Glass transition and water effects on sucrose inversion by invertase in a lactose-sucrose system

Article abstract of DOI:10.1021/jf9902999

Enzymatic changes are often detrimental to quality of low-moisture foods. In the present study, effects of glass transition and water on sucrose inversion in a lactose-sucrose food model were investigated. Amorphous samples were produced by freeze-drying lactose-sucrose (2:1)-invertase (20 mg invertase/49.4 g of carbohydrate) dissolved in distilled water. Sorption isotherms were determined gravimetrically at 24 °C. Sucrose hydrolysis was determined by monitoring glucose content using a test kit and the amounts of fructose, glucose, and sucrose using HPLC. The glass transition temperatures, T(g), at various water contents were measured using differential scanning calorimetry (DSC). The BET and the GAB sorption models were fitted to experimental data up to α(w) 0.444 and 0.538, respectively. Water sorption and DSC results suggested time-dependent crystallization of sugars at α(w) 0.444 and above. Significant sucrose hydrolysis occurred only above T(g), concomitantly with crystallization. Sucrose hydrolysis and crystallization were not likely in glassy materials.

Full text of DOI:10.1021/jf9902999

J. Agric. Food Chem. 2000, 48, 2461−2466
2461
Gla ss Tr a n sition a n d Wa ter Effects on Su cr ose In ver sion by
In ver ta se in a La ctose-Su cr ose System
Kouame Kouassi and Yrjo¨ H. Roos*
Department of Food Technology, P.O. Box 27 (Latokartanonkaari 7), FIN-00014 University of Helsinki,
Helsinki, Finland
Enzymatic changes are often detrimental to quality of low-moisture foods. In the present study,
effects of glass transition and water on sucrose inversion in a lactose-sucrose food model were
investigated. Amorphous samples were produced by freeze-drying lactose-sucrose (2:1)-invertase
(20 mg invertase/49.4 g of carbohydrate) dissolved in distilled water. Sorption isotherms were
determined gravimetrically at 24 °C. Sucrose hydrolysis was determined by monitoring glucose
content using a test kit and the amounts of fructose, glucose, and sucrose using HPLC. The glass
transition temperatures, Tg, at various water contents were measured using differential scanning
calorimetry (DSC). The BET and the GAB sorption models were fitted to experimental data up to
aw 0.444 and 0.538, respectively. Water sorption and DSC results suggested time-dependent
crystallization of sugars at aw 0.444 and above. Significant sucrose hydrolysis occurred only above
Tg, concomitantly with crystallization. Sucrose hydrolysis and crystallization were not likely in glassy
materials.
Keyw or d s: Crystallization; glass transition; invertase; sucrose hydrolysis; water
INTRODUCTION
was the temperature above which the diffusion of a
reactant increases exponentially with moisture, but the
glass transition theory did not explain the rate or the
rate constant maxima in the 0.6-0.8 water activity
range. Buera et al. (1995) found that the rate of acid-
catalyzed hydrolysis of sucrose was more dependent on
pH of the surrounding medium than the Tg.
Enzymatic reactions often cause deleterious changes
in foods. In some cases, the rate of these reactions may
be related to changes in the physical state, e.g., the glass
transition. The formation of a glassy state results in a
reduction of translational molecular motion and rates
of chemical and relaxation rates for various processes,
and rates of chemical reactions may become very low,
as stated by Cardona et al. (1997).
A number of studies have been carried out on the
hydrolysis of sucrose by invertase. The Michaelis and
Menten model is the basis for most of the theories of
enzyme kinetics using sucrose/invertase system. Cor-
responding studies have been conducted with main
regard to the enzyme-subtrate-water concentration
(Kertesz, 1935; McLaren, 1963; Ruchti and McLaren,
1964; Bowski, 1971). Since then, in some studies, more
interest has been paid to the effect of low moisture
content on rates of chemical/biochemical reactions (Sil-
ver and Karel, 1981; Bell and Labuza, 1994; Buera et
al., 1995). As the amorphous state is typical of low-
moisture foods, changes from glassy to rubbery states
may occur. Therefore, enzyme stability and reactivity
in such systems has been studied. Schebor et al. (1995)
reported that the extent of sucrose hydrolysis was
affected by moisture content but its effect was not
attributable to the plasticizing effect of water. Cardona
et al. (1997) found that the thermal stability of invertase
in reduced-moisture amorphous matrices and invertase
inactivation could not be predicted on the basis of Tg.
