Lebensm.-Wiss. u.-Technol., 30, 324–329 (1997)
Research Note
Protective Role of Trehalose on Thermal Stability of
Lactase in Relation to its Glass and Crystal Forming
Properties and Effect of Delaying Crystallization
Marı́a Florencia Mazzobre, Maria del Pilar Buera and Jorge Chirife
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, (1428) Buenos
Aires (Argentina)
(Received April 25, 1996; accepted July 23, 1996)
Thermal inactivation of β-galactosidase was investigated in dried matrices of poly (vinyl) pyrrolidone (PVP), maltodextrin and
trehalose. Significant lactase inactivation was observed in the polymeric matrices kept well below their glass transition temperature
(Tg). The stability of the enzyme in the anhydrous glassy matrices of maltodextrin and PVPs heated at 70 °C was directly related
to their Tg; i.e. systems with higher glass transition temperature afforded better thermal protection of lactase. However, the stability
of lactase in the heated trehalose matrix deviated from this behaviour since enzyme stability was higher than expected on the basis
of the results obtained with polymeric matrices.
In systems in which the trehalose matrix was rehumidified to conditions which allowed a high proportion of trehalose to
crystallize, the enzyme was rapidly inactivated upon heating. Addition of maltodextrin to trehalose matrix provided enhanced
protection to the enzyme, and this was probably due to delayed trehalose crystal formation.
©1997 Academic Press Limited
Keywords: trehalose; enzyme stability; lactase
Introduction
Thermal stability of enzymes is an important aspect
determining practical conditions for their application in
biotechnology and food processing (1–3). Enzyme
stability during storage could be increased by drying in
adequate matrices.
Trehalose is a nonreducing disaccharide found ubiquitously in fungi and widely distributed in both bacteria
and animals; it is specially common in anhydrobiotic
organisms capable of surviving extended periods of
dehydration (4). The role of saccharides as protectants
of membranes and proteins during drying had been
reported previously (5–8) and the effect of trehalose
has been particularly investigated since it was found to
be optimal in protecting membranes (liposomes, microorganisms) and proteins during freezing and drying (9,
10). The precise mechanism by which trehalose stabilizes biological molecules in dry systems has not been
yet elucidated, but two hypothesis have been suggested
(1). In one of the hypotheses the stabilization of
proteins provided by trehalose and other disaccharides
during drying was attributed to the formation of
hydrogen bonds between proteins and disaccharide
molecules when water is removed, replacing essential
water molecules to maintain the tertiary protein structure, thus preventing protein denaturation. The second
hypothesis is related to the ability of trehalose (like
other carbohydrates and polymers) to form a glassy
structure when drying in adequate conditions. The
glassy state, characterized by extremely low molecular
motion, could be the factor determining the long
stabilization of biological material (e.g. isolated
enzymes) which, in liquid solution have very limited
shelf-lives (11, 12). Since protein denaturation requires
a spacial reordering of the molecules, it could be
expected that protein stability (and hence enzymatic
activity) is enhanced in the glassy state, where molecular mobility is inhibited. Although thermal stability of
enzymes is expected to be related to mobility aspects,
the influence of glass transition has not yet been fully
demonstrated and the specific mechanism of enzyme
protection observed in trehalose matrices can not be
attributed entirely to the glass transition phenomenon
(3). The extent of protection provided by poly(vinyl)pyrrolidone (PVP) and maltodextrin (MD) matrices on the thermal stability of invertase (3) and lactase
(13) is somewhat related to their glass transition
temperatures. However, even when the matrices are in
the glassy state, the loss of enzymatic activity is
important if the temperature is sufficiently high (3,
13).
