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

2

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.

3

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

4

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

5

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

6

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.

7

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

8

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).

9

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

10

(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

11

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

12

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-

13

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).

14

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)

17

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

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