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Glassy state in relation to the thermal inactivation of the enzyme invertase in amorphous dried matrices of trehalose, maltodextrin and PVP
Glassy State in Relation to the Thermal Inactivation of the Enzyme Invertase in Amorphous Dried Matrices of Trehalose, Maltodextrin and PVP Carolina Schebor,’ Maria de1 Pilar Buera’ & Jorge Chirife’” Ikpartamento dc Industrias, Facultad dc Ciencias Exactas y Naturales, Universidad Buenos Aires, Ciudad Universitaria ( 1428). Buenos Aires, Argentina (Received
20 December
dc
IYW: acccptcd 3 August 1996)
ABSTRACT The stahilizution of invertuse by its incorporution in aqueous trehulose end polymer solutions, ,followrd by freeze-dying and desiccation to Zero’ moisture> content, was studied. The dried amorphous preparations of trehulose. maltodextrin (MD; DE = 10.9), and polv(vinyl)pyrrolidone (PVP), molecule weights 360000, 40 000 and 10000, greatly protected invertase-us compared with its hehalior in liquid solution-from heat inactivation at elellatcd tempemtures. Significant imqertuse inactivation wus observed in heated PVP and MD matrices kept well below their glass-transition temperature. Under glassy conditions the extent of enzq’mc protection by MD and PVP systems wus r&ted to their glass-transition temperature (T,) since systems of higher T, u,fforded better protection. Howevcl; the datu for trehnlose deviated from this behavior since invertase stabilization wus higher than e.xpected on the basis of the re.su1t.s obtained with polymer matrices. Present results suggest that invertuse inactivation in dried amorphous systems cannot be adequately explained by the glass-transition theoq and this is particularly true for trehalose, for which some additional mechanism c$’ enzyme protection is like!v to operate. Copyright 0 I996 Elsclier Science Lirnitc&
INTRODUCTION The stabilization of enzymes and other biomaterials by their incorporation aqueous carbohydrates or polymer solutions, followed by drying to low moisture .’ Author to whom correspondence
’ Fellow of CONICET, Argentina. ‘Member of CONICET, Argentina.
should hc addressed.
in in
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order to immobilize such labile materials in a solid matrix, has several practical implications for a large number of areas (Mugnier & Jung, 1985; Colaco et al., 1992). A relationship exists between temperature, moisture content and duration of thermal treatment on the stability of enzymes and other biomaterials (e.g. microorganisms) at low moisture contents. However, little is known about the precise role of water/matrix on the observed heat resistance (M&on & Guilbot, 1975; Corry, 1975; Harnulv et al., 1977; Mugnier & Jung, 198.5; Whitaker, 1995). Colaco et al. (1992) showed that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of the sugar trehalose with no loss of activity even after prolonged storage; they showed that the dried enzyme had the ability to withstand prolonged exposures to temperatures as high as 70°C. They stated that this stability was unique to trehalose and was not found with other sugars. Uritani et al. (1995) also found that disaccharides such as trehalose, maltose or sucrose protected restriction enzymes during vacuum desiccation and further storage in terms of both recognition and accurate cleavage of the substrate. In recent years, mainly as a result of the pioneering work of Slade and Levine (Slade & Levine, 1987, 1991a), the so-called food science polymer approach (or glass-transition theory) has been introduced to interpret the stability of low-moisture foods and biomaterials, and the important role of water as a plasticizer received increasing attention. This approach put emphasis on thermodynamically unstable states which achieve an apparent structural and chemical stability by virtue of their high viscosities. In such systems, depending on their chemical composition and on their actual temperature, the physical states range from brittle, elastic solids to viscoelastic, deformable rubbers. Water is usually a minor, yet very important component. Glass-transition theory applies to amorphous polymers and also has been found to be applicable to low molecular weight sugars (Slade & Levine, 1991a; Roos & Karel, 1991a). The glass-transition temperature, T,, is the temperature at which a glass-to-rubber transition takes place, and moisture acts to decrease the glass-transition temperature. Formation of a glassy solid state results in significant arrest of translational molecular motion and chemical reactions and relaxation rates of glassy matrices are low, especially when measured at temperatures well below T,: (Franks, 1993). Recently, various authors have suggested that the glass-forming properties of certain carbohydrates, e.g. trehalose, play a key role in the stabilization of biomaterials at low moisture content. Green & Angel1 (1989) related the efficacy of trehalose as a dehydroprotectant in both in-vivo and in-vitro biopreservation systems to the unusual glass-forming characteristic of this dry solute and its low-moisture solutions. In reviewing the role of carbohydrates to protect proteins from the denaturing effects of freezing and frozen storage, MacDonald & Lanier (1991) stated that in a glass all deteriorative processes are greatly slowed, thus helping to protect sugar-stabilized biomaterials. Sun & Leopold (1994) proposed that the cytoplasm of dry seeds exists in a glassy state and this has a retarding effect on deteriorative reactions leading to loss of viability. Koster (1990) also attributed to glass formation the desiccation tolerance of seed because glasses preclude chemical reactions requiring diffusion. Sapru & Labuza (1993) used polymer glass-transition theory to gain information about a possible general mechanism to explain the high heat resistance of bacterial spores and suggested that in a glassy state the configuration of vital macromolecules and supramolecular assemblies in the spore protoplast would change extremely slowly when heated. Levine & Slade (1992) suggested that not
only trehalose, but also other glass-forming carbohydrates such as maltodextrins (of elevated 7’,) can be usefully employed to preserve biological materials in lowmoisture glasses. The objective of the present work was to study the thermal stability of the enzyme invertase in dried model systems of trehalose, maltodextrin (MD) and poly(vinyl)pyrrolidone (PVP).
MATERIALS
AND METHODS
Preparation of model systems Amorphous systems were prepared by freeze-drying solutions containing 15% (h/w) poly(vinyl)pyrrolidone molecular weights 10000 (PVP- lo), 40000 (PVP-40) or 360000 (PVP-360) (S igma Chemical Co., St. Louis, MO); 20% (w/w) maltodextrin (MD), DE 10.9 (Refinacoes de Milho, (Corn Products Corp.), Sgo Paula, Brazil), or 20% (w/w) D( +) trehalose (Sigma Chemical Co.), in citrate buffer (pH = 5.0. 0.1 M). The aqueous model solutions were cooled over an ice bath and commercial invertase (/i-fructofuranosidasc; Solvay, Bioproducts Div., Buenos Aires, Argentina) was added. Aliquots of 1 ml of each model solution were placed in 2-ml vials, and immediately frozen using liquid air. A Stokes freeze-dryer model 21 (F.J. Stokes Company. Equipment Div., Pennsalt Chem. Corp., Philadelphia, PA) was used. which operated at a condenser plate temperature of -40°C and at a chamber pressure of less than 100 /lmHg. Following freeze-drying the samples were transferred into evacuated desiccators over P,Os for a period of 1 week at 35°C; these samples (with the exception of trehalose discussed elsewhere) were taken as zero ? moisture content (Karmas ct al., 1992; Roos & Karel, 1992; Buera 6i Karel, 1993).
