, and
2ml of acetonitrile immediately added. The final concentration of the solution was
acetonitrile:water (2:3). Two other mixtures were made with the same amount of
invertase solution and acetonitrile:water (5:5) and (3:2), respectively. The three
preparations were stored at room temperature (24°C) for 30min and filtered through a
0.45µm Sparttan 30/B filter (Dassel, Germany). Triplicate aliquots of each filtrate were
used for detection of glucose using the enzymatic kit and for fructose and glucose using
the HPLC method. The results of the tests showed that glucose and fructose were not
found in the mixtures. This was a proof that acetonitrile at a concentration ≥ 40% stopped
the activity of invertase.
4.4.1 Determination of glucose contents by spectrophotometry: enzymatic kit
The enzymatic method (Trinder kit, Sigma, St Louis, USA) is based on the oxidation of
glucose by glucose-oxidase to gluconic acid and hydrogen peroxide. Hydrogen peroxide
in the presence of peroxidase produces the oxidative coupling of phenol with 4-
aminophenazone, forming a red-colored chromagen absorbance of which is measured
spectrophotometrically at 505nm. The intensity of the color is directly proportional to the
glucose concentration in the sample. The mechanism of the reaction is outlined in Fig. 5.
Three sets of solution of glucose (5mg/ml) were prepared and their absorbance was
measured using a Perkin-Elmer LAMBDA 2 UV-Vis spectrophotometer at 505nm. The
37
absorbance of the sample was determined in the same way. Seven different
concentrations of glucose solutions over the concentration range of sucrose in samples
were used. A 10µl aliquot of the solution was mixed with 3ml of enzymatic kit. The
mixture was kept at room temperature for 18 min; thereafter the absorbance was
measured. Curves of the absorbance versus glucose concentration were made. The
coefficients of determination (R2) of the linear regression analysis were between 0.95 and
0.97. Triplicates samples stored over saturated salt solutions were dissolved each in 1.5ml
of acetonitrile/water (3:2), resulting in a solution that proved to inactivate invertase. The
mixture was filtrated through 0.45µm Sparttan 30/B filter (Dassel, Germany). 3 × 10µl
aliquots of each filtrate were taken for determination of the glucose content using the
enzymatic kit as described above. The concentration of glucose (GC) in the sample was
determined according to the equation (17):
[ ]StdofionConcentratAAAA
GCBlStd
Blsple
−−
= (17)
where ASple, ABl, and AStd were the absorbance of the sample, the absorbance of the blank,
and the absorbance of the standard solution of known concentration, respectively.
Figure 5. Mechanism of reaction of glucose with the enzymatic kit.
Glucose+ H2O + O2 Glucose OxidaseGluconic Acid + H2O2
H2O2 + O2 + 4-aminoantipyrine + p-Hydroxybenzene Sulfonate
Peroxidase
Quinoneimine Dye + H2O
38
Figure 6. First order plots for determination of rate constants of sucrose hydrolysis in
MDSI samples under various RH conditions.
The coefficients of variation between replicates were below 8%. Reaction rate and rate
constants of the curves of glucose formation were calculated from zero and first-order
kinetics using regression analysis.
4.4. 2 Determination of sugar content by HPLC
The determination of content of fructose, glucose, sucrose, and lactose in the sample was
done using High Performance Liquid Chromatography (HPLC) 1090 with RI detector
(Hewlett-Packard, GmbH, Germany). This study is presented in paper I. The column used
was a 25 × 4.6 mm S5NH2 (Spherisorb, UK) that was thermostated at 40°C. The mobile
phase was acetonitrile:water (3:2) with a flow rate of 1.8ml/min. The external standard
method was used for determination of the content of the different sugars present in the
samples. Six solutions of known concentrations of these sugars were prepared and linear
regression analysis of each sugar was done. The coefficients of determination (R2) were
between 0.97 and 0.99.
0 200 400 600 800
1.2
1.6
2.0
2.4
23.8 33.0 44.4 53.8 66.3 76.4
RH:%
ln(G
LUC
OSE
CO
NTE
NT,
g/1
00g
solid
s)
TIME (h)
39
Triplicate samples of the lactose-sucrose-invertase in Eppendorf tubes were removed
from desiccators over saturated salt solutions, corresponding to various RH, and
dissolved into 2mL of acetonitrile. The mixture was filtrated through 0.45µm Sparttan
30/B filter (Dassel, Germany). Triplicate aliquots of each the filtrates were prepared in
vials for HPLC analysis. The injection volume was 10µl. The amounts of fructose,
glucose, sucrose, and lactose in 100g of solids were calculated according the equation X
= (Y-b)/a. The equation was derived from the linear regression analysis, where X
represent the unknown amount of a sugar, Y the peak area, and a and b the slope and
intercept of the line, respectively. The lactose content was compared to the initial
concentration of the sample to evaluate the accuracy of the analytical procedure. Rate
constants for sucrose hydrolysis were calculated using first order plots. Fig. 6 is an
example of first order plots of sucrose hydrolysis in MDSI system at various RH.
5. RESULTS AND DISCUSSIONS
5.1 Water sorption isotherms
5.1.1 Lactose-sucrose-invertase and lactose-sucrose-invertase-carrageenan systems
(paper I and III)
Water sorption leveled off after 44h at RH below 44.4 % and indicated steady-state water
contents as also observed by Jouppila and Roos (1994). Above this RH, a reduction in
sorbed water was perceptible after 24-44h as an indication of crystallization (Linko et al.
1982; Roos and Karel, 1992; Saleki-Gerhardt and Zografi, 1994; Saleki-Gerhardt et al.,
1995). This was in agreement with the results of Vuataz (1988) who reported
crystallization of lactose at RH > 50% in skim milk. The GAB and BET models were
fitted to the average steady-state water contents in the 0.113-0.538 and 0.113-0.444 aw
ranges, respectively. The GAB model could not successfully fitted to experimental data
over the whole aw range as often found (Van den Berg and Bruin, 1975; Bell and Labuza,
2000), while the fitting to the BET model agreed fairly well with the aw range commonly
40
found for fitting most food materials. The restriction observed with the GAB model
resulted from structural changes in the microstructure of the system due to crystallization.
The sorption behavior of the system was consequently affected. This was supported by
the fact that the GAB sorption isotherm deviated from experimental data at aw where the
water content was sufficient to induce crystallization. It seemed that crystallization took
place in the 44.4-76.4% RH range causing discontinuity in the water sorption process.
Fig. 7 shows the deviation of the GAB model from the experimental data points as a
result of lactose crystallization
Figure 7. Relationships between water activity and water content in lactose-sucrose
invertase-system. The GAB deviated from experimental data from aw 0.444. The
monolayer value was 2.8g/100g solids.
Water sorption properties of LSI and LSI-carrageenan systems were used to evaluate
crystallization assuming that sorbed water is released during crystallization. The
evaluation was made for samples stored at RH 53.8 and 66.2 % since the systems
remained stable below these RH, and crystallization occurred concurrently with sucrose
hydrolysis (Fig.8).
