`
`ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: https://www.tandfonline.com/loi/ljfp20
`
`Glass Transition and Crystallization of Amorphous
`Trehalose-sucrose Mixtures
`
`K.D. Roe & T.P. Labuza
`
`To cite this article: K.D. Roe & T.P. Labuza (2005) Glass Transition and Crystallization of
`Amorphous Trehalose-sucrose Mixtures, International Journal of Food Properties, 8:3, 559-574
`To link to this article: https://doi.org/10.1080/10942910500269824
`
`Copyright Taylor and Francis Group, LLC
`
`Published online: 06 Feb 2007.
`
`Submit your article to this journal
`
`Article views: 3947
`
`View related articles
`
`Citing articles: 41 View citing articles
`
`Full Terms & Conditions of access and use can be found at
`https://www.tandfonline.com/action/journalInformation?journalCode=ljfp20
`
`Amneal v. Cubist
`IPR2020-00193
`Cubist Ex. 2002
`
`
`
`International Journal of Food Properties, 8: 559–574, 2005
`Copyright © Taylor & Francis Inc.
`ISSN: 1094-2912 print / 1532-2386 online
`DOI: 10.1080/10942910500269824
`
`GLASS TRANSITION AND CRYSTALLIZATION OF
`AMORPHOUS TREHALOSE-SUCROSE MIXTURES
`
`K.D. Roe and T.P. Labuza
`Department of Food Science and Nutrition, University of Minnesota, St. Paul, USA
`
`Our objective was to investigate the glass transition and crystallization of trehalose-sucrose
`mixtures at various moisture contents. Samples were freeze-dried, rehumidified, and
`scanned with Differential scanning calorimetry (DSC) to obtain Tg values for all mixtures
`and pure sugars. Amorphous cotton candy samples for crystallization studies were pre-
`pared, humidified, and monitored for crystallinity as a function of time using powder X-ray
`diffraction (XRD). The Tg of pure dry trehalose was found to be 106 °C, while sucrose had
`a Tg of 60 °C. Glass transition, as expected, occurred at an intermediate temperature for
`sucrose-trehalose mixtures. Of the dry samples, only those containing less than 16% treha-
`lose showed sucrose crystallization during scanning. In cotton candy made from a 25% tre-
`halose-75% sucrose mixture, humidified to 33%, sucrose did not crystallize after 30 days,
`whereas pure sucrose cotton candy at that humidity crystallized completely after 11 days.
`These data show that trehalose may be a useful crystallization inhibitor in foods with high
`sucrose content, although small amounts of trehalose did not significantly raise the Tg.
`
`Keywords: Glass transition, Trehalose, Sucrose, Crystallization.
`
`INTRODUCTION
`
`Knowledge of and the ability to manipulate sucrose-water state data are important in
`the effort to increase shelf-life of high-sucrose containing foods, such as cotton candy and
`soft cookies. Detrimental texture changes, such as stickiness and collapse, in high-sucrose
`food systems often accompanies the crystallization of the glassy sugar.[1,2] Furthermore, it
`is known that the crystallization rates of glasses depend on the temperature in relation to
`the glass transition temperature, Tg. Amorphous systems subjected to temperatures and
`humidities that fall on a point above the glass transition line can crystallize. Those systems
`with a large (T-Tg) will crystallize faster than systems close to or below their Tg.[3] There-
`fore knowledge of the Tg line of pure or mixed sugar systems is critical to stability predic-
`tion. Trehalose has been touted as a special molecule used in nature to protect biological
`systems in dry or cold environments, perhaps by slowing crystallization.[4] Thus, due to
`the relatively high glass transition temperature of trehalose, amorphous sucrose-trehalose
`mixtures deserve attention from the high-sucrose foods industry. If the presence of treha-
`lose can sufficiently slow crystallization of sucrose under ordinary storage conditions,
`which can exceed Tg, shelf life can be extended.
`
`Address correspondence to T.P. Labuza, Dept. of Food Science and Nutrition, University of Minnesota,
`St. Paul, MN 55108, USA; E-mail: tplabuza@umn.edu
`
`559
`
`
`
`560
`
`ROE AND LABUZA
`
`Fig. 1 is a composite state diagram of the trehalose-water system based on data
`reported in available sources,[5–25] including those compiled previously by Chen et al.,
`2000.[26] The equilibrium lines shown include the liquidus line (freezing point depression
`line), and the solidus line (solubility or solute crystal melting line). The glass transition
`curve shows a large variation among different studies, up to ±20 °C at any given solids
`content. In these studies the dry Tg of trehalose falls between 75 and 120 °C (see Table 1).
`As seen in Fig. 1, at low moistures the Tg rises dramatically. Therefore, the range of values
`in Table 1 could be due to unmeasured, and thus unreported, residual moisture. The same
`phenomenon can be seen in the case of sucrose-water systems, as shown in Fig. 2, where
`Tg values taken from publications also do not agree well with each other,[3,12,20,22,24, 27–41]
`particularly in the driest region where the glass transition curve has a steep slope.