Cardona et al. (14) demonstrated that if sufficient water
was present to allow crystalline trehalose dihydrate
formation in a high proportion, the protective action of
trehalose on invertase stability was lost. It is known that
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©1997 Academic Press Limited
lwt/vol. 30 (1997) No. 3
upon crystallization of amorphous sugar matrices, many
deteriorative reactions may occur, such as release and
oxidation of encapsulated lipids (15, 16), release of
volatiles (17) and acceleration of nonenzymatic browning reactions (18). Crystallization of amorphous sugars,
as a consequence of holding systems above the glass
transition temperature (Tg), is a time-dependent phenomenon which follows Williams, Landel and Ferry
(WLF, 19) equation.
The objective of the present work was to gain knowledge on the protective role of trehalose on thermal
stability of lactase (β-galactosidase) in relation to its
glass and crystal forming properties. The effect of
delaying crystallization of trehalose by incorporating
maltodextrin was particularly investigated.
Materials and Methods
Preparation of model systems
Amorphous matrices were obtained by freeze-drying
solutions containing 200 g/L total solids of each one of
the following substances: Poly(vinyl)pyrrolidone,
molecular weight 10,000 (PVP10) and 40,000 (PVP40)
(Sigma Chemical Co., St. Louis, MO); trehalose (T)
(Sigma Chemical Co., St. Louis, MO); maltodextrin
(MD), DE 10.9 (Refinacoes de Milho (Corn Products
Corp.), Sao Paulo, Brazil); or mixtures MD:T (80:20), in
phosphate buffer (0.1 mol/L, pH 6.9). The aqueous
solutions were cooled over an ice-bath and 10 mL/L
commercial lactase (β-galactosidase Maxilact 5000L,
from Gist Brocades, Holland, nv.) was added. Aliquots
of 1 mL of each model solution were placed in 3 mL
vials and immediately frozen using liquid air (temperature = 70 K). A Stokes freeze-dryer model 21 (F.J.
Stokes Company, Equipment Div., Pennsalt Chem.
Corp., Philadelphia, PA) was used which operated at
–40 °C condenser plate temperature and at a chamber
pressure of less than 100 µm Hg. After freeze-drying
the samples were transferred into vacuum desiccators
and equilibrated for 1 week over P2O5, for ‘zero’
moisture content, or over saturated salt solutions of
KCH3COO (for 22% relative humidity), K2CO3 (for
44% relative humidity) and NaCl (for 75% relative
humidity).
Determination of moisture content
The moisture contents of the equilibrated samples of
PVP and MD were determined by difference in weight
before and after drying in vacuum ovens at 70 °C for 48
h, and at 105 °C in a forced air circulating oven for
trehalose systems (14). These conditions had been
proved to be adequate to assess constant weight in each
case.
Heat treatment
After equilibration, the model systems were stored in
forced air convection ovens at selected temperatures
(45, 57 or 70 °C). At suitable intervals two samples were
removed from the oven and the remaining activity of
lactase was determined as described below, and the
average value reported.
Lactase activity
After heat treatment 1 mL of water was added to each
sample and the systems were kept at 5 °C until
complete dissolution was achieved. Then, 2 mL of 150
g/L lactose (substrate) was added and the vials were
incubated for 90 min at 37 °C. After incubation the
samples were exposed to 80 °C for 3 min to inactivate
the enzyme. Lactose hydrolysis was determined by
measuring the amount of glucose formed. An enzymatic method based on the oxidation of glucose by
glucose oxidase to gluconic acid and oxygen peroxide
was employed, as previously described (20). Two
replicates of each sample were analysed and, as two
samples were taken from the oven at each time, the
average of four measurements was reported for each
storage time. The relative error (95% confidence
interval), calculated from eight measurements of the
same sample from two separate runs, was 5%. The
amount of lactose hydrolysed by samples without
thermal treatment (L0) was considered to correspond
to 100% lactase activity; the amount of lactose hydrolysed after heat treatment (Lt) was referred to L0, and
the remaining activity (RA) was expressed as:
RA = 100 Lt/L0.