0.0 0.2 0.4 0.6 0.80
5
10
15 Experimental
GAB
2.8g
WATER ACTIVITY
WA
TER
CO
NTE
NT(
g/10
0g S
OLI
DS)
41
Figure 8. Water sorption properties of LSI (a) and LSI-carrageenan (b) at various RH
conditions. Crystallization and sucrose hydrolysis occurred competitively under various
RH conditions. Crystallization was delayed by κ-carrageenan (paper III).
After 48h of storage, the percentage of water lost from initial water at RH 53.8% was 9%,
then increased gradually to 52.4% after 296h for the LSI system while the LSI-
carrageenan system released from 0 to 10.8% of initial water. A similar behavior was
observed at RH 66.2% where the percentage of released water increased from 11 to 50%
for LSI while increasing only from 9.9 to 14.6% for LSI-carrageenan at 296h (paper III).
This loss of sorbed water was a prominent observation of the occurrence of crystallization
in lactose containing systems. Monosaccharides, fructose and glucose, produced in
hydrolysis of sucrose increased hygroscopic properties and water sorption of the systems.
At a RH as high as 76.4%, LSI contained more sorbed water than LSI-carrageenan at
196h (paper III). A possible explanation for LSI containing more sorbed water than to
LSI- carrageenan at 196h is that as carrageenan delayed crystallization in LSI-
0 50 100 150 200 250 300 3500
2
4
6
8
10
12
14
16a
RH%
23.933.0
44.4
53.8
66.2
76.4
WA
TER
CO
NTE
NT
(g/1
00g
solid
s)
TIME (h)0 50 100 150 200 250 300 350
0
2
4
6
8
10
12
14
16b
23.9
33.0
44.453.866.2
76.4
RH%
WA
TER
CO
NTE
NT
(g/1
00g
solid
s)
TIME (h)
42
carrageenan, crystallization became slower and more sucrose hydrolysis was observed.
These observations showed that the systems had undergone crystallization and sucrose
hydrolysis, but the order of occurrence of these changes depended on aw and probably on
the amount of water sorbed.
5.1.2 Maltodextrin-sucrose-invertase systems with and without lactose (paper II)
In systems containing maltodextrin, the steady-state water contents were reached after
24h at RH between 11.3 and 66.2%. At 76.4% RH, apparent equilibrium was attained
after about 54-72h but the water sorption properties changed after 96h showing an
increase in water uptake. The BET sorption model could be fitted to the experimental
data from 11.3 to 53.8% RH and the GAB sorption model over the 11.3–66.2% RH range
for both systems. The systems did not show any break in the sorption curves, which
indicated no crystallization during storage. It could be expected that the sugars present in
the system would undergo crystallization in the presence of water. The absence of
crystallization was probably related to the properties of the major matrix, maltodextrin,
since we observed crystallization in lactose-sucrose-invertase systems under the same
conditions. Roos (1995a) reported delayed crystallization by adding maltodextrin. It is
likely that sucrose and lactose crystallization was delayed by maltodextrin, as suggested
by the water sorption properties. It can be hypothesized that incorporation of maltodextrin
resulted in delayed crystallization. Further studies are needed to elucidate the mechanism.
5.1.3 Pullulan-sucrose systems: effects of glycerol
The water contents of PS and PSI at various RH over the 23.9-44.4% RH range were
approximately the same. At higher RH, PSI sorbed more water than PS. PSIG exhibited
higher water contents than PSI above 44.4% RH. The GAB and BET sorption isotherms
could be used to model water sorption. The GAB model showed a very good fit to
experimental water sorption data of PS, PSI, and PSIG over the whole aw range while the
BET model fitted the experimental data over the whole aw range only for PSIG and only
up to 0.444 aw for PS and PSI. The sorption isotherms had the sigmoid shape typical of
43
most food materials (Bell and Labuza, 2000; Serris and Biliaderis, 2001) and high
molecular weight hydrophilic polymers (Biliaderis et al., 1999). The GAB isotherms
indicated that PSI and PSIG sorbed more water than PS. A possible reason is the
hygroscopic property of fructose and glucose resulting from sucrose hydrolysis.
Furthermore, PSIG contained glycerol that is known to affect water sorption. Over the
44.4-76.4% RH range and especially at 76.4% RH, PSIG sorbed more water than PSI.
Forssell et al. (1997) discovered in their investigation of concentrated starch-glycerol-
water mixtures that glycerol limited the water sorption of the system at low aw while
promoting it at aw> 0.8. In the present study, the promotion of water uptake by glycerol
was observed at aw > 0.444. The different water sorption behavior of the glycerol
containing sample below and above 0.444 aw was probably a result of combined effects of
pullulan and glycerol molecules with other molecules, including water, sucrose, and the
monosaccharides produced by sucrose hydrolysis. Above 0.444 aw in both PSI and PSIG
systems, sucrose hydrolysis became obvious, probably affecting water sorption. The
higher water contents of PSIG compared to PS and PSI observed at 0.538 aw and above
suggested that at this aw many water molecules bind to glycerol molecules.
5.2 Glass transition
5.2.1 Lactose-sucrose-invertase and lactose-sucrose-invertase-carrageenan systems
(paper III)
In both systems, at RH exceeding 53.8%, determination of Tg became problematic,
because Tg was affected by crystallization that had taken place during sample storage
over saturated salt solutions, as suggested by water sorption properties. Tg values were
generally very close to each other in systems irrespective of whether they contained
carrageenan or not. The relationship of Tg with water content could be modeled using the
Gordon-Taylor equation. Tg values obtained experimentally at RH 66.2 and 76.4% did
not follow the curves predicted by the Gordon-Taylor equation (paper III). Only water
contents measured in the 0-53.8% RH range were used in calculation of the constant, k,
for the Gordon-Taylor equation. Glass transition and water sorption were time-dependent
44
phenomena that took place in systems where sucrose hydrolysis occurred. Fructose and
glucose resulting from sucrose hydrolysis favored plasticization. The systems therefore
became different from the original mainly in their carbohydrate composition.
5.2.2 Maltodextrin-sucrose-invertase systems with and without lactose
The thermograms obtained for MDSI and MDLSI by DSC showed a broad glass
transition range of about 15°C. The overall Tg in the systems seemed to be dependent on
plasticizers present such as water and sugars, but also on the maltodextrin content of the
systems. Indeed, the systems containing the highest amount of maltodextrin, MDSI
exhibited higher Tg values. This confirms the dependence of Tg on molecular weight and
branching (Slade and Levine, 1987). Fig. 9 shows DSC thermograms of MDSI under
various RH conditions. In lactose-sucrose-invertase systems, crystallization was apparent
in the diffraction patterns of samples stored at 23.9-53.8 %RH range. That suggested that
lactose underwent crystallization since its tendency to crystallization is well known
(Saleki-Gerhardt and Zografi, 1994; Roos, 1995a). In maltodextrin containing systems,
the thermograms did not suggest crystallization. Also the glass transition was apparent
over the whole aw range (paper II). This suggested that as maltodextrin delayed
crystallization, the glass transition could be more effectively determined.