Glass-transition
temperature of MD, PVPs and trehalose
Glass-transition temperatures of maltodextrin, PVPs and trehalose model systems were not determined here but were estimated from the data reported by Buera rotrrl. ( 1092) for PVP matrices of the same molecular weight, by Roos & Karel ( 199 1b) for maltodextrin of similar DE number (DE IO), and by Roos (1993) for trehalose. These authors employed differential scanning calorimetry (DSC) measurements at a rate of 5”Cimin in freeze-dried samples humidified in the same conditions followed in the present work. It is known that maltodextrins contain different amounts of monosaccharides, disaccharides, trisaccharides, tetrasaccharides and larger saccharides (Nelson, 1993) and their relative proportions determine their average molecular weight. The T, values measured by Roos & Karel (1991b), and used for the present MD model system, corresponded to Maltrin Ml00 (DE 10, Grain Processing Corp., Mascatine, IA) which has a saccharide composition (carbohydrate profile) similar to the maltodextrin used in the present work. For example, the average content of the larger saccharides (pentasaccharides and above) was 88.1% (dry basis) for Maltrin MIOO, and 89% for present maltodextrin (Refinacoes de Milho, S5o Paulo, Brazil).
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Heat treatment in the dried state After drying over P205 the vials containing the dried matrices were hermetically sealed and placed in air-forced ovens operated at 90, 110, 130, 150 and 170°C (k 1°C). At suitable intervals two samples were removed from the oven and the activity of invertase determined as described below. It was observed that trehalose samples still contained some residual moisture (about 2% dry basis determined gravimetrically by drying at 90°C in a forced convection oven overnight) after desiccation over P205. For this reason the vials containing trehalose were kept uncovered during heating to remove simultaneously this small amount of moisture, since it is known that in amorphous sugars the glass-transition temperature is extremely sensitive to water (Roos, 1995). This fact may explain the different dry Tp values for trehalose reported in the literature (see Table 1). The freeze-dried polymer systems (MD and PVP) also contained some small residual water after desiccation over PZOs but it was below 1%; and this was not a problem for assignment of their dry T, values since the adopted literature values (see Table 1) corresponded to samples subjected to the same desiccation procedure (1 week over P20s after freeze-drying) as employed here. Invertase activity After heat treatment the dried samples were dissolved in 1 ml of water; 1 ml of 40% (w/w) sucrose (substrate) was added and the vials were incubated for 1 h in a bath at 37°C. After incubation the enzyme was inactivated with 1 ml of Na,CO, 0.33 M. Sucrose hydrolysis was followed by glucose determination using the enzymatic method based on the oxidation of glucose by glucose-oxidase to gluconic acid and oxygen peroxide, and has been described previously (Buera et al., 1995). Two replicates of each sample were analyzed and the average was reported for each storage time. The amount of sucrose hydrolyzed by samples without thermal treatment (S,) was considered to correspond to 100% invertase activity; sucrose hydrolyzed after heat treatment (S,) was referred to S,, to obtain remaining activity (&I), RA = S&,100. It was determined that the standard deviation for remaining activity in replicates of the different dried heated matrices varied between 4 and 6%.
TABLE 1
Literature
Glass-transition
Temperature
of Dried MD, PVP and Trehalose
Matrices
Matrix
Tp “C (onset)
PVP-10 PVP-40 PVP-360 Maltodextrin Trehalose
93”” 137 “.s 1SY’ 160 hg 100(.h, -86”“,
(DE = 10.9)
79’, -79
“Buera et al. (1992), hRoos & Karel (1991b), ‘Roes (1993). “Buera (1991) (unpublished), ‘Slade & Levine (1991b), ‘Green & AnFell (1989). PDried over PzOs after freeze-drying, ‘dried by heating, ‘estimated from the Fox and Flory equation (Buera et al., 1992).
RESULTS
AND DISCUSSION
Figure 1 shows thermal inactivation of invertase in liquid buffer of pH 5.0 at 9O’C; the activity of the enzyme was almost completely lost after about 10 min heating time. Figure 2 compares the thermal inactivation of invertase at 90°C in dried (zero !? moisture content) preparations of trehalose, PVP of various molecular weights (360000, 40000 and lOOO0) and maltodextrin. In all cases the heat resistance is dramatically increased as compared with the liquid system (Fig. 1). For example, the half-life (time to reduce invertase activity to 50% of its original value) is 3 min in liquid buffer, but increased to 2400 min in dried maltodextrin heated at the same temperature (90°C). As reviewed by Whitaker (1995), the enzymes lysozymc and ribonucleasc heated at low moisture content were far more stable than in aqueous buffer at the same temperature. The order of relative stability in the different systems is as follows, trehalose MD, PVP 360. PVP 40. PVP 10. It is known that a glass heated to T>T, undergoes a physical state transformation to unstable, rubbery material; according to Levine & Slade (1992). in this state all translational diffusionlimited relaxation processes arc free to occur. Table I shows the dry r, of trehalosc. maltodextrin and PVP matrices taken from several literature sources: the .r: for PVP-360 was estimated from the Fox and Flory equation (Buera et al., 1992). The Tg values for trehalose range from 75 to 100°C according to the source, and as mentioned before, it is likely that these differences are due to small amounts of residual water (Roes, 1995). Roos (1993) reported a value of 100°C for anhydrous
YU~b pH= 5.0
I
10
15
20
Time, min. Fig. 1.
Remaining activity of invcrtase in a liquid system (pH = 5) as a function of heating time at 00°C.
C. Schehor
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et al.
80
e
Maitodextrin
60
I
0’
0
20
I
I
I
40
60
80
100
Time, hour Fig. 2. Remaining activity of invertase in freeze-dried
matrices of zero % moisture as a function of heating time at 90°C.