Chen et al. (1999) studied the effects of water activity
and Tg on the stability and reactivity of invertase in two
PVP systems. However, there are no existing data on a
commonly used food system, such as the sucrose/lactose
system, in which the effects of Tg and water on enzyme
activity have been studied over a large aw range.
Transition from the glassy into the rubbery state has
the characteristics of a second-order phase transition,
but the glass transition occurs over a temperature
range. Moisture, the main plasticizer in foods, is an
important factor affecting the food stability (Nelson and
Labuza, 1994). It is well-known that plasticization
decreases the glass transition temperature, Tg, which
refers to the temperature range over which the transi-
tion occurs (Slade and Levine, 1991; Roos and Karel,
1991; Roos, 1993, 1995). Nelson and Labuza (1994)
suggested that reactant mobility and diffusion within
a matrix could be related to water activity, aw, and glass
transition. Viscous flow and diffusion above Tg of
amorphous sugars, resulting in stickiness and structural
collapse, precede crystallization, which occur as a func-
tion of temperature, moisture content, and time (Slade
and Levine, 1991). However, relationships between glass
transition and chemical and enzymatic changes have
been difficult to establish. For example, the molecular
arrangement of aspartame in solid PVP systems at
constant temperature, pH, and buffer concentration
were affected more by aw than Tg (Bell and Hageman,
1994). Bell and Labuza (1994) considered that the Tg
* Address correspondence to this author at Food Technology,
University College Cork, Cork Ireland. Tel: +353.21.902386.
Fax: +353.21.27.0213. E-mail: yrjo.roos@ucc.ie.
10.1021/jf9902999 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/23/2000

2462 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Kouassi and Roos
desiccators were removed at various time intervals for analy-
sis: one set for enzymatic determination of glucose using the
glucose Trinder kit, and the other for determination of glucose,
fructose, sucrose, and lactose by HPLC.
The purpose of this study was to investigate the
effects of glass transition and water on the hydrolysis
of sucrose by invertase, in an amorphous carbohydrate
system.
Testin g of In ver ta se In h ibition by Aceton itr ile. In
order to monitor the activity of invertase, it was essential to
inhibit its action at any desired time, so that the sugar
concentration did not change further. According to Folkes and
J ordan (1996), acetonitrile deactivates any enzyme. This has
been proved in this study for invertase. Thus, 20 mg/mL of
sucrose in distilled water was prepared. One milliliter of
freshly prepared invertase (0.5 mg/mL) solution was added to
2 mL of the sucrose solution, and 2 mL of acetonitrile (99.7%,
HPLC grade, Merck, Germany) was immediately added, so
that the solution concentration was acetonitrile:water (40:60).
Two other mixtures were made with the same amount of the
invertase solution and acetonitrile:water (50:50) and (60:40),
respectively. The three preparations were kept for 30 min and
filtered through a 0.45 µm Sparttan 30/B filter (Dassel,
Germany). Triplicate aliquots of each filtrate were used for
detection of glucose using the Trinder kit, and for detection of
fructose and glucose, products of sucrose hydrolysis using
HPLC. No hydrolysis product was detected by the Trinder kit
analysis as well as from the HPLC. This showed that a
concentration of acetonitrile higher than 40% stopped the
activity of invertase.
Deter m in a tion of Su cr ose Hyd r olysis by th e Tr in d er
Kit. Six solutions of glucose of known concentrations over the
concentration range of sucrose in samples were used. A 10 µL
aliquot of the solution was mixed with 3 mL of Trinder kit.
The mixture was kept at room temperature for 18 min;
thereafter the absorbance was measured using a Perkin-Elmer
Lambda 2 UV-vis spectrometer at 505 nm. Linear regression
analysis of the absorbance vs glucose concentration was made.