45
Figure 9. DSC thermograms of maltodextrin-sucrose-invertase (MDSI) under various
RH conditions.
5.2.3 Pullulan-sucrose systems: effects of glycerol
The thermograms of PS, PSI, and PSIG samples showed complex transitions over the 0-
53.8% RH range. Above 53.8% RH a clear Tg was obtained. At lower RH, the
thermograms showed both endothermal and exothermal enthalpy relaxations (Roos,
1995a). Rescanning of the samples after quenching to room temperature gave a clear
glass transition for PS and most PSI and PSIG samples. However, in some PSI and PSIG
samples the relaxations were also observed in the immediate rescans of the samples.
Upon the second heating, the appearance of the endothermal and exothermal events
associated with the transition was dependent on water content (Thiewes and Steeneken,
1997; Le Bail et al., 1999). The relaxations reflect the relative rate of glass formation and
aging and, thereby, the thermodynamic state of the amorphous material. The broad
-100 -50 0 50 100 150
76.4
66.2
0
53.844.4
33.023.9
RH%
END
OTH
ERM
AL
HEA
T FL
OW
TEMPERATURE (°C)
46
temperature range of the glass transition became bigger as RH increased. This agreed
with the results obtained by Biliaderis et al. (1999). The broad temperature range of the
glass transition was probably related to the composition of the systems and water content
at various RH. The Tg of the anhydrous PS and PSI were very similar, while PSIG had a
lower Tg. The similarity of the Tg of anhydrous PS and PSI indicated that the systems
were not much different in their carbohydrate composition. However, the Tg of anhydrous
PSIG was lower than that of PS and PSI. This difference resulted from glycerol that was
an additional plasticizer in PSIG. At higher RH, also fructose and glucose resulting from
sucrose hydrolysis contributed to plasticization. The presence of these monosaccharides
contributed to the plasticization of PSI and explained the difference in Tg between PS and
PSI as RH increased. The temperature range of the glass transition was much wider in the
sample containing glycerol especially above 23.9% RH. Ward and Hardley (1993) found
that plasticizers lowered the temperature of the glass transition. Also, the transition
seemed broader. Such a broadening could be dependent on the nature of the interaction
between the polymer and the plasticizers. The results of the present study are in
agreement with this statement and suggested that glycerol tended to increase the
cooperation of different components involved in the transition in the pullulan-sucrose
systems (Fig10). The Gordon-Taylor equation was used to predict Tg. However, water
activities giving complex transitions in the second thermograms were not considered in
the prediction of Tg because of the occurrence of successive endothermic events in the
thermograms. The values of the constant, k, were 4.7 ± 0.6, 4.4 ± 0.3, and 5.3 ± 0.5 for
PS, PSI, and PSIG, respectively.
47
Figure 10. DSC thermograms of PSI system at RH 23.9 % showing successive
endothermic events.
5.3 Sucrose hydrolysis
5.3.1 Effect of glass transition
It has been suggested that the glassy state of an amorphous matrix could be responsible
for the rate control of diffusion-limited rates of chemical reactions that take place at low
moisture contents (Slade and Levine, 1994). The limitation in diffusion disappears and a
drastic increase in the reaction rates as a result of increased water content (Buera et al.,
1995) and plasticizer effects may be noticeable as the glass-rubbery transition occurs
(Fig. 11).
-20 0 20 40 60 80 100 120 140
Endothermic heat flow
TEMPERATURE (°C)
2nd scan
1st scan
END
OTH
ERM
AL
HEA
T FL
OW
48
Figure 11. Relationship of Tg at T = 24°C with rate of sucrose hydrolysis in PSI and
PSIG systems. Sucrose hydrolysis occurred in the supercooled liquid matrix.
When rate of sucrose hydrolysis is plotted against T-Tg, no apparent change was found at
T-Tg = 0. Furthermore, although sucrose hydrolysis occurred mainly above the glass
transition, a significant increase in the rate was observed much above Tg. That suggested
that Tg did not directly control the rate of sucrose hydrolysis. However, the high diffusion
and molecular mobility prevailing above Tg could serve possibly as a bridge between
rates of sucrose hydrolysis and Tg. Sucrose hydrolysis surely is affected by diffusion and
mobility of enzyme and substrate molecules. A study of these phenomena at a molecular
level in the vicinity of Tg could provide a step toward understanding the relationship of Tg
and enzymatic reactions in low moisture foods.
-60 -40 -20 0 20 40 60 80 100
0.0
5.0x10-4
1.0x10-3
1.5x10-3
2.0x10-3
2.5x10-3
Supercooled liquid matrixGlassy state
PSI PSIG
RA
TE C
ON
STA
NT
(h-1)
T - T g(°C)
49
5.3.2 Effect of water
Whitaker (1995) elucidated the role of water on enzyme activity. He stated that water
plays at least four important functions in all enzyme-catalyzed reactions: (1) folding of
the protein, (2) acting as a transport medium for the substrate and enzyme, (3) hydration
of the protein, and (4) ionization of prototropic groups in the active sites of the enzyme.
In a lactose-sucrose-invertase system (paper I), sucrose hydrolysis did not occur below
44.4 %RH. At 44.4% and 53.8% RH, sucrose hydrolysis was moderate but occurred
concomitantly with crystallization. At 66.2% and 76.4% RH, significant hydrolysis was
found, with a rate increasing with increasing water content. The occurrence of sucrose
hydrolysis together with crystallization suggested that crystallization might affect the rate
of hydrolysis on the basis of the water repelling ability of crystallized materials. Indeed,
upon crystallization, water in the crystallized regions might have been released to the
system enhancing the hydrolysis process. Reactants were also concentrated in the non-
crystalline medium. In maltodextrin-sucrose matrices, where crystallization was not
found, the rate of sucrose hydrolysis was low below 53.8 % RH compared to above this
RH, as found earlier by Silver and Karel (1981). In the LSI and LSI-carrageenan as well
as in pullulan-sucrose systems, significant sucrose hydrolysis occurred also above 66.2%
RH except that at 53.8% RH sucrose hydrolysis become considerable in a systems
containing glycerol. Table 1 summarizes rate constants of sucrose hydrolysis from
different systems. The overall trend show that sucrose hydrolysis increased with water
activity. In systems where no crystallization was observed, and containing no glycerol,
the enzyme activity seemed consistent with 66.2 % RH. It is likely that in the vicinity of
66.2 %, water is available in the form that allows its function in ionizing the active sites
of the enzymes and acting as transport medium for molecules. Also, the activity of the
enzyme might depend more on the mobility and diffusivity of the constituents of the
systems. Such an action may be devoted to water that appears as a key factor in
controlling the activity of the enzyme and possibly plasticization of the enzyme itself.