amorphous trehalose which was produced by heating the crystalline compound above its melting point. Dehydration during melting was assumed since a temperature well above 100°C was used, and for this reason we adopted here Roos’ value as representative of the anhydrous T, for trehalose. The reliability of using anhydrous Tg data determined by others instead of a direct measurement, as well as the presence of buffer salts in present model systems, deserves consideration. As mentioned above, the 7’,: values measured by Roos & Karel (1991b)-and used for present maltodextrin systems-corresponded to Maltrin Ml00 (DE 10, Grain Processing Corporation). We have confirmed the similarity between both maltodextrins (Maltrin Ml00 and Refinacoes de Milho) by freezing point determination of their aqueous solutions and also by differential scanning calorimetry on samples equilibrated at 22Y0 RH (Cardona et al., 1996). Various authors reported good agreement between their Tg determinations for maltodextrins. Nelson (1993) reported that the difference between her Tg values measured for maltodextrin DE 15 and those measured by Roos & Karel (1991b) was quite small over a moisture range of interest to the present study. Chuy & Labuza (1994) measured (DSC) the Tg of maltodextrin DE 10 at low moisture contents and their values were in good agreement with those reported previously by Roos & Karel (1991b) for a similar maltodextrin. Roos & Karel (1991b) found that the anhydrous Tg value of maltodextrin MW 1800 (Maltrin MlOO, DE 10; larger saccharides SS.lYo) was 160°C as compared to 141°C for maltodextrin MW 900 (Maltrin M200, DE 20; larger saccharides 74.4%). Thus, we may safely assume that small variations in the carbohydrate profile would not significantly affect the reliability of the estimated anhydrous Tg value for the present maltodextrin; at least for the purposes of the present work. Buffer salts may (or may not) modify actual Tg values since small molecular weight compounds may act as plasticizers or anttplasticizers. However, we have
observed (Cardona et al., 1996) that the measured T,: for samples of trehalose and PVP-10 including citrate buffer (equilibrated at 22% RH) was only slightly lower than that of the trehalose and PVP-10 without citrate buffer (equilibrated at the same RH %). In both cases (trehalose and PVP) the difference between the 7’, with or without citrate buffrer was within the experimental error usual in the determination of Tg. Nelson (1993) measured (using DSC) the T, of a maltodextrin DE IS model system with and without sodium phosphate buffer salts and found that the measured T, for maltodextrin including the buffer salts was slightly higher than that of maltodextrin without buffer salts, but the effect was not considered significant in view of the usual error involved in T, measurement. Nelson (1993) also reported that the difference between the T, measured for her maltodextrin DE IS model systems with added phosphate buffer salts and those measured by Karel & Roos (1991b) for maltodextrin DE 15 without added buffer salts was quite small over a low-moisture range. Bell & Hageman (1994) used DSC to determine the T, of’ PVP40 model systems with added phosphate buffer and aspartame; at 11.1% (dry basis) moisture content the measured Tg was 63°C a value which compares very well with 63°C found by Buera et al. (1992) for PVP-40 without buffer salts (at identical moisture content). Both values were also in good agreement with a value of 59°C reported by Karmas (1994) for the Tg of a model system of PVP-40 lxyloseilysine (98:l:l). Several data are available in the literature about the plasticizing effect of small additions of low molecular weight compounds on the T, of PVP and trehalose freeze-dried systems. Buera et al. (1992) found that dialyzed PVP samples (to remove low molecular weight compounds) had a *rg only 5°C higher than nondialyzed ones. They also measured the effect of adding different low molecular weight organic plasticizers, namely, xylose (MW = 150) glucose (MW = 180) and lysine (MW = 146) on the anhydrous Tg of PVP-10 and found that the T, was depressed by only 4-6°C by the addition of 5% (dry basis) of one or another of the above compounds. Karmas (1994) determined (using DSC) the anhydrous T, of freeze-dried trehalose with added 5% (dry basis) xylose plus 1% (dry basis) lysine and found that the anhydrous (1 week over P20s) T, of his model system was 82°C as compared with 85°C for pure trehalose as determined by Cardona ct rd. (1996). Figure 3 shows the remaining activity of invertase in the different model systems after 10 or 20 h heating at 90°C versus the difference between the temperature of treatment (i”) and the estimated glass-transition temperature (T-T,). An inverse correlation is observed to exist between remaining activity and (T-T,) for MD and the various PVPs mostly in the glassy state, indicating that higher enzyme activities are associated with higher T, values. According to glass-transition theory (Levine & Slade, 1992) stability should improve as positive (T-T,) values approach zero but no further improvement in stability is expected at (T- T,) < 0. Thus, present results cannot be adequately explained by glass-transition theory. It is noteworthy that collapse (dramatic shrinkage of the matrix) was not observed for any of the model systems during heating at 90°C. Collapse is a physical phcnomenon caused by the viscous flow that results from decreasing viscosity above glass transition (Roos, 1995) and the loss of structure occurs since the material is unable to support its own weight. Since glassy systems are stable to collapse (Nelson. 1993), the absence of collapse noted in the present experiments is to some extent confirmatory of the glassy nature (suggested by their estimated (T-T,) values) of most of the model systems under the conditions shown in Fig. 3. This agreement is
C. Schebor
276 100
et al.
T
TREHALOSE
90°c
P
MD
80
60
“0
‘/
. .
t-~
40
PVP40 l
.
.. .
_
PVP 10 0
.-.
20 i
PWlO --‘a
I~---
0 10 hours
j .’ 0 L -80
-7
-,o
-;o
20 hours .---~
0
1 ~
1 20
(T-Tg), “C Fig. 3. Effect of estimated
(T-T,) values on activity loss of invertase in freeze-dried of zero 9%moisture heated at 90°C for 10 or 20 h.
matrices
qualitative because collapse is also a time-dependent phenomenon. As suggested by their estimated (T-T,) values, the conditions at which thermal stability of invertase was measured (Fig. 3) corresponded to very glassy states in all polymeric systems, with the exception of PVP-10 (for which heating temperature is very close to its estimated T&. Therefore, even if a difference as high as, say, 10°C existed between estimated and actual Tg values, it would not modify significantly the interpretation of the present findings. The data for trehalose (Fig. 3) fall outside the observed empirical correlation for the polymers (MD and PVP); enzyme stabilization is higher than expected from its dry Tg value and this suggests an additional mechanism for enzyme stabilization in this sugar. The precise molecular mechanism by which trehalcse stabilizes biological molecules is unknown, but two hypotheses have been suggested (Colaco et al., 1992). The water-replacement hypothesis states that trehalose can make multiple external hydrogen bonds and could therefore replace the essential water molecules that are involved in the maintenance of the tertiary structure. The glassy-state theory states that the tendency of trehalose (and also other carbohydrates and polymers) solutions to undergo gIass transformation results in an amorphous vitreous phase in which molecular motion would be kinetically insignificant, Levine & Slade (1992) postulated that the glassy state is the sole important factor in the long-term stabilization of stored restriction enzymes in a dried trehalose matrix. Franks (1993) also stated that amorphous glassy states pIay a special role in the long-term stabilization of biological products (e.g. isolated enzymes), which in liquid solution have very limited shelf lives. Figure 4 shows the effect of 3 h heating at increasing temperatures (90, 110, 130, 150 and 170°C) on remaining activity of invertase entrapped in a dried maltodextrin
system (Fig. 4 (top)); a photograph (Fig. 4 (bottom)) of the heated systems is also shown. The anhydrous T,: of the maltodextrin system is estimated to be 160°C (Table 1); if enzyme inactivation were entirely (or largely) dependent on the glass transition a dramatic change in invertase stabilitv would be observed at T near T... However, such a change ig not observed (Fig. 4’ (top)), but the enzyme is almolt inactivated when heating at 130°C (Fig. 4 (top)), which is still well below the
170
150
130
110
Heating temperature
170°C
15ooc
130°C
110°C
Fig. 4. Remaining activity of invertase in freeze-dried moi sture after 3 h heating at the spccilkd temperature shows the aspect of dried
90
(“C)
matrices
90°C
maltodextrin matrices of zero r i (top). The after heating.
photograph
(bottom)
278
C. Schebor et al.
estimated anhydrous T, of the MD system. After heating for 3 h at 170°C the sample had collapsed (Fig. 4 (bottom)); since the estimated Ts of the MD system is 160°C the observed physical change appears to be in good agreement with predictions of glass-transition theory (Slade & Levine, 1987). However, this agreement is only qualitative because collapse at different temperatures is also a time-dependent phenomenon. Photographs of MD matrices also evidence darkening (e.g. nonenzymatic browning) after 3 h heating at increasing temperatures (Fig. 4b). It is noteworthy that browning does occur in the glassy state, well below the estimated glass-transition temperature of the MD system and in the absence of moisture (zero % moisture). Colaco et al. (1992) studied the storage stability of DNA restriction enzymes dried in trehalose and other sugars and suggested that the Maillard reaction may be an important factor during stabilization and storage of their dried enzyme preparations. Although the most protective sugars were non-reducing while the rest were reducing ones, this is not sufficient to conclude that Maillard’s reaction was a key factor in enzyme stabilization, mainly because Colaco et al. (1992) did not report the actual moisture content of their dried sugar matrices. It is well known that the Tg of sugars is dramatically reduced by small amounts of residual moisture (Roos, 1995), and this precludes comparison between Tg values (either dry or wet) of the different matrices. Figure 5 shows residual invertase activity in dried trehalose preparations after 3 h heating at temperatures between 90 and 170°C. It can be seen that at llO”C, which is above the glass-transition temperature of trehalose (collapse of the matrix was observed), significant activity is retained and the most dramatic decrease is observed when heating at 130°C. Figure 6 shows similar results for the PVPs of different molecular weights, and for which the order of decreasing of their estimated dry Tg
100
80
s
.a .2_
60
0 m .E
.s E L?