The R2 values were between 0.96 and 0.99. Triplicate samples
of lactose/sucrose/invertase stored over saturated salt solutions
were removed at intervals and dissolved each in 2 mL of
acetonitrile:H2O (60:40) (as proved to stop totally invertase
action). 3 × 10 µL aliquots were taken for determination of
the glucose content using the Trinder kit solution as described
above. The amount of glucose in the sample was determined
according to the equation
MATERIALS AND METHODS
P r ep a r a tion of th e F ood Mod el. R-Lactose monohydrate
and sucrose (2:1) from Sigma (USA) were successively dis-
solved in distilled water (200 mL) under mild heating to obtain
a clear solution. Invertase V grade from bakers yeast (Sigma,
USA) (20 mg of invertase/49.4 g of carbohydrate) was added
after cooling at +5 °C and mixed (cooling was essential to
minimize sucrose hydrolysis during mixing). Sucrose (2 g) was
dissolved in 10 mL of distilled water and 5 mg of invertase
was added. After 1 h, the glucose kit was used to test the
presence of glucose formed in the solution. This was to check
if the invertase used truly possesses its hydrolytic function.
The invertase /carbohydrate solution was rapidly prepared in
20 mL vials (5 mL aliquots) and frozen at 20 °C for 2 h. The
frozen material was stored at 80 °C about 10 h and freeze-
dried at a pressure <0.1 mbar for 3 days using a Lyovac GT
2, Amsco Finn-Aqua GmbH freeze-dryer (Germany). After
freeze-drying, the material was stored over P2O5 in a vacuum
desiccator to keep it anhydrous. The anhydrous samples were
used for sorption isotherms, differential scanning calorimetry
(DSC), and kinetic studies.
Sor p tion Isoth er m s. Sorption isotherms for the lactose/
sucrose/invertase food model were determined gravimetrically
at 24 °C. Triplicate samples of 1 g of the freeze-dried material
in the 20 mL vials were stored over saturated salt solutions
until the sample weight leveled off, a sign of the steady-state
water content. The salts used were LiCl, CH3COOH, MgCl2,
K2CO3, Mg(NO3), NaNO2, and NaCl (E. Merck, Darmstadt,
Germany); the respective relative humidities (RH) were 11.3,
23.9, 33.3, 44.4, 53.8, 66.2, and 76.4% (Labuza et al. 1985),
giving water activity values 0.01 × RH % at equilibrium.
Sample weights were measured at intervals during storage.
The Brunauer-Emmett-Teller (BET) and Guggenheim-
Anderson-Deboer (GAB) sorption isotherm models were fitted
to the water sorption data, according to Roos (1993).
Differ en tia l Sca n n in g Ca lor im etr y (DSC). The glass
transition temperatures for lactose/sucrose /invertase model
stored at various water activities were determined using DSC
(Mettler TA 4000 system with TC 15 TA processor, DSC 30
measuring cell, and STAR Thermal Analysis System version
3.1 software; Mettler-Toledo AG, Switzerland). The instrument
was calibrated using n-pentane (mp -129.7 °C; ∆H ) 116.7
J /g), n-hexane (mp (94.0 °C; ∆H ) 151.8 J /g), mercury (mp
-38.8 °C ∆H ) 11.4 J /g), distilled water (mp 0.0 °C; ∆H )
334.5 J /g), gallium (mp 29.8 °C; ∆H ) 80 J /g), and indium (mp
156.6 °C; ∆H ) 28.45 J /g). Samples were prepared in 40 µL
aluminum pans (Mettler ME-2733). Triplicate samples in open
pans were stored in vacuum desiccators over saturated salts
solutions, as for the sorption isotherm. After 24 or 44 h
(depending on time for leveling off) the pans were hermetically
sealed and steady-state water contents were determined
gravimetrically. The samples (10-20 mg) were scanned at 5
°C/min from at least 50 °C below the glass transition temper-
ature range with an empty pan as the reference. An immediate
rescan was run for each sample to verify the endothermic
baseline shift associated with the glass transition. The average
onset temperature of the change in heat capacity was consid-
ered as the glass transition temperature.
Kin etic Stu d ies. The freeze-dried materials in glass vials
were ground and the amorphous powder was transferred into
Eppendorf polypropylene test tubes (Greiner, Germany). The
distribution was performed quickly and vials and test tubes
were immediately closed after filling or removal of sample
materials, to avoid moisture uptake. Sample weights were
between 80 and 100 mg. The tubes were placed on supports
made of cardboard and stored in desiccators under vacuum at
24 °C over saturated salt solutions (RH 23.9-76.4%). Two sets
of triplicate samples in test tubes equilibrated in closed
Asample - Ablank
× concentration of standard
Astandard - Ablank
where Asample, Ablank, and Astandard are the absorbance of the
sample, the absorbance of a standard solution of known
concentration, and the absorbance of a blank, respectively. The
coefficient of variation between samples was less than 8%.