50
Table 1. Rate constants of sucrose hydrolysis in lactose-sucrose-invertase, MDSI,
MDLSI, LSI, LSI-carrageenan, PSI, and PSIG at various RH.
k (h-1) Systems LSI a MDSI MDLSI LSIb LSI-Carr. PSI PSIG
RH %
23.9 5.01 .10-5 5.95 .10-4 5.99 .10-4 9.48 .10-5 1.64 .10-4 33.0 5.31 .10-5 8.40 .10-4 6.03 .10-4 5.94 .10-5 2.28 .10-4 44.4 5.28 .10-4 9.26 .10-4 7.23 .10-4 9.14 .10-5 8.72 .10-5 53.8 5.31 .10-4 9.55 .10-4 6.15 .10-4 8.80.10-4 8.34 .10-4 3.84 .10-4 1.11 .10-3 66.2 9.17 .10-4 1.75 .10-3 1.42 .10-3 1.22.10-3 1.13 .10-3 1.79 .10-3 1.84 .10-3 76.4 7.98 .10-3 1.92 .10-2 2.05 .10-2 9.56.10-3 9.26 .10-3 2.46 .10-3 2.47 .10-3
a lactose-sucrose-invertase system in paper I blactose-sucrose-invertase system in paper III
5.3.3 Effects of nonaqueous plasticizers, additives, and composition change
Glycerol was found to decrease Tg of PSIG system. Fig. 12 shows the effect of water and
glycerol on rate of sucrose hydrolysis in PSI and PSIG systems.
Figure 12. Combined effects of water and glycerol on rate of sucrose hydrolysis. Sucrose
hydrolysis increased in the presence of glycerol at 0.538 aw.
0.2 0.3 0.4 0.5 0.6 0.7 0.8-5.0x10-4
0.0
5.0x10-4
1.0x10-3
1.5x10-3
2.0x10-3
2.5x10-3
3.0x10-3
PSI PSIG
RA
TE C
ON
STA
NT
(h-1)
WATER ACTIVITY
51
The rate of sucrose hydrolysis in PSIG was higher than in PSI especially at aw 0.538 with
a rate constant of 3.84 × 10-4h-1 for PSI and 11.11×10-4h-1 for PSIG. The difference in the
rate constants of these systems was due to glycerol favoring the activity of the enzyme. It
seems that at this aw, the presence of glycerol favored the activity of the enzyme by
increasing its mobility and probably that of sucrose so that the overall mobility of
molecules was facilitated. Labuza (1980) reported that liquid-like humectants might act
as aqueous phases by dissolving reacting species at very low moisture contents resulting
in higher reaction rates. Glycerol, as a plasticizer can affect enzyme activity even at very
moderate RH, which should be taken into account in processing and storage of food and
pharmaceutical systems. κ-Carrageenan was added to LSI system as network forming
polysaccharide to regulate mobility of the systems. It was found that although the overall
rates of sucrose hydrolysis were close to each other in both carrageenan and non-
carrageenan systems, at 76.4% RH the rate of sucrose hydrolysis was only little faster in
the system containing carrageenan. A probable explanation was that at a higher RH
carrageenan delayed crystallization allowing more time for sucrose hydrolysis. Glucose
and fructose were not components of the systems during their preparation. As their
amount increased progressively in the systems with sucrose hydrolysis, water contents
increased and the extent of hydrolysis also increased. This was observed in most of the
systems at 76.4% RH after a certain time of storage. The increase in water contents
concurrently with crystallization in LSI and LSI-carrageenan, with relatively higher water
content in LSI was explained possibly that hydrolysis favored water uptake by the
systems. In pullulan containing systems, the difference in the sorption behavior of PS
compared to PSI and PSIG was due to the absence of fructose and glucose in the PS
system, since it included no invertase. Sucrose hydrolysis was therefore not expected.
The changes in composition resulting from the sucrose hydrolysis provided the systems
with more water that enhanced sucrose hydrolysis.
52
5.3.3 Crystallization and sucrose hydrolysis
The establishment of crystallinity versus hydrolysis is a complicated task because of
possible interference of both changes at such RH where mobility is high. The present
study aimed at evaluating this competitiveness at RH 76.4%, since at this RH changes
were quite distinguishable from the water sorption curves. The extent of crystallization
was evaluated by measuring the percentage of loss of sorbed water and by X-ray
diffraction while the extent of sucrose hydrolysis was evaluated as the percentage of
sorbed water. The analysis of the percentage of released water at RH 53.8 and 66.2%
suggested that the LSI released more water than LSI-carrageenan in the same duration of
storage. That means that the extent of crystallization was higher in LSI compared to LSI-
carrageenan. The ratio of crystallinity to hydrolysis was 1.04 for LSI and 1.95 for LSI-
carrageenan. This confirmed that at this RH the extent of crystallization of lactose
overtook sucrose hydrolysis in LSI, in which lactose crystallized faster than in LSI-
carrageenan. The X-ray diffraction patterns showed that crystallization begun at RH
44.4% in both systems. The major peaks of the X-ray patterns appeared in samples stored
at RH higher than 44.4%. The intensities of peaks in the diffraction patterns of LSI were
more developed than those of LSI-carrageenan and were characteristic of lactose in
different crystal forms. This is understandable since in the systems, the amount of lactose
was at least two times bigger that of sucrose. Sucrose was hydrolyzed completely or at
least extensively leaving mainly lactose as a residual matrix. The X-ray results confirmed
the results obtained by the sorption study. The presence of κ-carrageenan delayed
crystallization. The competitive occurrence of sucrose hydrolysis and crystallization was
described in paper III. The water uptake increased in both systems at 76.4% RH from
about 88h to 196h of storage where it reached a peak. This was a result of water binding
by fructose and glucose. The peak in moisture content suggested that after 196h, sucrose
hydrolysis was probably completed and binding of water molecules to fructose and
glucose terminated. Water that enabled the hydrolysis of sucrose might have favored the
crystallization of the lactose existing in the system. The amount of released water
observed after 196h was the measure of crystallinity in the systems. The sorption
53
behavior of the systems 96h before and after the peak indicated that sucrose hydrolysis
and crystallization occurred competitively. However, these two phenomena shared certain
compatibility since upon crystallization the released water was capable of inducing
sucrose hydrolysis. On the other hand, the natural attraction of fructose and glucose to
water may increase the water content of the system to induce crystallization.
6 CONCLUSIONS
In the present study, the physical characterization of freeze-dried lactose-sucrose-
invertase, LSI, LSI-carrageenan, MDSI, MDLSI, PS, PSI and PSIG systems was done by
determining water sorption properties and glass transition temperature under various RH
conditions. Rates of sucrose hydrolysis by invertase were determined under those RH
conditions. The relationships between aw, water content, glass transition, and rates of
sucrose hydrolysis were examined. Water sorption studies were used to determine
parameters, such as the monolayer values and aw and RH, as well as moisture content
above which a number of changes including crystallization and sucrose hydrolysis
occurred.