1
40
20
0 110
130 Heating temperature,
150
170
“C
Fig. 5. Remaining activity of invertase in freeze-dried trehalose of zero % moisture after 3 h heating at specified temperatures.
80
110 Heating temperature, PPVP360
Fig. 6. Remaining content
after
activity .7 h heating
lPVP40
“C
BPIPVP 101
of invertase in freeze-dried PVP matrices of zero ‘1; moisture at speciticd temperatures: R: rubbery. Ci: glassy (from their cstimatcd Tc values).
values is: PVP-360 (T, = 1SYC) PVP-40 (T, = 137°C) PVP-10 (r, = 9OYY). PVP-360 and PVP-40 generally afforded better enzyme stabilization than PVP-IO and this is correlated with their higher ‘f? values. The glassy state does not prevent enzyme inactivation since PVP systems (PVP-360 and PVP-40) heated well below their estimated glass-transition temperatures allowed significant invertase inactivation. It is noteworthy that collapse was observed for all systems identified as rubbery (R) in Fig. 6; the glassy nature assigned to the PVP-10 system heated at 90°C is only tentative and cannot be confirmed here because the heating temperature (90°C) is very close to its estimated Tg value and collapse is a time-dependent property. In reviewing basic aspects of glass transition, Simatos et al. (1995) noted that some mobility persists at temperatures below T,, which may result in significant changes if the time scale of observation is enough. Present results for enzyme inactivation (e.g. Fig. 3) appear to confirm these statements.
CONCLUSIONS Amorphous dried matrices of maltodextrin, trehalose and PVP of various molecular weights, greatly protected invertase-as compared with its behavior in liquid media-from thermal inactivation when heated at 90°C and above. Since protein denaturation requires a spatial reordering of the molecules, it seemed reasonable to expect that protein (enzyme) stability should be greatly enhanced in a glassy matrix. However, it was here determined that significant invertase inactivation occurred in
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maltodextrin and PVP systems in the glassy state. Enzyme stability under glassy conditions was observed to be related to the Tg value of the dried matrices, since systems with higher T, yielded better enzyme stabilization. Trehalose exerted a high degree of protection which did not correlate with its T, value, as observed with the polymers. Present results suggest that enzyme inactivation in dried amorphous systems cannot be predicted on the basis of the glass-transition theory, and this is particularly true for trehalose for which it is evident that an additional mechanism of protection exists. The food science polymer approach introduced new and valuable concepts for interpreting the behavior of low moisture biomaterials (Slade & Levine, 1995); however, the stability of biomolecules in a polymeric (or carbohydrate) matrix is not only dependent on mobility but also on other factors as shown in the present work. For this reason the definition of stability maps as proposed by Slade et al. (1989) and Slade & Levine (1994) in which the T,s of major constituents provide a boundary between regions of low mobility (glasses) and increased mobility (rubbers) cannot be used as the sole important parameter for predicting the stabilization of invertase in amorphous dried systems. Simatos et al. (1995) suggested that although glass transition is an important phenomenon to understand some of the effects of water on properties of foods and related biomaterials, it cannot be used by itself to characterize a material in relation to temperature/water conditions prevailing during storage; and that Tg cannot be considered as an absolute threshold of stability. ACKNOWLEDGEMENTS The authors acknowledge financial support from Universidad de Buenos Aires (Secretaria de Ciencia y Tecnica) and International Foundation for Science. They are also grateful to Departamento de Fisica (Bajas Temperaturas), Facultad de Ciencias Exactas y Naturales for providing liquid air. REFERENCES Bell, L. N. & Hageman, M. J. (1994). Differentiating between the effects of water activity and glass transition dependent mobility on a solid state chemical reaction: aspartame degradation. J. Agric. Food Chem., 42, 2398-2401. Buera, M. P. & Karel, M. (1993). Application of the WLF equation to describe the combined effects of moisture and temperature on noncnzymatic browning rates in food systems. J. Food Proc. Pres., 17, 3 I-45.
Bucra, M. P., Levi, G. & Karel, M. (1992). Glass transition in poly(vinyl)pyrrolidone: effect of molecular weight and diluents. Biotechnol. Prog., 8, 144-148. Buera, M. P., Chirife, J. & Karel, M. (1995). A study of acid catalyzed sucrose hydrolysis in an amorphous polymeric matrix at reduced moisture content. Food Res. Int., 28, 359-36.5. Cardona, S., Schebor, C., Buera, M. P., Karel, M. & Chirife, J. (1996). Thermal stability of invcrtase in reduced-moisture amorphous matrices in relation to glassy state and role of trehalose crystallization. J. Food Sci. (in press). Chuy, L. E. & Labuza, T. P. (1994). Caking and stickiness of dairy-based food powders as related to glass transition. J. Food Sci.. 59, 43-46. Colaco, C., Sen, S., Thangavelu, M., Pinder, S. & Roser, B. (1992). Extraordinary stability of enzymes dried in trehalose: simplified molecular biology. BiolTechnologv, 10, 1007-1010. Corry, J. E. L. (1975). The effect of water activity on the heat resistance of bacteria. In Water Relations of Foods, ed. R. B. Duckworth. Academic Press, New York, pp. 325-337.