Deter m in a tion of Su cr ose Hyd r olysis by HP LC. High-
performance liquid chromatography (HPLC) 1090 (Hewlett-
Packard) with a RI detector (Hewlett-Packard) was used for
the determination of fructose, glucose, sucrose, and lactose.
The column used was 25 × 4.6 mm S5NH2 (Spherisorb, U.K)
thermostated at 40 °C. Folkes and J ordan (1996) suggested
acetonitrile-water (75:25)-(85:15) as an appropriate mobile
phase. We observed that at this concentration range, although
the sugars eluted rapidly, peaks tailing was very significant.
Therefore, acetonitrile:water (60:40) was used as mobile phase,
as it provided a better peak symmetry. The flow rate was 1.8
mL/min. The external standard method was used to determine
the content of fructose, glucose, sucrose, and lactose in the
samples. Six solutions of known concentration of these sugars
were used and linear regression analysis of each sugar was
done. R2 values of glucose, fructose, sucrose, and lactose were
0.98, 0.99, 0.97, and 0.99, respectively. Triplicate samples of
the lactose/sucrose/invertase in Eppendorf tubes were taken
from desiccators over saturated salt solutions, at various RH,
and dissolved into 2 mL of acetonitrile:water (40:60). This
solvent system was chosen for two reasons. First, lactose did
not dissolved easily when the amount of acetonitrile in the
solvent was higher than 40%. Second, we tested that this
solution stopped the action of invertase. The injection volume

Sucrose Inversion by Invertase
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2463
F igu r e 1. Water content of samples at various RH as a
function of storage time.
F igu r e 2. Relationships between water activity, rate constant,
and water content.
was 10 µL. The amounts of glucose and fructose released by
hydrolysis of sucrose, the remaining sucrose, and lactose per
100 g of solids were calculated according to the equation X )
(Y - b)/a obtained from the linear regression analysis, where
X represents the unknown amount of sugar, Y is its peak area,
and a and b are the slope and the intercept of the linear
regression curve, respectively. The coefficient of variation was
less than 5.5%. The analytical lactose content was compared
to the initial concentration in samples. This was to evaluate
the accuracy of the analytical procedure. Rate constants were
calculated from first-order plots (Villota and Hawkes, 1989)
using single regression analysis with 95% confidence limit.
Points below 0.75 were not considered for the determination
of these rate constants.
Ta ble 1. Gla ss Tr a n sition Tem p er a tu r e (Tg) a n d
Cr ysta lliza tion Tem p er a tu r e a t Va r iou s a w
Tg (°C)
water content
(g/100 g)
crystallization
onset (°C)
aw
onset
midpoint
0
0
72.60 ( 0.44 81.6 ( 0.38 145.30 ( 0.47
0.239
0.333
0.444
0.538
0.662
1.63 ( 0.23 35.38 ( 2.57 40.07 ( 2.57 102.01 ( 2.30
3.14 ( 0.05 21.32 ( 2.67 29.46 ( 3.06 87.63 ( 2.02
4.85 ( 0.05
8.17 ( 0.19
8.86 ( 0.12
4.82 ( 0.50 12.70 ( 0.23 63.81 ( 0.23
8.18 ( 0.16
0
0.764 12.99 ( 0.14
RESULTS AND DISCUSSION
Sor p tion Isoth er m s. Figure 1 shows the water
sorption at 24 °C at various RH. The water sorption
leveled off within a day at 11.3, 44.4, 53.9, and 66.2%
RH. At 33.3% RH the system leveled off after 2 days.
Water sorption at RH g 44.4% showed a reduction in
sorbed water after 24-44 h, as an indication of crystal-
lization (Linko et al., 1982; Roos and Karel, 1992). Below
this RH, water sorption indicated steady-state water
contents as also observed by J ouppila and Roos (1994).
This agreed with Vuataz (1988) who reported that
R-lactose monohydrate above aw 0.57 crystallized at
room temperature. As the system contained 2/3 of
lactose, this sugar might be responsible for the crystal-
lization suggested by the sorption curves at RH > 44.4%.