Glass transition temperatures
Glass transition temperatures (onset) of anhydrous
maltodextrin and PVP model systems were not determined here, but were estimated from data reported by
Buera et al. (21) for PVP of various molecular weights
and from Roos and Karel (22) for maltodextrin of
similar DE number (Maltrin 100, DE 10, Grain
Processing Corporation; Muscatine, IA). The anhydrous Tg values (onset) were 137 °C, 93 °C and 160 °C
for PVP40, PVP10 and MD, DE 10, respectively. Values
for Tg of anhydrous trehalose reported in the literature
range from 75 to 100 °C (3). It is most likely that in
some cases these differences may arise from some small
amount of water (10 to 20 g/kg) left in the ‘anhydrous’
matrices (even after desiccation over P2O5), since water
is known to depress Tg greatly. In a previous study (14)
we have determined a Tg value of 85 °C after
desiccation over P2O5 and this value was used here. The
Tg values for rehumidified trehalose systems were those
obtained previously (14). In all cases the data were
obtained by differential scanning calorimetry (DSC)
measurements at a rate of 5 °C/min, in samples
equilibrated at the same conditions followed in the
present work.
The reliability of using Tg data determined by others
instead of a direct measurement, as well as the presence
of phosphate buffer salts in the present systems,
deserves consideration.
The addition of buffer salts in present model systems
may (or may not) modify their actual Tg values, since
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lwt/vol. 30 (1997) No. 3
Results and Discussion
After thermal treatment of lactase in glassy polymeric
matrices (PVP and MD) of ‘zero’ moisture content for
10 or 15 h at 70 °C, the remaining enzymatic activity
was found to be markedly dependent on the difference
between the storage temperature (T) and the estimated
glass transition temperature of the system (Tg) (Fig. 1).
Also, a significant enzyme inactivation was observed as
a result of heating in these glassy systems, mainly in
PVP. Although the polymeric matrix (i.e. PVP) is
considered immobile (as suggested by its estimated
(T – Tg) value) this did not present a blockade to lactase
denaturation. In the trehalose system the remaining
100
Remaining activity (%)
small molecular weight compounds may act as plasticizers or antiplasticizers. However, Nelson (23) determined the effect of sodium phosphate buffer salts on
the measured glass transition temperature of maltodextrin (DE 15) at various moisture values and found that
the effect was not significant considering the error in
the Tg measurements. Bell and Hageman (24) determined the Tg of a model system composed by PVP 40
with added phosphate buffer and aspartame; at 110
g/kg (dry basis) moisture content the measured Tg was
almost identical to that measured by Buera et al. (21)
for PVP M.W. 40,000 without buffer at identical
moisture content. Both values were also in good
agreement with that reported by Karmas (18) for the Tg
of a model system of PVP M.W. 40,000/xylose/lysine
(98:1:1) of same moisture content.
The similarity of both maltodextrins, Maltrin 100
(GPC) and that used in the present work (Refinacoes
de Milho) was confirmed by comparing their relative
proportions of saccharides of different M.W., by freezing point depression of an aqueous solution, and by
DSC measurement (Polymer Laboratories LTD. PLDSC., Thermal Sciences Division, U.K.) of a sample
equilibrated at 22% RH. It is known that the saccharide
composition (carbohydrate profile) of maltodextrins
influences their Tg (22). However, the average content
of larger saccharides (pentasaccharides and above) was
881g/kg (dry basis) for Maltrin M100 and 890g/kg for
the present maltodextrin (Refinacoes de Milho). The
anhydrous Tg value of maltodextrin MW 1800 (Maltrin
100, DE 10; larger saccharides 881g/kg) was 160 °C as
compared to 141 °C for maltodextrin M.W. 900
(Maltrin 200, DE 20; larger saccharides 74.4%) (22).
Thus, we may safely assume that small variations in the
carbohydrate profiles would not affect significantly the
reliability of our estimated Tg values. The similarity
between average molecular weights of both maltodextrins was confirmed by a freezing point determination
(using a standard milk cryoscope) performed on 40g/kg
(w/w) solutions; average values were found to be
almost identical, i.e. –2.77 °C (σ = ± 0.08) and –2.71 °C
(σ = ± 0.06) for GPC and present maltodextrin, respectively. Both maltodextrins also showed similar thermograms (and hence Tg) as determined by DSC of samples
equilibrated at 22% RH.