The investigation of lactose-sucrose-invertase systems showed that crystallization was
likely to start at RH as low as 44.4% and became substantial above RH 53.8% where it
occurred concomitantly with sucrose hydrolysis. It was also observed that as
crystallization took place during storage. In some cases, determination of the glass
transition was prevented by interference of crystallization and glass transition. The
concomitant occurrence of crystallization with sucrose hydrolysis suggested that the
moisture content that allowed crystallization was capable to allow sucrose hydrolysis.
Furthermore, the crystallizing portion of the systems at 76.4% RH released sorbed water
allowing more sucrose hydrolysis. Although sucrose hydrolysis was perceptible at 53.8%
RH and above in crystallizing systems, in non-crystallizing systems the appearance of
sucrose hydrolysis seemed consistent above 66.2 % RH suggesting that this RH was a
more important factor controlling sucrose hydrolysis.
54
The present study has evidenced that in non-crystallizing system sucrose hydrolysis
occurred mainly in the rubbery state. However, sucrose hydrolysis occurred well above
the glass transition. This indicated that the rate of sucrose hydrolysis was not directly
related to the change in state. In systems based on a maltodextrin matrix, crystallization
was not observed from water sorption properties although the systems contained
crystallizing materials such as sucrose and lactose. This absence of crystallization
indicated that maltodextrin could be used in preparation of food materials where
crystallization needs to be delayed.
The evaluation of crystallinity by water sorption study and by X-ray diffraction in LSI
and LSI-carrageenan demonstrated that κ-carrageenan used as a network forming
polysaccharide was effective in delaying crystallization. This result is of great importance
for industrial processes for example in confectionery or ice cream production where the
control of crystallization is crucial for acceptance of the final product.
The investigation of pullulan containing systems showed a complex glass transition in the
DSC thermograms at some RH. This probably resulted from the interactions of pullulan
with sucrose and other components. This complexity suggested possible phase separation
that needs to be further studied. However, relationships of these complicated transitions
with the rate of sucrose hydrolysis were not established. The presence of fructose and
glucose as hydrolysis products of sucrose increased water sorption especially at high RH.
This contributed to increase rate of sucrose hydrolysis. Glycerol, when added at the level
of 5 %, increased the water sorption of PSIG system and enhanced the rate of sucrose
hydrolysis. Glycerol may affect substrate and enzyme mobility. This suggests that
plasticizers, such as glycerol, may affect the enzyme activity even at moderate aw, which
should be taken into consideration in processing and storage of food and pharmaceutical
systems.
In the systems studied, water seems to be the most important plasticizer, but also a factor
affecting sucrose hydrolysis. Since mobility and diffusivity are recognized as factors
55
controlling time-dependent and diffusion controlled changes in foods, water plays a
significant role in these phenomena. Water is a key factor controlling the activity of an
enzyme and possibly plasticization of the enzyme itself.
56
7 REFERENCES
Acker, I. 1969. Water activity and enzyme activity. Food Technol. 23:1257-61, 69.
Andersen, B., Thiesen, N. and Broe, E.P. 1969. The transferring action of β-
fructofuranosidase from yeast; A quantitative description. Acta Chem. Scand. 23:
2367-2374.
Andronis, V., Yoshioka M. and Zografi, G. 1997. Effects of sorbed water on
crystallization of indomethacin from the amorphous state. J. Pharm. Sci. 86: 346-
351.
Andronis, V. and Zografi, G. 1998. The molecular mobility of supercooled amorphous
indomethacin as a function of temperature and relative humidity. J. Pharm. Sci.
15: 835-842.
Antonov, Y.A. and Gonçalves, M.P. 1999. Phase separation in aqueous gelatin-K-
carrageenan systems. Food Hydrocoll. 13: 517-524.
Arvanitoynnis, I. and Biliaderis, C.G. 1998. Physical properties of polyol-plasticized
edible films made from sodium caseinate and soluble starch blends. Food Chem.
62: 333-342.
Bell, L. and Hageman, M.J. 1994. Differenciating between the effects of water activity
and glass transition dependent mobility on a solid-state chemical reaction:
aspartame degradation. J. Agric. Food Chem. 42: 2398-2401.
Bell, L. and Labuza, P.T. 1992. pH of low-moisture solids. Trends Food Sci. Technol. 3:
271-274.
Bell, L. and Labuza, T.P. 1994. Influence of the low-moisture state on pH and its
implication for reaction kinetics. J. Food. Eng. 22: 291-312.
Bell, L. and Labuza, T.P. 2000. Moisture sorption. Practical Aspects of Isotherm
Measurement and Use. 2nd Ed. St Paul MN. USA: American Association of
Cereals Chemists.
Bhargava, A. and Jelen, P. 1996. Lactose solubility and crystal growth as affected by
impurities. J. Food Sci. 61: 180-184.
57
Bigelis, R. 1993. Carbohydrases. In: Enzymes in Food Processing. 3rd edition. T.
Nagodawithana, (ed). p. 121-147. Academic Press, San Diego.
Biliaderis, C.G., Lazaridou, A. and Arvanitoyannis, I.A. 1999. Glass transition and
physical properties of polyol-plasticized pullulan-starch blends at low moisture.
Carbohydr. Polym. 40: 29-47.
Bosma, M., Brinke, G.T. and Ellis, S.T. 1988. Polymer-polymer miscibility and enthalpy
relaxation. Macromolecules 21: 1465-1470.
Bowski, L., Saini, R., Ryu, Y. and Vieth, R. 1971. Kinetic modeling of the hydrolysis of
sucrose by invertase. Biotechnol. Bioeng. 8: 641-656.
Brunauer, S., Emmett, P.H. and Teller, E. 1938. Adsorption of gases in multimolecular
layers. J. Am. Chem. Soc. 60: 309-319.
Buckton, G. and Darcy, P. 1995. The use of gravimetric studies to assess the degree of
crystallinity of predominantly crystalline powders. Int. J. Pharm. 123: 265-271.
Buera, M., Chirife, J. and Karel, M. 1995. A study of acid-catalyzed sucrose hydrolysis in
amorphous polymeric matrix at reduced moisture contents. Food Res. Int. 128:
359-365.
Burin, L., Buera, M.P., Hough, G. and Chirife, J. 2002. Thermal resistance of β-
galactosidase in dehydrated dairy model systems as affected by physical and
chemical changes. Food Chem. 76: 432-430.
Cardona, S., Schebor, C., Buera, M.P., Karel, M. and Chirife, J.1997. The thermal
stability of invertase in reduced-moisture amorphous matrices in relation to glassy
state of trehalose. J. Food Sci. 62: 105-112.
Champion, D., Le Meste, M. and Simatos, D. 2000. Towards an improved understanding
of glass transition and relaxations in foods: molecular mobility in the glass
transition range. Trends Food Sci. Technol. 11: 41-55.