This observation is in agreement with J ouppila and
Roos (1994) who reported crystallization of lactose in
milk powders within 24 h at RH > 50%.
Average steady-state water contents of all samples
were used in fitting the BET and GAB sorption models
to the data over aw 0.113-0.444 and 0.113-0.538,
respectively. The BET constant, C, was 15.16 and the
monolayer value, mm, 3.58 g of H2O/100 g of solids. The
GAB constants, C′ and K were 15.16 and 0.53, respec-
tively, and the GAB monolayer value, mm, was 2.85 g
of H2O /100 g of solids. The GAB model has been shown
to fit experimental data over almost the whole aw range
(Van den Berg et al., 1975), and to be applicable to
predict water sorption of most foods (Roos, 1995; J oup-
pila and Roos, 1994; Maskan et al., 1997). However,
Bizot (1993) pointed out that the GAB model does not
apply to all shapes of isotherms, and indicated the
example of starch for which this model did not fit the
data at aw 0.6-0.7 due to crystallization. Our results
seem to obey the same restriction. Indeed, crystalliza-
tion seemed to occur at room temperature at aw 0.4-
F igu r e 3. Relationships between water activity, glass tran-
siton temperature Tg, and reaction rates.
0.7, causing a discontinuity during the sorption process
as observed in the shape of the sorption isotherm
(Figure 2).
DSC An a lysis. The onset temperature of the glass
transition (Tg) of the lactose/sucrose/invertase system,
and the onset of the crystallization temperature de-
creased with increasing water activity and water con-
tent (Table 1). Above aw 0.538 it was very difficult to
determinine the Tg. This was probably due to interfer-
ence from crystallization that probably had taken place
during sample storage under saturated solutions as
suggested by the water sorption isotherm. This is in
agreement with Hagiwara and Hartel (1996) who
reported that crystallization took place during sample
storage. Thus, at this aw range, no exact values of Tg
and crystallization could be found. Values of Tg and
crystallization temperatures of single sugars, such as
lactose, sucrose, and glucose, have been reported (Roos,
1991; Roos, 1993). The system studied here contained
lactose and sucrose and eventually fructose and glucose
formed by sucrose inversion. Figure 3 shows the rela-

2464 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Kouassi and Roos
6 shows an example of the first-order plot of glucose
content vs time. The curves for sucrose hydrolysis as
determined by glucose kits and HPLC followed the same
trend. No hydrolysis was observed at aw 0.239 and
0.333. Above aw 0.333 sucrose hydrolysis was moderate
with a corresponding rate at aw 0.444 and 0.538. Above
aw 0.538 the rate of sucrose hydrolysis increased with
aw. At aw 0.764, the rate was highly significant, and the
reaction completed after 25 days approximately. The
variation between the initial amount of lactose in
sample and the calculated values from the HPLC was
between 0.36 and 4.9%. This low variation indicated
that the analytical procedure was relevant. The first-
order reaction rate and rate constants determined from
the kinetics plots with 95% confidence limits are dis-
played in Tables 2 and 3, respectively. The rate con-
stants were very low in the aw 0.239-0.538 range. Above
aw 0.538 the rate constant increased markedly with
increasing water activity. Silver and Karel (1981) found
that not sucrose hydrolysis was measurable in Avicel/
sucrose/invertase, agar/sucrose/invertase, and starch/
sucrose/invertase model systems at aw lower than 0.58.
That is the impression that emerges at first sight from
the curve of kinetics of hydrolysis products vs time.
However, the values of the reaction rates and the rate
constants, showed that a slight hydrolysis occurred at
aw 0.444 and 0.538 concomitantly with sugars crystal-
lization. This result did not totally meet those reported
by Chen et al. (1999), who indicated that no sucrose
hydrolysis was found at aw < 0.62. Above aw 0.662, our
results agreed with those of Silver and Karel (1981) who
F igu r e 4. DSC thermal curves for amorphous lactose/sucrose/
invertase samples, after equilibration at different water activi-
ties (24 °C).
tionships between water activity, Tg, and reaction rates,
and Figure 4 shows DSC thermal curves of samples at
various aw.
Su cr ose Hyd r olysis. The extents of sucrose hydroly-
sis (g/100 g solids/h) were determined at various aw.