Franks, F. ( IYY3). Solid aqueous solutions. Pure & Appl. Chem., 65, 2527-2537. Green. 81 Angell, C. A. (1989). Phase relations and vitrification in saccharidc-water solutions and the trehalose anomaly. J. Phys. Chem., 93, 2880-2882. Harnulv. B. G.. Johansson, M. & Snygg, B. G. (1977). Heat resistance of Bucil1n.s .stwrothermophilus spores at different water activities. J. Food Sci., 42, 91-93. Karmas, R. (19Y4). The effect of glass transition on non-enzymatic browning in dchydratcd food systems. Ph.D. Thesis, University of New Jersey, New Brunswick, NJ. Karmas, R., Buera. M. P. & Karel. M. (1992). Effect of glass transition on rates of nonenzymatic browning in food systems. J. Agric. & Food Chem.. 40. 873-87’). Koster, K. L. (1990). Glass formation and desiccation tolerance in seeds. Plunt Plysiof.. 96. 302-304. H. & Sladc. L. (lYY2). Another view of trehalose for drying and stabilizing biological materials. BioPhurm. 5, 36-40. MacDonald, G. A. CG Lanicr, T. (1091). Carbohydrates as cryoprotectants t’w meats e~nd surimi. Footi Tcchrrol., March. pp. 150-Y. Mugnicr. J. & Jung, G. (19%). Survival of bacteria and fungi in relation to water activity JIKI the properties of water in biopolymer gels. Appl. Em: Microhiol., 50, 108-I 14. Multon, J. L. & Guilbot. A. (1975). Water activity in relation to the thermal inactivation of cnzymic proteins. In Wutcr wlutions of Foods, cd. R. B. Duckworth. Academic Press, New York, pp. 379-96. Nelson. K. A. (1903). Reaction kinetics of food stability: comparison of glass transition and classical models for temperature and moisture dcpcndcncc. Ph.D. Thesis, University of Minnesota, U.S.A. Roes. Y. & Karcl. M. ( IYYla). Plasticizing effect of water on thermal behavior and crystallization of amorphous food models. J. Food Sci.. 34, 324-320. Roes, Y. & Karcl, M. (1991b). Phase transitions of mixtures of amorphous polyaccharidcs and sugars. Biotechnof. Prog.. 7. 49-U. Roes. Y. & Karel, M. (1992). Crystallization of amorphous lactose. J. Ebod Sci.. 57. 775-777. Roes, Y. (1993). Melting and glass transitions of low molecular weight carbohydrates. (‘rr&ofydrute Res., 238, 39-48. Roes, Y. (lY95). Phase Trunsition.s in Foods. Academic Press, New York. Sapru. V. Kr Labuza, T. P. (1903). Glassy state in bacterial spores predicted by polymer glasstransition theory. J. Food Sci., 58. 445-448. Simatos. D.. Blond, G. & Perez, J. (1995). Basic physical aspects of glass transition. III f?ml Ptwenvtion hy Moisture Control. eds G. V. Barbosa-C&novas & J. Welti-Chants. Tcchnomic. Lancaster, PA, pp. 3-3 I. Sladc, L. & Levine. H. (1087). Structural stability of intermediate moisture foods--a new understanding. In Food Structuw - Its Creation und E~wluution. eds J. R. Mitchell & J. M. V. Blanshard. Butterworths, London, pp. 115-147. Slade, L., Levine, H. & Finley, J. W. (lY8Y). Protein-water interactions: water as a plasticizer of gluten and other protein polymers. In Protein Q&it\! und the i?ffects of’ f’roccwin,y. ccis Ii. Dixon Phillips & J. W. Finley. Marcel Dekkcr, Inc., New York. pp. Y-124. Sladc, L. & Levine, H. (l9Y la). A food polymer science approach to structure-property relationships in aqueous food systems: non equilibrium behavior of carbohydrate-water systems. In Water R&tionships in Foods. eds H. Levine & L. Slade. Plenum Prc:,\, New, York. pp. 29- IO I. Slade. L. & Lcvinc. H. (IVY lb). Beyond water activity: recent advances based on an ;.ilternativc approach to the assessment of food quality and safety. (‘r-it. Rev Food Sci. Nutr:. 30. I IS-360. Slade, L. CyrLevine, H. (1994). Glass transitions and water-food structure interaction. In IMmc~~s in Food und Nutrition Research. Vol. 38, cd. J. E. Kinsella. Academic Press. San Diego, CA. Levine,
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Slade, L. & Levine, H. (1995). Polymer science approach to water relationships in foods. In Food Preservation by Moisture Control, eds G. V. Barbosa-Canovas & J. Welti-Chanes. Technomic, Lancaster, PA, pp. 33-132. Sun, W. Q. & Leopold, C. (1994). Glassy state and seed storage stability: a viability equation analysis. Ann. Botany, 74, 601-604. Uritani, M., Takai, M. & Yoshinaga, K. (1995). Protective effect of disaccharides on restriction endonucleases during drying under vacuum. J. Biochem., 117, 774-779. Whitaker, J. R. (1995). Enzyme action in aqueous systems with special emphasis on intermediate and high moisture foods. In Food Preservation by Moisture Control, eds G. V. Barbosa-Canovas & J. Welti-Chanes. Technomic, Lancaster, PA, pp. 255-77.
20 December
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IYW: acccptcd 3 August 1996)
ABSTRACT The stahilizution of invertuse by its incorporution in aqueous trehulose end polymer solutions, ,followrd by freeze-dying and desiccation to Zero’ moisture> content, was studied. The dried amorphous preparations of trehulose. maltodextrin (MD; DE = 10.9), and polv(vinyl)pyrrolidone (PVP), molecule weights 360000, 40 000 and 10000, greatly protected invertase-us compared with its hehalior in liquid solution-from heat inactivation at elellatcd tempemtures. Significant imqertuse inactivation wus observed in heated PVP and MD matrices kept well below their glass-transition temperature. Under glassy conditions the extent of enzq’mc protection by MD and PVP systems wus r&ted to their glass-transition temperature (T,) since systems of higher T, u,fforded better protection. Howevcl; the datu for trehnlose deviated from this behavior since invertase stabilization wus higher than e.xpected on the basis of the re.su1t.s obtained with polymer matrices. Present results suggest that invertuse inactivation in dried amorphous systems cannot be adequately explained by the glass-transition theoq and this is particularly true for trehalose, for which some additional mechanism c$’ enzyme protection is like!v to operate. Copyright 0 I996 Elsclier Science Lirnitc&
INTRODUCTION The stabilization of enzymes and other biomaterials by their incorporation aqueous carbohydrates or polymer solutions, followed by drying to low moisture .’ Author to whom correspondence
’ Fellow of CONICET, Argentina. ‘Member of CONICET, Argentina.
should hc addressed.
in in
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order to immobilize such labile materials in a solid matrix, has several practical implications for a large number of areas (Mugnier & Jung, 1985; Colaco et al., 1992). A relationship exists between temperature, moisture content and duration of thermal treatment on the stability of enzymes and other biomaterials (e.g. microorganisms) at low moisture contents. However, little is known about the precise role of water/matrix on the observed heat resistance (M&on & Guilbot, 1975; Corry, 1975; Harnulv et al., 1977; Mugnier & Jung, 198.5; Whitaker, 1995). Colaco et al. (1992) showed that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of the sugar trehalose with no loss of activity even after prolonged storage; they showed that the dried enzyme had the ability to withstand prolonged exposures to temperatures as high as 70°C. They stated that this stability was unique to trehalose and was not found with other sugars. Uritani et al. (1995) also found that disaccharides such as trehalose, maltose or sucrose protected restriction enzymes during vacuum desiccation and further storage in terms of both recognition and accurate cleavage of the substrate. In recent years, mainly as a result of the pioneering work of Slade and Levine (Slade & Levine, 1987, 1991a), the so-called food science polymer approach (or glass-transition theory) has been introduced to interpret the stability of low-moisture foods and biomaterials, and the important role of water as a plasticizer received increasing attention. This approach put emphasis on thermodynamically unstable states which achieve an apparent structural and chemical stability by virtue of their high viscosities. In such systems, depending on their chemical composition and on their actual temperature, the physical states range from brittle, elastic solids to viscoelastic, deformable rubbers. Water is usually a minor, yet very important component. Glass-transition theory applies to amorphous polymers and also has been found to be applicable to low molecular weight sugars (Slade & Levine, 1991a; Roos & Karel, 1991a). The glass-transition temperature, T,, is the temperature at which a glass-to-rubber transition takes place, and moisture acts to decrease the glass-transition temperature. Formation of a glassy solid state results in significant arrest of translational molecular motion and chemical reactions and relaxation rates of glassy matrices are low, especially when measured at temperatures well below T,: (Franks, 1993). Recently, various authors have suggested that the glass-forming properties of certain carbohydrates, e.g. trehalose, play a key role in the stabilization of biomaterials at low moisture content. Green & Angel1 (1989) related the efficacy of trehalose as a dehydroprotectant in both in-vivo and in-vitro biopreservation systems to the unusual glass-forming characteristic of this dry solute and its low-moisture solutions. In reviewing the role of carbohydrates to protect proteins from the denaturing effects of freezing and frozen storage, MacDonald & Lanier (1991) stated that in a glass all deteriorative processes are greatly slowed, thus helping to protect sugar-stabilized biomaterials. Sun & Leopold (1994) proposed that the cytoplasm of dry seeds exists in a glassy state and this has a retarding effect on deteriorative reactions leading to loss of viability. Koster (1990) also attributed to glass formation the desiccation tolerance of seed because glasses preclude chemical reactions requiring diffusion. Sapru & Labuza (1993) used polymer glass-transition theory to gain information about a possible general mechanism to explain the high heat resistance of bacterial spores and suggested that in a glassy state the configuration of vital macromolecules and supramolecular assemblies in the spore protoplast would change extremely slowly when heated. Levine & Slade (1992) suggested that not
only trehalose, but also other glass-forming carbohydrates such as maltodextrins (of elevated 7’,) can be usefully employed to preserve biological materials in lowmoisture glasses. The objective of the present work was to study the thermal stability of the enzyme invertase in dried model systems of trehalose, maltodextrin (MD) and poly(vinyl)pyrrolidone (PVP).