80
T
T
MD
MD
60
40
PVP40
20
PVP40
0
–100 –90
–80
–70
–60 –50 –40
T – Tg (°C)
PVP10
–30
–20
–10
Fig. 1 Remaining activity of lactase in freeze-dried systems of
‘zero’ moisture content as a function of estimated (T – Tg)
values after 10 (m) or 15 (d) h of heating at 70 °C. (None of
the samples had collapsed during heating)
enzyme activity was higher than expected from its
estimated (T – Tg) value, according to the curve defined
by MD and PVP, as also shown in Fig. 1. Schebor et al.
(3) reported similar results for experiments performed
with invertase.
As expected for glassy materials heated below their
glass transition temperature (11, 17) collapse was not
observed after heating any of the systems shown in Fig.
1. In spite of any difference between estimated Tg
values and actual ones for present model systems, the
absence of collapse of PVP, MD and trehalose systems
shown in Fig. 1 was an additional, although qualitative,
proof that they were definitely in the glassy state. The
polymer systems shown in Fig. 1 are so glassy (as
suggested by their low (T – Tg) values) that a difference,
i.e. as high as 10 °C, between actual and estimated Tg
values would not modify the interpretation of the
aforementioned findings.
Crowe et al. (7) formulated the ‘water replacement
hypothesis’, based on the possibility that polyhydroxy
compounds can replace the structural water of cellular
components thereby preventing a variety of potential
lethal events from taking place. Differences have been
observed, however, among sugars in protecting proteins
against denaturation, but there is no clear explanation
for the relative efficacy of different sugars, except that
it is not related to the number of positions of hydroxyl
groups available for hydrogen bonding (9). Another
hypothesis, proposed by Green and Angell (25), indicated that the order of efficacy of different sugars is
linked to their glass forming ability. Results shown in
Fig. 1, and those reported previously for invertase
activity (3), indicated that although the enzyme was
very much more stable than in liquid systems heated at
an identical temperature, the trehalose protecting effect
cannot be based solely on its glass forming properties,
since dried molecular weight polymers with higher Tg
(i.e. PVP 40,000) than trehalose exerted less protective
effect than this sugar. Thus, a specific protective
mechanism, related to the maintainance of the tertiary
structure is suggested (14).
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lwt/vol. 30 (1997) No. 3
Table 1 Characteristics of the rehumidified model systems
Matrix
RH%
Moisture content
(g/kg d.b.)
22
44
75
40
70
111
22
44
75
45
84
90
75
97
Maltodextrin
(DE 10.9)
T
MD : T
(80 : 20)
Table 1 shows the moisture content obtained for the
freeze-dried systems humidified at different relative
humidities. Figure 2 shows the remaining activity of
lactase in trehalose matrices equilibrated at 22, 44 and
Remaining activity (%)
100
80
60
40
20
0
20
40
60
80 100 120
Time (h)
140
160
180
Fig. 2 Remaining activity of lactase in trehalose systems
equilibrated at specified RH, as a function of heating time at
57 °C (Tg data are from (14)). (All samples collapsed).
(n) = 22% RH, (T – Tg) = 12 °C; (*) = RH 44%, (T – Tg) =
43 °C; (e) = RH 75%, (T – Tg) very large
100
Remaining activity (%)
80
Incipient collapse
Uncollapsed
60
Uncollapsed
40
20
Crystallized
very collapsed
0
MD
T
Matrix
Fig. 3 Remaining activity of lactase in different systems
(maltodextrin and trehalose) equilibrated to 22 (B) and 44%
RH (h) after 30 d of storage at 45 °C
75% RH, as a function of storage time at 57 °C.