`Fig. 3 shows that in general the reported Tg curve for trehalose is higher than
`that of sucrose using only those references that reported either onset or midpoint or
`
`Figure 1 Literature values for the state diagram of the trehalose-water system, including the glass transition line.
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`561
`
`Table 1 Literature values for the glass transition temperature of dry amorphous trehalose and sucrose
`
`Reported Tg values in literature for pure,
`dry trehalose
`Tg (°C)
`
`Reference
`
`75
`79
`80
`85
`90
`100
`110
`112
`113.9
`114.9
`115
`115
`115
`116.9
`120
`
`Green and Angell (1989)
`Aldous et al (1995)
`Suzuki and Okazaki (1999)
`Cardona et al (1997)
`Saleki-Gerhardt (1993)
`Roos (1993)
`Crowe, J. et al (1996)
`Mehl (1997)
`Crowe, L. et al (1996)
`Miller et al (1997)
`Miller et al (1998)
`Ding et al (1996)
`Saleki-Gerhardt and Zografi (1994)
`Miller and de Pablo (2000)
`Sussich et al (1998)
`
`Reported Tg values in literature for pure,
`dry sucrose
`
`Tg (°C)
`
`52
`56.6
`57
`58.8
`59.6
`60.6
`62
`67.7
`68.5
`70
`70.2
`73.85
`74
`75.9
`
`Reference
`
`Slade and Levine (1988)
`Roos and Karel (1990)
`Finegold et al (1989)
`Suzuki and Okazaki (1999)
`Blond et al (1997)
`Blond et al (1997)
`Roos and Karel (1991a)
`Sun et al (1996)
`Urbani et al (1997)
`Orford et al (1990)
`Sun et al (1996)
`Hancock and Zografi (1994)
`Saleki-Gerhardt and Zografi (1994)
`Shamblin and Zografi (1999)
`
`both. In no case was the onset of glass transition for trehalose reported as less than the
`onset for sucrose. The Tg values for dry, amorphous sucrose reported in the literature
`is also listed in Table 1. As noted above, a factor contributing to inaccuracy in Tg ver-
`sus moisture reporting may arise from failure to remove all water from the dry sam-
`ples. In this case presumed dry samples would actually have moisture contents greater
`than that reported, and thus the reported Tgs would be lower than their true values.
`Using the Gordon-Taylor model for the trehalose-water system with values derived in
`Chen et al. (2000), (k = 5.2), the Tg falls more than 12 °C as the moisture content
`increases from 0% to 1%.
`A variety of methods have been reported for the drying of trehalose and sucrose
`samples, most of which involve freeze-drying over several days with an increasing tem-
`perature program. For trehalose, reports of a dry Tg between 105 and 120 °C—which con-
`stitute the upper range of the reported values—come from samples that were dried at high
`temperature (∼60 °C) during the final day of drying.[10,14,16,17,20,37] Typically, reports of Tg
`for trehalose below 105 °C were from studies where the samples were dried only at
`temperatures up to and including room temperature[3,7,22,24] or even just equilibrated over
`P2O5 at room temperature.[34] Because inadequate drying is the most prominent source of
`error in reporting a Tg versus moisture relationship, it follows logically that of the values
`reported in the literature the lower ones are less likely to be reliable, and the higher ones
`are perhaps more realistic.
`Several papers[3,7,20,22,37,42,43] have reported data for the moisture content of
`amorphous sucrose and trehalose as a function of water activity. Fig. 4 shows these
`data at 25 °C along with a linear regression. The purpose of this study was to evalu-
`ate the overall Tg for combined trehalose-sucrose mixtures, as well as the influence
`of relative humidity and added trehalose on the recrystallization of sucrose in these
`mixtures.
`
`
`
`562
`
`ROE AND LABUZA
`
`Figure 2 Literature values for the state diagram of the sucrose-water system, including the glass transition line.
`
`METHOD
`
`Freeze-drying
`
`Samples were prepared by first dissolving 10 grams of a pure crystalline sugar mix-
`ture in roughly 30 mL of distilled water, and poured into a plastic 40 mm diameter dish.
`The mixtures consisted of 0, 4, 8, 12, 16, 20, 40, 60, 80 and 100% trehalose mixtures with
`the balance being sucrose. Each dissolved mixture was rapidly frozen in liquid nitrogen
`and placed in a Dura-Top bulk tray dryer with a Dura-Dry condenser module (FTS Sys-
`tems, Inc., Stone Ridge, NY) running at 13.3 Pa vacuum, with a temperature program of −
`40 °C for 24 hours, −20 °C for 12 hours, 0 °C for 12 hours, 25 °C for 24 hours, followed
`by 50 °C for 36 hours. The high-temperature step was not done in the initial trials, result-
`ing in samples with some residual moisture.