Figure 5 shows the curves of the extent of sucrose
hydrolysis A, B, C, and D corresponding to glucose
content by enzymatic assay (test kit), glucose, fructose,
and sucrose remaining, by HPLC, respectively. Figure
F igu r e 5. Extent of sucrose hydrolysis in g/100 g (dry weight) as a function of time for glucose by test kit (A), and by HPLC for
glucose (B), fructose (C), and sucrose (D).

Sucrose Inversion by Invertase
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2465
Ta ble 2. Ra te Con sta n ts w ith 95% CL for Su cr ose Hyd r olysis a t Va r iou s a w
rate constant (h-1
)
aw
glucose kit
glucose HPLC
fructose HPLC
sucrose HPLC
0.239
0.333
0.444
0.538
0.662
0.764
0.00005 ( 0.00001
0.00005 ( 0.00001
0.0005 ( 0.00002
0.0005 ( 0.0001
0.0009 ( 0.0002
0.0079 ( 0.0003
0.00005 ( 0.00001
0.00005 ( 0.00002
0.00050 ( 0.0001
0.0006 ( 0.0001
0.0009 ( 0.0002
0.0089 ( 0.0003
0.00005 ( 0.00002
0.00005 ( 0.00001
0.0006 ( 0.0001
0.0006 ( 0.0001
0.0009 ( 0.0001
0.0081 ( 0.0003
0.00006 ( 0.00001
0.000060 ( 0.00001
0.00060 ( 0.0001
0.0005 ( 0.0002
0.0009 ( 0.0001
0.0070 ( 0.0004
Ta ble 3. Ra te of Su cr ose Hyd r olysis Ca lcu la ted fr om Glu cose, F r u ctose, a n d Su cr ose Con cen tr a tion s a t Va r iou s a w w ith
95% CL
HPLC
glucose kit
glucose
fructose
sucrose
aw
g/100 g h-1 mmol/100 g h-1
g/100 g h-1
mmol/100 g h-1
g/100 g h-1
mmol/100 g h-1
g/100 g h-1
mmol/100 g h-1
0.239 1.59 × 10-3
0.333 1.31 × 10-3
0.444 3.75 × 10-2
0.538 4.64 × 10-2
0.662 22.98 × 10-2
0.764 58.98 × 10-1
8.87 × 10-3
7.30 × 10-3
2.08 × 10-2
2.57 × 10-2
7.28 × 10-2
3.22 × 10-1
1.16 × 10-3
6.45 × 10-3
7.50 × 10-3
2.03 × 10-2
2.08 × 10-2
8.41 × 10-2
2.97 × 10-1
1.017 × 10-3
1.051 × 10-3
3.750 × 10-2
3.314 × 10-2
31.250 × 10-2
42.697 × 10-1
5.65 × 10-3
5.83 × 10-3
2.08 × 10-2
1.73 × 10-2
6.87 × 10-2
2.37 × 10-1
3.026 × 10-3
3.053 × 10-3
8.116 × 10-2
8.928 × 10-2
54.00 × 10-2
79.546 × 10-1
8.884 × 10-3
8.917 × 10-3
2.608 × 10-2
2.608 × 10-2
6.886 × 10-2
2.32 × 10-1
1.35 × 10-3
3.65 × 10-2
3.75 × 10-2
34.375 × 10-2
53.544 × 10-1
noticeable as the glass-rubber transition occurs due to
increased moisture content (Buera et al., 1995). Water,
therefore, may facilitate the structural organization and
provide a better diffusibility to the enzyme and the
substrate through the matrix. Figure 3 shows the
reaction rates as related to the Tg and aw. Our results
show that there is no sudden change in reaction rate at
T - Tg ) 0, corresponding approximately to aw 0.333,
but the effective change appears above 0.538 aw, con-
comitant with crystallization. Our Tg determined below
aw 0.538 confirmed this tendency. Crystallization may
favor the diffusibility and mobility of the enzyme or the
substrate, increasing, therefore, the rate of sucrose
hydrolysis in the water solute phase.
CONCLUSIONS
Sucrose hydrolysis was perceptible at aw 0.444. Above
this value sucrose inversion increased with water activ-
ity and reached a maximum at aw 0.764. It was also
observed that sucrose inversion did not occur in the
glassy state but rather in the rubbery state where
crystallization also occurred. The increased sucrose
hydrolysis may be presumably due to increase molecular
mobility of the enzyme or substrate molecules in the
phase-separated, partially crystalline system. Further
studies should be addressed to mobility of the enzyme
and substrate through the glassy-rubbery transition.