Collapse was observed as a dramatic visible shrinkage
in all systems during storage. Since collapse is known to
occur only above the glass transition temperature
(glassy systems are stable to collapse) the observed
collapse is confirmation of the rubbery state of these
systems. The remaining lactase activity dropped markedly in the systems of 44 and 75% RH, as compared to
22% RH, and this behaviour was related to trehalose
crystallization (14). It is known that crystallization of
amorphous sugars occurs above the glass transition
temperature (26), at a rate which depends on the
(T – Tg) value. Based only on the estimated (T – Tg)
values of systems shown in Fig. 2, trehalose could
crystallize in the three humidified systems. However,
since trehalose crystallizes as a dihydrate, 105 g of water
per kg dry matter is required to allow complete
crystallization. In the rehumidified systems, in which
the amount of water was enough to allow a high
proportion of trehalose to crystallize (44 and 75% RH
(see Table 1)), the enzyme was rapidly inactivated.
Amorphous trehalose humidified to RH 22% would
not crystallize completely due to lack of water, and the
enzyme is more protected.
Figure 3 compares the remaining lactase activity after
30 d of storage at 45 °C in MD and T systems
humidified to 22 and 44% RH. The physical aspect of
the samples reflected their expected glassy or rubbery
state. At 22% RH lactase activity was retained at a
similar level in both systems (as occurred at ‘zero’
moisture content, shown in Fig. 1). However, at 44%
RH the retention was very low in the crystallized
trehalose matrix, while it was still considerably high in
the amorphous MD system.
As the protective effect of trehalose was observed to be
related to the extent of trehalose crystallization, experiments were developed with mixtures in which maltodextrin was incorporated to the matrix to delay
trehalose crystallization. The retardation of crystallization by the addition of high molecular weight compounds has been demonstrated by Berlin et al. (27) for
lactose and by Iglesias and Chirife (28) for sucrose.
Tsourouflis et al. (29) added maltodextrins and gums to
orange juice to delay collapse, and Gerschenson et al.
(30) added pectin to freeze-dried tomato juice to
increase the collapse temperature and to enhance the
retention of encapsulated volatiles. Karmas et al. (31)
observed delayed lactose crystallization by incorporating amioca, carboxymethylcellulose and/or trehalose.
Figure 4 shows remaining lactase activity as a function
of time in samples of 75% RH, stored at 45 °C (Fig. 4a)
or 57 °C (Fig. 4b). The incorporation of MD to a
trehalose matrix delays thermal inactivation of lactase,
which can be attributed to a delayed trehalose crystallization (18). At a given storage time the remaining
activity of lactase was higher in the MD:T mixture than
in the T or MD systems.
One of the effects of adding high molecular weight
compounds to sugar is to increase the Tg of the system.
However, the effect of high molecular weight compounds to retard sugar crystallization is not entirely due
327
lwt/vol. 30 (1997) No. 3
to raising the Tg (18, 32). Adding any substance to a
sugar system (another sugar, for instance (18)) may
retard crystallization by affecting the environment of
the crystallizing sugar molecules, but without changing
Tg significantly. Incorporation of MD to a trehalose
matrix improved the stability of the enzyme at relatively high RH (75%). This protection may be due to
any of the above mechanisms, but it is not associated
with maintaining a glassy structure, since under the
above moisture conditions and storage temperature (45
or 57 °C) the MD:T system was in the rubbery state as
indicated by collapse of the matrix (dramatic shrinkage
was observed).
Acknowledgements
The authors acknowledge financial support from Universidad de Buenos Aires (Secretarı́a de Ciencia y
Técnica) and from International Foundation for Science (Sweden).
Remaining activity (%)
100
(a)
80
60
40
20
0
Remaining activity (%)
100
200
400
600
Time (h)
800
1000
1200
(b)
80
60
40
20
0
100
200
300
Time (h)
Fig. 4 Remaining activity of lactase in different systems
(maltodextrin (j), trehalose ( + ), and mixture MD:T 80:20
(*)) equilibrated at 75% RH, as a function of storage time at
(a) 45 °C and (b) 57 °C. Collapsed was observed in all
systems
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