MATERIALS
AND METHODS
Preparation of model systems Amorphous systems were prepared by freeze-drying solutions containing 15% (h/w) poly(vinyl)pyrrolidone molecular weights 10000 (PVP- lo), 40000 (PVP-40) or 360000 (PVP-360) (S igma Chemical Co., St. Louis, MO); 20% (w/w) maltodextrin (MD), DE 10.9 (Refinacoes de Milho, (Corn Products Corp.), Sgo Paula, Brazil), or 20% (w/w) D( +) trehalose (Sigma Chemical Co.), in citrate buffer (pH = 5.0. 0.1 M). The aqueous model solutions were cooled over an ice bath and commercial invertase (/i-fructofuranosidasc; Solvay, Bioproducts Div., Buenos Aires, Argentina) was added. Aliquots of 1 ml of each model solution were placed in 2-ml vials, and immediately frozen using liquid air. A Stokes freeze-dryer model 21 (F.J. Stokes Company. Equipment Div., Pennsalt Chem. Corp., Philadelphia, PA) was used. which operated at a condenser plate temperature of -40°C and at a chamber pressure of less than 100 /lmHg. Following freeze-drying the samples were transferred into evacuated desiccators over P,Os for a period of 1 week at 35°C; these samples (with the exception of trehalose discussed elsewhere) were taken as zero ? moisture content (Karmas ct al., 1992; Roos & Karel, 1992; Buera 6i Karel, 1993).
Glass-transition
temperature of MD, PVPs and trehalose
Glass-transition temperatures of maltodextrin, PVPs and trehalose model systems were not determined here but were estimated from the data reported by Buera rotrrl. ( 1092) for PVP matrices of the same molecular weight, by Roos & Karel ( 199 1b) for maltodextrin of similar DE number (DE IO), and by Roos (1993) for trehalose. These authors employed differential scanning calorimetry (DSC) measurements at a rate of 5”Cimin in freeze-dried samples humidified in the same conditions followed in the present work. It is known that maltodextrins contain different amounts of monosaccharides, disaccharides, trisaccharides, tetrasaccharides and larger saccharides (Nelson, 1993) and their relative proportions determine their average molecular weight. The T, values measured by Roos & Karel (1991b), and used for the present MD model system, corresponded to Maltrin Ml00 (DE 10, Grain Processing Corp., Mascatine, IA) which has a saccharide composition (carbohydrate profile) similar to the maltodextrin used in the present work. For example, the average content of the larger saccharides (pentasaccharides and above) was 88.1% (dry basis) for Maltrin MIOO, and 89% for present maltodextrin (Refinacoes de Milho, S5o Paulo, Brazil).
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Heat treatment in the dried state After drying over P205 the vials containing the dried matrices were hermetically sealed and placed in air-forced ovens operated at 90, 110, 130, 150 and 170°C (k 1°C). At suitable intervals two samples were removed from the oven and the activity of invertase determined as described below. It was observed that trehalose samples still contained some residual moisture (about 2% dry basis determined gravimetrically by drying at 90°C in a forced convection oven overnight) after desiccation over P205. For this reason the vials containing trehalose were kept uncovered during heating to remove simultaneously this small amount of moisture, since it is known that in amorphous sugars the glass-transition temperature is extremely sensitive to water (Roos, 1995). This fact may explain the different dry Tp values for trehalose reported in the literature (see Table 1). The freeze-dried polymer systems (MD and PVP) also contained some small residual water after desiccation over PZOs but it was below 1%; and this was not a problem for assignment of their dry T, values since the adopted literature values (see Table 1) corresponded to samples subjected to the same desiccation procedure (1 week over P20s after freeze-drying) as employed here. Invertase activity After heat treatment the dried samples were dissolved in 1 ml of water; 1 ml of 40% (w/w) sucrose (substrate) was added and the vials were incubated for 1 h in a bath at 37°C. After incubation the enzyme was inactivated with 1 ml of Na,CO, 0.33 M. Sucrose hydrolysis was followed by glucose determination using the enzymatic method based on the oxidation of glucose by glucose-oxidase to gluconic acid and oxygen peroxide, and has been described previously (Buera et al., 1995). Two replicates of each sample were analyzed and the average was reported for each storage time. The amount of sucrose hydrolyzed by samples without thermal treatment (S,) was considered to correspond to 100% invertase activity; sucrose hydrolyzed after heat treatment (S,) was referred to S,, to obtain remaining activity (&I), RA = S&,100. It was determined that the standard deviation for remaining activity in replicates of the different dried heated matrices varied between 4 and 6%.
TABLE 1
Literature
Glass-transition
Temperature
of Dried MD, PVP and Trehalose
Matrices
Matrix
Tp “C (onset)
PVP-10 PVP-40 PVP-360 Maltodextrin Trehalose
93”” 137 “.s 1SY’ 160 hg 100(.h, -86”“,
(DE = 10.9)
79’, -79
“Buera et al. (1992), hRoos & Karel (1991b), ‘Roes (1993). “Buera (1991) (unpublished), ‘Slade & Levine (1991b), ‘Green & AnFell (1989). PDried over PzOs after freeze-drying, ‘dried by heating, ‘estimated from the Fox and Flory equation (Buera et al., 1992).
RESULTS
AND DISCUSSION
Figure 1 shows thermal inactivation of invertase in liquid buffer of pH 5.0 at 9O’C; the activity of the enzyme was almost completely lost after about 10 min heating time. Figure 2 compares the thermal inactivation of invertase at 90°C in dried (zero !? moisture content) preparations of trehalose, PVP of various molecular weights (360000, 40000 and lOOO0) and maltodextrin. In all cases the heat resistance is dramatically increased as compared with the liquid system (Fig. 1). For example, the half-life (time to reduce invertase activity to 50% of its original value) is 3 min in liquid buffer, but increased to 2400 min in dried maltodextrin heated at the same temperature (90°C). As reviewed by Whitaker (1995), the enzymes lysozymc and ribonucleasc heated at low moisture content were far more stable than in aqueous buffer at the same temperature. The order of relative stability in the different systems is as follows, trehalose MD, PVP 360. PVP 40. PVP 10. It is known that a glass heated to T>T, undergoes a physical state transformation to unstable, rubbery material; according to Levine & Slade (1992). in this state all translational diffusionlimited relaxation processes arc free to occur. Table I shows the dry r, of trehalosc. maltodextrin and PVP matrices taken from several literature sources: the .r: for PVP-360 was estimated from the Fox and Flory equation (Buera et al., 1992). The Tg values for trehalose range from 75 to 100°C according to the source, and as mentioned before, it is likely that these differences are due to small amounts of residual water (Roes, 1995). Roos (1993) reported a value of 100°C for anhydrous
YU~b pH= 5.0
I
10
15
20
Time, min. Fig. 1.