F igu r e 6. Example of first-order plots of sucrose hydrolysis.
reported a maximum reaction rate or rate constant at
0.6-0.7 water activity range.
The BET mm we found corresponded to aw between
0.25 and 0.33. As Roos (1993) emphasized, the critical
water content is often slightly higher than the BET mm,
the critical water content corresponds to aw close to 0.33,
value above which hydrolysis becomes noticeable. Whi-
taker (1995) considered the same water activity when
he described the effect of water on enzyme activity.
Similarly, Acker (1969) reported that phospholipase
activity on lecithin was not noticeable after almost 2
months until aw reached 0.35. Our results suggest that
a correlation between water activity and invertase
activity exists, and sucrose hydrolysis and crystalliza-
tion take place mainly in the rubbery state.
LITERATURE CITED
Acker, L. The practical approach to better low-moisture
foods: water activity-enzyme activity. Food Technol. 1969,
23, 1257-1259.
Bell, L. N, Hageman, M. J . Differentiating between the effects
of water activity and glass transition dependent mobility
on a solid-state chemical reaction: aspartame degradation.
J . Agric. Food Chem. 1994, 42, 2398-2401.
Bell, L.N and Labuza, T. P. Influence of the low moisture state
on pH and its implication for reaction kinetics. J . Food Eng.
1994, 22, 291-312.
Bizot, H. Using the G. A. B. Model to construct sorption
isotherms. In Physical Properties of Foods; J owit, R., Escher,
F., Hallstro¨m, B., Meffert, H., Spiess, W., Gilbert, V., Eds.;
Applied Science Publishers: London, 1983; pp 43-54.
Buera, M.; Chirife, J .; Karel, M. A study of acid-catalyzed
sucrose hydrolysis in amorphous polymeric matrix at re-
duced moisture contents. Food Res. Int. 1995, 28, 359-365.
Whitaker (1995) stated that water plays at least four
important functions in all enzyme-catalyzed reactions:
folding of the protein, acting as a transport medium for
the substrate and enzyme, hydration of the protein, and
ionization of prototropic groups in the active sites of the
enzyme. It is also known from the polymer science
approach (Slade and Levine, 1994) that the glassy state
of the amorphous matrix in which the reactants are
embedded could be responsible for the diffusion-limited
rates of the chemical reactions that take place at low
moisture content. This diffusional limitation disappears
and a drastic increase in the reaction rate may be

2466 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Kouassi and Roos
Cardona, S.; Schebor, C.; Buera, M. P.; Karel, M.; Chirife, J .
The thermal stability of invertase in reduced-moisture
amorphous matrices in relation to glassy state of trehalose.
J . Food Sci. 1997, 62, 105-112.
Chen, Y. H.; Aull, J . L.; Bell, L. N. Invertase storage stability
and sucrose hydrolysis in solids as affected by water activity
and glass transition. J . Agric. Food Chem. 1999, 47, 504-
509.
Folkes, D. J .; J ordan, M. A. Mono- and disaccharides: analyti-
cal aspects. In Carbohydrates in Foods; Eliasson, A. C., Ed.;
M. Dekker: New York, 1996; pp 1-37.
Hagiwara, T.; Hartel, R. W. Effect of sweetener, and storage
temperature on ice recrystallization in ice cream. J . Dairy
Sci. 1996, 79, 735-744
J ouppila, K.; Roos, Y. Water sorption and time dependent
phenomena of milk powders. J . Dairy Sci. 1994, 77, 1798-
1808.
J ouppila, K.; Roos, Y. The physical state of amorphous corn
starch and its impact on crystallization. Carbohydr. Polym.
1997, 32, 95-104.
Kertesz, Z. I. Water relation to enzymes: water concentration
required for invertase action. J . Am. Chem. Soc. 1935, 57,
1277-1280.
Labuza, T.; Kaanane, A, Chen, J . Y. Effect of temperature on
the moisture sorption isotherm and water activity shift of
two dehydrated foods. J . Food Sci. 1985, 50, 585-391.
Le Meste, M. Mobility of small molecules in low and interme-
diate moisture foods. In Food Preservation by Moisture
Control: Fundamentals and Applications; Barbosa-Ca´novas,
G., Ed.; Technomic: Lancaster, PA, 1995; pp 210-223.