Chang, R. 1981. Physical Chemistry with Applications to Biological Systems, 2nd ed.
Macmillan Publishing Co., Inc., New York.
Chen, Y.H., Aull, J.L. and Bell, L.N. 1999. Invertase storage stability and sucrose
hydrolysis in solids as affected by water activity and glass transition. J. Agric.
Food Chem. 47: 504-509.
58
Contreras-Lopez, E., Champion, D., Hervet, H., Blond, G. and Le Meste, M. 2000.
Rotational and translational mobility of small molecules in sucrose plus
polysaccharides solutions. J. Agric. Food Chem. 48: 1009-1015.
Couchman, P.R. and Karasz, F.E. 1978. A classical thermodynamic discussion of the
effect of composition theory to compatible polymer blends. Macromolecules. 11:
117-119.
De Graaf, L.A., Albers, P.J.W.and Möller, M. 1996. Influence of the bulk and surface
morphology on adhesion of polystyrene-inter-poly-cross-2-ethylhexyl-
methacrylate films and particles. J. Polym. Sci. 34: 1839-1852.
Donatelli, A. A.and Sperling, L.H. 1976. Interpenetrating polymer networks based on
SBR/PS. Control of morphology by level of cross-linking. Macromolecules 9:
671-675.
Drapon, R. 1985. Enzyme activity as a function of water activity. In: Properties of Water
in Foods in Relation to Quality and Stability. D. Simatos (ed). p. 171-189.
Nighoff, M. Publishers, Lancaster.
Farhat, I.A., Mitchel, J.R, Blanshard, J.M, and Derbishire, W. 1996. A pulse H1 NMR
study of the hydration properties of extruded maize-sucrose mixtures. Carbohydr.
Polym. 30: 219-227.
Ferry, J.D. 1980. Viscoelastic Properties of Polymers, 3rd edition. John Wiley and Sons,
New York.
Fennema, O.R. 1996. Water in ice. In: Food Chemistry. O.R. Fennema (ed). p. 17-94. M.
Dekker, New York.
Flink, J.M. 1983. Structure and structure transitions in dried carbohydrate materials. In:
Physical Properties of Food. M. Peleg and E.B. Bagley (ed.), p. 473-521. AVI
Publishing, Co. Wesport, CT.
Folkes, D.J. and Jordan, M. A. 1996. Mono- and disaccharides analytical aspects. In:
Carbohydrates in Foods. A.-C. Eliasson (ed.), p.1-37. M. Dekker, New York.
Forssell, P.M., Mikkilä, J.M., Moates, K.G. and Parker, R. 1997. Phase transition
behaviour of concentrated barley starch-glycerol-water mixtures, a model for
thermoplastic starch. Carbohydr. Polym. 34: 275-282.
59
Garbarra, P. and Hartel, R.W. 1998. Corn syrup solids and their saccharide fractions
affect crystallization of amorphous sucrose. J. Food Sci. 63: 523-528.
Gillies, D.G, Sutcliffe, L.H. Wu, X. and Belton, P.S. 1996. Molecular motion of a water-
soluble nitrosyl radical in gelatin gels. Food Chem. 55:349-352.
Goff, H.D., Cadwell, K.B., Stanley, D.W. and Maurice, T.J.1993. The influence of
polysaccharides in the glass transition in frozen sucrose solutions and ice cream.
J. Dairy Sci. 76: 1268-1277.
Gontard, N., Guilbert, S. and Cuq, J.-L. 1993. Water and glycerol as plasticizers affect
mechanical and water vapor barrier properties of an edible wheat gluten film. J.
Food. Sci. 58: 206-211.
Gordon, M. and Taylor, J.S. 1952. Ideal copolymers and the second-order transitions of
synthetic rubbers. I. Non-crystalline copolymers. J. Appl. Chem. 23: 493-500.
Griffin, W.C. and Lynch, M.J. 1972. Polyhydric alcohols. In: Handbook of Food
Additives. T.E. Furia, (ed), p. 431-456. CRC Press, Florida.
Gunning, Y., MacDougall, A., Noel, T., Parker, R. and Ring, S. 1999. Plant
polysaccharides and food quality homogeneous, heterogeneous and phase-
separated systems. In: Water Management in the Design and Distribution of
Quality Foods. Y.H. Roos, B.R. Leslie, and P.J. Lillford (ed.), p. 455-479.
Technomic Publishing, Lancaster.
Hallbrucker, A., Mayer, E. and Johari, G.P. 1989. Glass liquid transition and the
enthalphy of devitrification of annealed vapor-deposited amorphous solid water.
A comparaison with hyperquenched glassy water. J. Phys. Chem. 93: 4986-4990.
Hancock, B.C. and Zografi, G. 1997. Characteristics of significance of amorphous state
in pharmaceutical systems. J. Pharm. Sci. 86: 1-12.
Hartel R.W. and Shastry, A.V. 1991. Sugar crystallization in food products. Crit. Rev.
Food Sci. 30: 49-112.
Hermans, P.H. and Weidinger, A. 1950. Quantitative X-ray investigation on the
crystallinity of cellulose fibers. A background analysis. J. Appl. Phys. 52: 491-
505.
60
Isayeva, I., Kyu, T. and Manley, J. 1998. Phase transition, structure evolution and
mechanical properties of blends of two crystalline polymers: poly(vinylidene
fluoride) and poly(butylene adipate). Polymer 39: 4599-4608.
Johari, G.P., Hallbrucker, A. and Mayer, E. 1987. The glass-transition of hyperquenched
water. Nature 330: 552-553.
Jouppila, K. and Roos, Y.H. 1994. Glass transitions and crystallization in milk powders.
J. Dairy Sci. 77: 2907-2915.
Jouppila , K., Kansikas, J. and Roos, Y. H.1998. Crystallization and X-ray diffraction of
crystals formed in water-plasticized amorphous lactose. Biotechnol. Prog. 14:
347-350.
Kalichevsky, M.T. and Blanshard, J.M.V. 1993. The effect of fructose and water on glass
transition of amylopectin. Carbohydr. Polym. 20: 107-113.
Kalichevsky, M.T. Jaroszkiewicz, E.M., Ablett, S. and Blanshard, J.M.V. 1992. Glass
transition of amylopectin sugar mixtures by DSC, DMTA, and NMR. Carbohydr.
Polym. 18: 77-88.
Kang, Y., Marangoni, A.G. and Yada, R.Y. 1994. Effect of two polar organic-aqueous
solvent systems on the structure-function relationships of proteases. III. Papain
and trypsin. J. Food Biochem. 17: 389-405.
Karel, M. 1985. Effects of water activity and water content on mobility in food
components, and their effect on phase transition in food systems. In: Poperties of
Water in Foods. D. Simatos. and J.L. Multon (ed.), p.153-169. M. Nijhoff
Publishers. Dordrecht, Netherlands.