Remaining activity of invcrtase in a liquid system (pH = 5) as a function of heating time at 00°C.
C. Schehor
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et al.
80
e
Maitodextrin
60
I
0’
0
20
I
I
I
40
60
80
100
Time, hour Fig. 2. Remaining activity of invertase in freeze-dried
matrices of zero % moisture as a function of heating time at 90°C.
amorphous trehalose which was produced by heating the crystalline compound above its melting point. Dehydration during melting was assumed since a temperature well above 100°C was used, and for this reason we adopted here Roos’ value as representative of the anhydrous T, for trehalose. The reliability of using anhydrous Tg data determined by others instead of a direct measurement, as well as the presence of buffer salts in present model systems, deserves consideration. As mentioned above, the 7’,: values measured by Roos & Karel (1991b)-and used for present maltodextrin systems-corresponded to Maltrin Ml00 (DE 10, Grain Processing Corporation). We have confirmed the similarity between both maltodextrins (Maltrin Ml00 and Refinacoes de Milho) by freezing point determination of their aqueous solutions and also by differential scanning calorimetry on samples equilibrated at 22Y0 RH (Cardona et al., 1996). Various authors reported good agreement between their Tg determinations for maltodextrins. Nelson (1993) reported that the difference between her Tg values measured for maltodextrin DE 15 and those measured by Roos & Karel (1991b) was quite small over a moisture range of interest to the present study. Chuy & Labuza (1994) measured (DSC) the Tg of maltodextrin DE 10 at low moisture contents and their values were in good agreement with those reported previously by Roos & Karel (1991b) for a similar maltodextrin. Roos & Karel (1991b) found that the anhydrous Tg value of maltodextrin MW 1800 (Maltrin MlOO, DE 10; larger saccharides SS.lYo) was 160°C as compared to 141°C for maltodextrin MW 900 (Maltrin M200, DE 20; larger saccharides 74.4%). Thus, we may safely assume that small variations in the carbohydrate profile would not significantly affect the reliability of the estimated anhydrous Tg value for the present maltodextrin; at least for the purposes of the present work. Buffer salts may (or may not) modify actual Tg values since small molecular weight compounds may act as plasticizers or anttplasticizers. However, we have
observed (Cardona et al., 1996) that the measured T,: for samples of trehalose and PVP-10 including citrate buffer (equilibrated at 22% RH) was only slightly lower than that of the trehalose and PVP-10 without citrate buffer (equilibrated at the same RH %). In both cases (trehalose and PVP) the difference between the 7’, with or without citrate buffrer was within the experimental error usual in the determination of Tg. Nelson (1993) measured (using DSC) the T, of a maltodextrin DE IS model system with and without sodium phosphate buffer salts and found that the measured T, for maltodextrin including the buffer salts was slightly higher than that of maltodextrin without buffer salts, but the effect was not considered significant in view of the usual error involved in T, measurement. Nelson (1993) also reported that the difference between the T, measured for her maltodextrin DE IS model systems with added phosphate buffer salts and those measured by Karel & Roos (1991b) for maltodextrin DE 15 without added buffer salts was quite small over a low-moisture range. Bell & Hageman (1994) used DSC to determine the T, of’ PVP40 model systems with added phosphate buffer and aspartame; at 11.1% (dry basis) moisture content the measured Tg was 63°C a value which compares very well with 63°C found by Buera et al. (1992) for PVP-40 without buffer salts (at identical moisture content). Both values were also in good agreement with a value of 59°C reported by Karmas (1994) for the Tg of a model system of PVP-40 lxyloseilysine (98:l:l). Several data are available in the literature about the plasticizing effect of small additions of low molecular weight compounds on the T, of PVP and trehalose freeze-dried systems. Buera et al. (1992) found that dialyzed PVP samples (to remove low molecular weight compounds) had a *rg only 5°C higher than nondialyzed ones. They also measured the effect of adding different low molecular weight organic plasticizers, namely, xylose (MW = 150) glucose (MW = 180) and lysine (MW = 146) on the anhydrous Tg of PVP-10 and found that the T, was depressed by only 4-6°C by the addition of 5% (dry basis) of one or another of the above compounds. Karmas (1994) determined (using DSC) the anhydrous T, of freeze-dried trehalose with added 5% (dry basis) xylose plus 1% (dry basis) lysine and found that the anhydrous (1 week over P20s) T, of his model system was 82°C as compared with 85°C for pure trehalose as determined by Cardona ct rd. (1996). Figure 3 shows the remaining activity of invertase in the different model systems after 10 or 20 h heating at 90°C versus the difference between the temperature of treatment (i”) and the estimated glass-transition temperature (T-T,). An inverse correlation is observed to exist between remaining activity and (T-T,) for MD and the various PVPs mostly in the glassy state, indicating that higher enzyme activities are associated with higher T, values. According to glass-transition theory (Levine & Slade, 1992) stability should improve as positive (T-T,) values approach zero but no further improvement in stability is expected at (T- T,) < 0. Thus, present results cannot be adequately explained by glass-transition theory. It is noteworthy that collapse (dramatic shrinkage of the matrix) was not observed for any of the model systems during heating at 90°C. Collapse is a physical phcnomenon caused by the viscous flow that results from decreasing viscosity above glass transition (Roos, 1995) and the loss of structure occurs since the material is unable to support its own weight. Since glassy systems are stable to collapse (Nelson. 1993), the absence of collapse noted in the present experiments is to some extent confirmatory of the glassy nature (suggested by their estimated (T-T,) values) of most of the model systems under the conditions shown in Fig. 3. This agreement is
C. Schebor
276 100
et al.
T
TREHALOSE
90°c
P
MD
80
60
“0
‘/
. .
t-~
40
PVP40 l
.
.. .
_
PVP 10 0
.-.