Linko, P.; Pollari, M.; Heikonen, M. Water sorption properties
and the effects of moisture on structure of dried milk
products. Lebensm.-Wiss. Technol. 1982, 15, 26-30.
Maskan, M.; Go¨gus, F. The fitting of various models to water
sorption isotherms of pistachio nut paste. J . Food Eng. 1997,
33, 227-237.
Michaelis, L. Menten, M. L. The kinetics of invertase. Biochem.
Z. 1913, 49, 333-369.
Nelson, K. A.; Labuza, T. P. Water activity and food polymer
science: implications on the state of Arrhenius and WLF
models in predicting shelf life. J . Food Eng. 1994, 22, 271-
289.
Preservation by Moisture Control: Fundamentals and Ap-
plications; Barbosa-Ca´novas, G., Ed.; Technomic: Lancaster,
PA, 1995; pp 133-168.
Roos, Y. Glass transition-related physicochemical changes in
foods. Food Technol. 1995, 49, 97-102.
Ruchti, J .; McLaren, A. Enzymes reaction in structurally
restricted systems, V. Further observations on the kinetics
of yeast (-fructofuranoside (invertase activity in viscous
media). Enzymologia 1964, 27, 185-198.
Schebor, C.; Buera, M.; Chirife, J .; Karel, M. Sucrose hydroly-
sis in a glassy starch matrix. Lebensm.-Wiss. Technol. 1995,
28, 245-248.
Shimada, Y. Roos, Y. Oxidation of methyl linoleate encapsu-
lated in amorphous lactose-based food model. J . Agric. Food
Chem. 1991, 39, 637-641.
Silver. M.and Karel, M. The behavior of invertase in model
systems at low moisture contents. J . Food Biochem. 1981,
5, 283-311.
Slade, L and Levine, H. Beyond water activity: recent
advances based on an alternative approach to the assess-
ment of food quality and safety. Crit. Rev. Food. Sci. Nutr.
1994, 30, 115-360.
Slade, L and Levine, H. Polymer science approach to water
relationships in foods. J . Food Eng. 1994, 22, 143-189.
Van Den Berg, C.; Kaper, F. S.; Weldring, A. G.; Wolters, I.
Water binding by potato starch. J . Food Technol. 1975, 10,
589-602.
Villota, R.; Hawkes, J . G. Reaction kinetics in food systems.
In Handbook of Food Engineering; Heldman, D. R., Lund,
D. B., Eds.; M. Dekker: New York, 1989; pp 30-144.
Vuataz, G. Preservation of skim-milk powders: role of water
activity and temperature in lactose crystallization and lysine
loss. In Food Preservation by Water Activity Control; Seow,
C. C., Ed.; Elsevier Science: Amsterdam, The Netherlands,
1988; p 73.
Whitaker, J . R. Enzymes action in aqueous systems with
special emphasis on intermediary and high moisture foods.
In Food Preservation by Moisture Control: Fundamentals
and Applications; Barbosa-Ca´novas, G. Ed.; Technomic:
Lancaster, PA, 1995; pp 255-273.
Roos, Y.; Karel, M. Plasticizing effect of water on thermal
behavior and crystallization of amorphous food models. J .
Food Sci. 1991, 56, 38-43.
Received for review March 23, 1999. Revised manuscript
received February 28, 2000. Accepted March 13, 2000. The
study was carried out with financial support from the Com-
mission of the European Communities, Agriculture and Fish-
eries (FAIR) specific RTD program, CT96-1085, Enhancement
of Quality of Food and Related Systems by Control of Molec-
ular Mobility (Molecular Mobility in Foods). It does not
necessarily reflect its views and in no way anticipates the
Commission’s future policy in the area. The study was also
supported by the Academy of Finland, Project no. 32578.
Roos, Y.; Karel, M. Crystallization of amorphous lactose. J .
Food Sci. 1992, 57, 775-777.
Roos, Y. Water activity and physical states effects on amor-
phous food stability. J . Food Processing and Preservation.
1993, 16, 433-447.
Roos, Y. Phase Transition in Foods; Academic Press: San
Diego, CA, 1995; pp 73-103.
Roos, Y. Water activity and glass transition temperature; how
do they complement and how do they differ. In Food
J F9902999