Kasapis, S., Al Marhoobi, I.M.A. and Giannouli, P. 1999. Molecular order versus
vitrification in high-sugar blends of gelatin and κ-carrageenan. J. Agric. Food
Chem. 47: 4944-4949.
Kedward, C.J., Macnauthtan, W and Mitchel, J. R 1998. Crystallization kinetics of
lactose and sucrose based on isothermal differential scanning calorimetry. J. Food
Sci. 63: 192-197.
Keller, A. and Cheng, S.Z.D. 1998. The role of metastability in polymer phase transition
Polymer. 30: 4487-4461.
61
Kwei, K.T., Nishi, T. and Roberts, R.F. 1974. A study of compatible polymer mixtures.
Macromolecules 7: 667-674.
Labuza. T.P. 1980. The effect of water activity on reaction kinetics of food deterioration.
Food Technol. 56: (15) 36-43.
Le Bail, P., Bizot, H., Ollivon, G.K. and Bourgaux, C. 1999. Monitoring the
crystallization of amylose-lipid complexes during maize starch melting by
synchrom X-ray diffraction. Biopolymers 50: 99-110.
Le Meste, M. 1995. Mobility of small molecules in low and intermediate moisture foods.
In: Food Preservation by Moisture Control. Fundamental and Applications, G.
Barbosa-Cánovas (ed.), p. 210-223. Technomic: Lancaster, USA.
Levine, H. and Slade, L. 1986. A polymer physico-chemical approach to the study of
commercial starch hydrolysis products (SHPs). Carbohydr. Polym. 6: 213-244.
Levine, H. and Slade, L. 1990. Influence of the glassy and rubbery states on the thermal,
mechanical and structural properties of doughs and baked products. In: Doughs
Rheology and Baked Product Texture. H. Faridi and J.M. Faubion (ed.), p. 157-
330. AVI Publishing. New York.
Lim, M.H. and Reid, D.S. 1991. Studies of reaction kinetics in relation to the Tg' of
polymers in frozen models systems. In: Water Relationships in Foods. H. Levine
and L. Slade (ed.), p. 103-122. Plenium Press. New York.
Linko, P., Pollari, T., Harju, M. and Heikonen, M. 1982. Water sorption properties and
the effect of moisture on structure of dried milk products. Food Sci. Technol. 15:
26-30.
Lund, D.B., Fennema, O. and Powrie, D. 1969. Enzymic and acid hydrolysis of sucrose
as influenced by freezing. J. Food Sci. 34: 378-382.
Mathewson, P.R. 1998. Enzymes. Eagan Press, St Paul Minnesota, USA.
Matveev, Y.I.V., Grinberg, V.B. and Tolstoguzov, V.B. 2000. The plasticizing effect of
water on proteins, polysaccharides, and their mixtures. Glassy state of polymer,
foods ands seeds. Food Hydrocoll. 14: 425-437.
Mazzobre, M.F., Buera, M.P. and Chirife, J. 1997 (a). Protective role of trehalose on
stability of lactase in relation to its glass and crystal forming properties and effect
of delaying crystallization. Lebensm.-Wiss. u. Technol. 30: 324-329.
62
Mazzobre, M.F., Buera, M.P. and Chirife, J. 1997 (b). Glass transition and thermal
stability of lactase in low-moisture amorphous polymeric matrices. Biotechnol.
Prog. 13: 195-199.
McCurdy, R.D., Goff, H.D., Stanley, D.W. and Stone, A.P. 1994. Rheological properties
of dextran related to food application. Food Hydrocoll. 8: 606-623.
McLaren, A.D. 1963. Enzyme reaction in structurally restricted systems III. Yeast β-
fructofuranosidase. Invertase activity in concentrated sucrose solutions.
Enzymologia. 26: 1-11.
Michaelis, L. and Menten, M.L. 1913. The kinetics of invertase. Biochem. Z. 49: 38-43.
Moates, G.K., Noel, T.R., Parker, R. and Ring, S.G. 2001. Dynamic mechanical and
dielectric characterization of amylose-glycerol films. Carbohydr. Polym. 44: 247-
253.
Nelson, J.M. and Schubert, M.P. 1928. Water concentration and the rate of hydrolysis of
sucrose by invertase. J. Am. Chem. Soc. 50: 2188-2193.
Nelson, K.A. and Labuza, T.P.1994. Water activity and food polymer science:
Implications of state on Arrhenius and WLF models in predicting shelf life. J.
Food Eng. 22: 271-289.
Noel, T.R., Ring, S.G. and Whittam, P.A. 1990. Glass transition in low moisture food.
Trends Food Sci. Technol. 1: 62-67.
Parkin, K.L. 1993. General characteristics of enzymes. In: Enzymes in Food Processing.
3rd edition. T. Nagodawithana and G. Reed (ed.), p. 7-38. Academic Press, USA.
Olabisi O., Robeson L.M. and Shaw, M. 1979. Properties of miscible polymer systems.
In: Polymer-Polymer Miscibility. O. Olabisi (ed.), p. 113-197. Academic press,
New York.
Rahman, S. and Labuza, T.P. 1999. Water activity and food preservation. In: Handbook
of Food Preservation. S.Rahman. (ed.), p. 339-382. M. Dekker, New York.
Ruchti, J. and McLaren, A.D. 1964. Enzyme reactions in structurally restricted systems
V. Further observations on the kinetics of yeast β-fructofuranosidase (Invertase)
activity in viscous media.
Roos, Y.H. 1987. Effect of moisture on the thermal behavior of strawberries studies using
differential scanning calorimetry. J. Food Sci. 52: 146-149.
63
Roos, Y.H. 1993. Water activity and physical state effects on amorphous food stability. J.
Food Preservation. 16: 433-447.
Roos, Y.H. 1995a. Phase transition in foods. Academic Press, San Diego, CA.
Roos, Y.H. 1995b. Glass transition-related physicochemical changes in foods. Food
Technol. 49: (10) 97-102.
Roos Y.H. 1998. Phase transition and structure of solid food matrices. Current opinion on
colloid and interface Sci. 3: 651-656.
Roos, Y.H. and Karel, M. 1990. Differential scanning calorimetry study of phase
transition affecting the quality of dehydrated materials. Biotechnol. Prog. 6: 159-
163.
Roos, Y.H. and Karel, M. 1991. Phase transitions of mixtures of amorphous
polysaccharides and sugars. Biotechnol. Prog. 7: 49-53.
Roos, Y.H and Karel, M. 1992. Crystallization of amorphous lactose. J. Food Sci. 57:
775-777.
Roos, Y.H., Karel, M. and Kokini, J.L. 1996. Glass transition in low-moisture and frozen
foods. Effects on shelf life and quality. Food Technol. 50: 95-108.
Roozen, M.J.G.W., Hemminga, M.A and Walstra, P. 1991. Molecular motion in glassy
water-malto-oligosaccharides (maltodextrin) mixtures as studied by conventional
and saturation transfer spin probe ESR. Carbohydr. Res. 215: 229-237.