20 i
PWlO --‘a
I~---
0 10 hours
j .’ 0 L -80
-7
-,o
-;o
20 hours .---~
0
1 ~
1 20
(T-Tg), “C Fig. 3. Effect of estimated
(T-T,) values on activity loss of invertase in freeze-dried of zero 9%moisture heated at 90°C for 10 or 20 h.
matrices
qualitative because collapse is also a time-dependent phenomenon. As suggested by their estimated (T-T,) values, the conditions at which thermal stability of invertase was measured (Fig. 3) corresponded to very glassy states in all polymeric systems, with the exception of PVP-10 (for which heating temperature is very close to its estimated T&. Therefore, even if a difference as high as, say, 10°C existed between estimated and actual Tg values, it would not modify significantly the interpretation of the present findings. The data for trehalose (Fig. 3) fall outside the observed empirical correlation for the polymers (MD and PVP); enzyme stabilization is higher than expected from its dry Tg value and this suggests an additional mechanism for enzyme stabilization in this sugar. The precise molecular mechanism by which trehalcse stabilizes biological molecules is unknown, but two hypotheses have been suggested (Colaco et al., 1992). The water-replacement hypothesis states that trehalose can make multiple external hydrogen bonds and could therefore replace the essential water molecules that are involved in the maintenance of the tertiary structure. The glassy-state theory states that the tendency of trehalose (and also other carbohydrates and polymers) solutions to undergo gIass transformation results in an amorphous vitreous phase in which molecular motion would be kinetically insignificant, Levine & Slade (1992) postulated that the glassy state is the sole important factor in the long-term stabilization of stored restriction enzymes in a dried trehalose matrix. Franks (1993) also stated that amorphous glassy states pIay a special role in the long-term stabilization of biological products (e.g. isolated enzymes), which in liquid solution have very limited shelf lives. Figure 4 shows the effect of 3 h heating at increasing temperatures (90, 110, 130, 150 and 170°C) on remaining activity of invertase entrapped in a dried maltodextrin
system (Fig. 4 (top)); a photograph (Fig. 4 (bottom)) of the heated systems is also shown. The anhydrous T,: of the maltodextrin system is estimated to be 160°C (Table 1); if enzyme inactivation were entirely (or largely) dependent on the glass transition a dramatic change in invertase stabilitv would be observed at T near T... However, such a change ig not observed (Fig. 4’ (top)), but the enzyme is almolt inactivated when heating at 130°C (Fig. 4 (top)), which is still well below the
170
150
130
110
Heating temperature
170°C
15ooc
130°C
110°C
Fig. 4. Remaining activity of invertase in freeze-dried moi sture after 3 h heating at the spccilkd temperature shows the aspect of dried
90
(“C)
matrices
90°C
maltodextrin matrices of zero r i (top). The after heating.
photograph
(bottom)
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estimated anhydrous T, of the MD system. After heating for 3 h at 170°C the sample had collapsed (Fig. 4 (bottom)); since the estimated Ts of the MD system is 160°C the observed physical change appears to be in good agreement with predictions of glass-transition theory (Slade & Levine, 1987). However, this agreement is only qualitative because collapse at different temperatures is also a time-dependent phenomenon. Photographs of MD matrices also evidence darkening (e.g. nonenzymatic browning) after 3 h heating at increasing temperatures (Fig. 4b). It is noteworthy that browning does occur in the glassy state, well below the estimated glass-transition temperature of the MD system and in the absence of moisture (zero % moisture). Colaco et al. (1992) studied the storage stability of DNA restriction enzymes dried in trehalose and other sugars and suggested that the Maillard reaction may be an important factor during stabilization and storage of their dried enzyme preparations. Although the most protective sugars were non-reducing while the rest were reducing ones, this is not sufficient to conclude that Maillard’s reaction was a key factor in enzyme stabilization, mainly because Colaco et al. (1992) did not report the actual moisture content of their dried sugar matrices. It is well known that the Tg of sugars is dramatically reduced by small amounts of residual moisture (Roos, 1995), and this precludes comparison between Tg values (either dry or wet) of the different matrices. Figure 5 shows residual invertase activity in dried trehalose preparations after 3 h heating at temperatures between 90 and 170°C. It can be seen that at llO”C, which is above the glass-transition temperature of trehalose (collapse of the matrix was observed), significant activity is retained and the most dramatic decrease is observed when heating at 130°C. Figure 6 shows similar results for the PVPs of different molecular weights, and for which the order of decreasing of their estimated dry Tg
100
80
s
.a .2_
60
0 m .E
.s E L?
1
40
20
0 110
130 Heating temperature,
150
170
“C
Fig. 5. Remaining activity of invertase in freeze-dried trehalose of zero % moisture after 3 h heating at specified temperatures.
80
110 Heating temperature, PPVP360
Fig. 6. Remaining content
after
activity .7 h heating
lPVP40
“C
BPIPVP 101
of invertase in freeze-dried PVP matrices of zero ‘1; moisture at speciticd temperatures: R: rubbery. Ci: glassy (from their cstimatcd Tc values).
values is: PVP-360 (T, = 1SYC) PVP-40 (T, = 137°C) PVP-10 (r, = 9OYY). PVP-360 and PVP-40 generally afforded better enzyme stabilization than PVP-IO and this is correlated with their higher ‘f? values. The glassy state does not prevent enzyme inactivation since PVP systems (PVP-360 and PVP-40) heated well below their estimated glass-transition temperatures allowed significant invertase inactivation. It is noteworthy that collapse was observed for all systems identified as rubbery (R) in Fig. 6; the glassy nature assigned to the PVP-10 system heated at 90°C is only tentative and cannot be confirmed here because the heating temperature (90°C) is very close to its estimated Tg value and collapse is a time-dependent property. In reviewing basic aspects of glass transition, Simatos et al. (1995) noted that some mobility persists at temperatures below T,, which may result in significant changes if the time scale of observation is enough. Present results for enzyme inactivation (e.g. Fig. 3) appear to confirm these statements.
CONCLUSIONS Amorphous dried matrices of maltodextrin, trehalose and PVP of various molecular weights, greatly protected invertase-as compared with its behavior in liquid media-from thermal inactivation when heated at 90°C and above. Since protein denaturation requires a spatial reordering of the molecules, it seemed reasonable to expect that protein (enzyme) stability should be greatly enhanced in a glassy matrix. However, it was here determined that significant invertase inactivation occurred in
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maltodextrin and PVP systems in the glassy state. Enzyme stability under glassy conditions was observed to be related to the Tg value of the dried matrices, since systems with higher T, yielded better enzyme stabilization. Trehalose exerted a high degree of protection which did not correlate with its T, value, as observed with the polymers. Present results suggest that enzyme inactivation in dried amorphous systems cannot be predicted on the basis of the glass-transition theory, and this is particularly true for trehalose for which it is evident that an additional mechanism of protection exists. The food science polymer approach introduced new and valuable concepts for interpreting the behavior of low moisture biomaterials (Slade & Levine, 1995); however, the stability of biomolecules in a polymeric (or carbohydrate) matrix is not only dependent on mobility but also on other factors as shown in the present work. For this reason the definition of stability maps as proposed by Slade et al. (1989) and Slade & Levine (1994) in which the T,s of major constituents provide a boundary between regions of low mobility (glasses) and increased mobility (rubbers) cannot be used as the sole important parameter for predicting the stabilization of invertase in amorphous dried systems. Simatos et al. (1995) suggested that although glass transition is an important phenomenon to understand some of the effects of water on properties of foods and related biomaterials, it cannot be used by itself to characterize a material in relation to temperature/water conditions prevailing during storage; and that Tg cannot be considered as an absolute threshold of stability. ACKNOWLEDGEMENTS The authors acknowledge financial support from Universidad de Buenos Aires (Secretaria de Ciencia y Tecnica) and International Foundation for Science. They are also grateful to Departamento de Fisica (Bajas Temperaturas), Facultad de Ciencias Exactas y Naturales for providing liquid air. REFERENCES Bell, L. N. & Hageman, M. J. (1994). Differentiating between the effects of water activity and glass transition dependent mobility on a solid state chemical reaction: aspartame degradation. J. Agric. Food Chem., 42, 2398-2401. Buera, M. P. & Karel, M. (1993). Application of the WLF equation to describe the combined effects of moisture and temperature on noncnzymatic browning rates in food systems. J. Food Proc. Pres., 17, 3 I-45.
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