Saleki-Gerhardt, A. and Zografi, G. 1994. Non isothermal and isothermal crystallization
of sucrose from the amorphous state. Pharm. Res. 11: 1166-1173.
Saleki-Gerhardt, A., Stowell, J.G., Byrn, S.R. and Zografi, G. 1995. Hydration and
dehydration of crystalline and amorphous forms of raffinose. J. Pharm. Sci. 84:
318-323.
Saltmarch, M., Vagnini-Ferrari, M. and Labuza, T.P. 1981. Theoretical basis and
application of kinetics to browing in spray-dried whey food systems. Prog. Food
Nutr. Sci. 5: 331-344.
Schebor, C., Buera, M.P. and Chirife, J. 1996. Glassy state in relation to thermal
inactivation of the enzyme Invertase in amorphous dried matrices of trehalose,
maltodextrin and PVP. J. Food Eng. 30: 269-282.
64
Schmitt, E.A., Law, D., Geoff, G. and Zhang, Z. 1999. Nucleation and crystallization
kinetics of hydrated amorphous lactose above the glass transition temperature. J.
Pharm. Sci. 88: 291-296.
Schwimmer, S. 1980. Influence of water activity on enzyme reactivity and stability. Food
Technol.34: (15) 64, 66-68, 70, 72-74, 82-83.
Sears, J.K. and Darby, J.R.1982. Technology of Plasticiers. Wiley-Interscience, New
York.
Serris, G.S. and Biliaderis, G. 2001. Degradation kinetics of beetroot pigment
encapsulated in polymeric matrices. J. Sci. Food. Agric. 81: 691-700.
Shamblin, S.L., Taylor, S.L. and Zografi, G. 1998. Mixing behavior of colyophilized
binary systems. J. Pharm. Sci. 87: 694-701.
Shimada, S., Hori, Y. and Kashiwabara, H. 1988. Distribution of molecular orientation
and stability of peroxyl radicals in the noncrystalline region of elongated
polypropylene. Macromolecules 2: 979-982.
Shimada, Y., Roos, Y. and Karel, M. 1991. Oxidation of methyl linoleate encapsulated in
amorphous lactose-based food model. J. Agric. Food Chem. 39: 637-641.
Shneidman, V.A. and Uhlman, D.R. 1998. The fast cooling/heating rate effects in
devitrification of glasses. II Crystallization kinetics. J. Chem. Phys. 109: 186-195.
Shogren, R.L. 1992. Effect of moisture-content on the melting and subsequent physical
aging of corn starch. Carbohydr. Polym. 19: 83-90.
Silver, M. and Karel, M. 1981. The behavior of invertase in model systems at low
moisture contents. J. Food. Biochem. 5: 283-311.
Silver, M.S. and Haskell, J.H. 1990. Chemistry in a microenvironment of low pH,
generated with aid of an immobilized proteinase. Biochim. Biophys. Acta 1039:
25-32.
Slade, L. and Levine, H. 1987. Recent advances in starch retrogradation. In: Industrial
polysaccarides: The Impact of Biotechnology and Advanced Methodologies, S.S.
Stivala,. V. Crescenzi, and I.C.M. Dea (ed.), p. 387-430. Gordon and Breach
Science Publishers, New York, USA.
65
Slade, L. and Levine, H. 1991. Beyond water activity: recent advances based on an
alternative approach to the assessment of food quality and safety. Crit. Rev. Food
Sci. Nutr. 2: 874-895.
Slade, L. and Levine, H. 1994. Polymer science approach to water relationship in foods.
J. Food Eng. 22: 143-189.
Slade, L. and Levine, H. 1995. Glass transition and water-food structure interactions.
Adv. Food Res. 38: 103-269.
Sloan, A.E. and Labuza, T.P. 1975. Investigating alternative humectants for use in foods.
Food Product Developement 9 (10): 68-73.
Sperling, L.H. 1986. Introduction to Physical Polymer Science. 1st edition, Wiley, J. and
Sons, New York.
Sperling, L.H. 1992. Introduction to Physical Polymer Science. 2nd Edition. Wiley, J. and
Sons, New York.
Taylor, L. and Zografi, G. 1998. Sugar-polymer hydrogen bond interactions in
lyophilized amorphous mixtures. J. Pharm. Sci. 87: 1615-1621.
Thiewes, J.H. and Steeneken, A.M. 1997. The glass transition and the sub-Tg endotherm
of amorphous and native potato starch at low moisture content. Carbohydr.
Polym. 32: 123-130.
Tipton, K.F. and Dixon, H. 1983. Effect of pH on enzymes. In: Contemporary Enzymes
Kinetics and Mechanism. D. Purich (ed.), p.97-139. Academic press, Sidney.
Tjuradi, P. and Hartel, R.W. 1995. Corn syrup oligosaccahrides effects on sucrose
crystallization. J. Food. Sci. 60: 1353-1356.
Tolstoguzov, V.B. 1999. Origins of globular proteins. Letters. 444: 145-148.
Tolstoguzov, V.B. 2000a. Composition and phase diagrams for aqueous systems based on
proteins and polysaccharides. Inter. Rev. Cytol. 192: 3-30.
Tolstoguzov, V.B. 2000b. Phase behavior of macromolecular components in biological a
food systems. Nahrung 44: 299-308.
Van den Berg, C. and Bruin, S. 1981. Water activity and it estimation in food systems.
Theoretical aspects. In: Water Activity: Influences on Food quality. L.B.
Rockland, and G.F. Stewart (ed.), p. 1-61. Academic press, New York.
66
Van den Berg, C., Kaper, F.S., Weldring, J.A.G. and Wolters, I. 1975. Water binding by
potato starch. J. Food Technol. 10: 589-602
Vuataz, G. 1988. Preservation of skim-milk powders: role of water activity and
temperature in lactose crystallization and lysine loss. In: Food Preservation by
Water Activity Control. C.C. Seow (ed.), p. 3-101. Elsevier Science, Amsterdam,
Netherlands.
Ward, I.M. and Hadley, D.W. 1993. Experimental studies of linear viscoelastic behavior
as a function of frequency and temperature: the time-temperature equivalence. In:
An Introduction to the Mechanical Properties of Solids Polymers. p.84-108.
Wiley, J and Sons, Chechester.
Whitaker, J.R. 1972. Principles of Enzymology for the Food Scientists. O.R. Fennema.
(ed.), p. 1-30. M, Dekker, New York.
Whitaker, J.R. 1995. Enzymes action in aqueous systems with emphasis on intermediary
and high moisture foods. In: Food Preservation by Moisture Control:
Fundamentals and Application G. Barbosa-Canovas (ed), p. 255-273. Technomic
Lancaster.
Whitaker, J.R. 1996. Enzymes. In: Food Chemistry. O.R. Fennema. (ed.), p. 431-530. M.
Dekker, New York.
White, G.W. and Cakebread, S.H. 1966. The glassy state in certain sugar-containing food
products. J. Food Technol. 1: 73-82.