`After drying, the samples were placed into four desiccators for re-humidification at
`25 °C. One set of samples were kept dry over P2O5 powder, while the other three sets were
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`563
`
`Figure 3 Trehalose-water and sucrose-water Tg data points from literature sources, as measured by DSC, showing
`onset and midpoint values as a function of solids percent.
`
`stored over the following saturated salt solutions: LiCl (aw = 0.11), CH3COOK (aw =
`0.23), and MgCl (aw = 0.33). Samples were equilibrated for 3 weeks.
`
`Cotton Candy Preparation
`
`Amorphous cotton candy mixtures were created using an Econo Floss Model 3017
`cotton candy machine (Gold Medal Products, Co., Cincinnati, OH). About 100 g of pure,
`crystalline sugar, or trehalose-sucrose mixtures, was heated in the machine’s sugar reser-
`voir. When necessary, in order to melt the anhydrous trehalose (Tm ∼215 °C.) additional
`heat was supplied by a space heater positioned above the sugar reservoir. All cotton candy
`samples were placed in cups and stored in desiccators humidified to 23%, 33%, and 54%
`relative humidity.
`
`Differential Scanning Calorimetry
`
`Glass transition temperatures was determined on a Perkin Elmer differential scan-
`ning calorimeter, model DSC 7 (Norwalk, CT). Roughly 10 to 20 mg of each sample from
`the various desiccators was transferred into aluminum pans, sealed, and scanned from −40 °C
`to 230 °C at a rate of 10 °C/min. All samples were prepared in a dry nitrogen glove box to
`minimize moisture absorption from the air. Samples that had undergone crystallization
`
`
`
`564
`
`ROE AND LABUZA
`
`Figure 4 Moisture sorption isotherm of amorphous trehalose and sucrose reported in literature (25 to 30°C.)
`
`during storage exhibited a visible change in volume and texture, and were not scanned.
`The onset of Tg was recorded. In all cases two replicates were made. Fig. 5 shows sample
`DSC plots for dry sucrose and trehalose. In some cases the Tg zone was accompanied by
`an enthalpic relaxation (ΔHrelax).
`This phenomenon occurs when the amorphous molecules reorient themselves to a
`lower energy amorphous state. Since this relaxation is made possible by the onset of glass
`transition, it does not affect the onset of glass transition. This assumption was confirmed with
`DSC by heating a sample to a temperature just above the Tg, and thereby allowing it to anneal,
`and then recooling it below the Tg, followed by again heating it above Tg. In this case the
`onset of Tg did not appear to change, but the peak associated with relaxation enthalpy disap-
`peared. Because enthalpic relaxation was neither influential nor vital to the focus of this study,
`no further attempts were made to suppress it. It should be clarified, however, that enthalpic
`relaxation can affect the midpoint of Tg (depending on how midpoint is determined.)
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`565
`
`Figure 5 DSC thermogram of amorphous, freeze-dried sucrose and trehalose.
`
`Moisture and Water Activity Determinations
`
`The water activity of each sample was done on an AquaLab model 3 TE (Decagon
`Devices, Inc., Pullman, WA). The moisture content was measured by the Karl Fischer
`titration method.[44]
`
`X-ray Diffraction
`
`X-ray diffraction (XRD) patterns were then taken periodically to monitor crystalliza-
`tion using a Seimens D5005 Powder X-ray diffractor. Scans went from 10 to 27 2θ degrees
`with a 4-second dwell every 0.04 degrees. To verify the identity of the crystalline sugar, the
`diffraction angle of measured peaks were compared to database values using Jade 5.0 analy-
`sis software. One problematic feature of powder X-ray diffraction is the appearance of one
`or two peaks in an otherwise amorphous sample. This is most likely due to the presence of a
`single, small crystal in the sample that never completely melted in the cotton candy-making
`process. When the sample as a whole begins to crystallize all of the characteristic peaks
`appear at once, not just one or two. In addition, upon crystallization of the bulk sample, the
`intensity of the amorphous halo, typically above 500 counts, begins to diminish.
`
`RESULTS AND DISCUSSION
`
`Fig. 6 shows the moisture sorption data for the freeze-dried trehalose-sucrose mix-
`tures as a function of water activity. If both sugars had the same sorption properties, the
`lines would be flat, but as seen adding trehalose, which binds more water (see Fig. 4), a
`slight increase in water content results. The slope of the sucrose data was roughly 15.0,
`
`
`
`566
`
`ROE AND LABUZA
`
`Figure 6 Moisture sorption data for amorphous, freeze-dried trehalose-sucrose mixtures at different relative
`humidities, based on Karl Fischer titration.
`
`and for trehalose the slope was 17.9. In comparison, data collected for moisture sorption
`of sucrose at 25 °C by Makower and Dye,[43] had a slope of about 18.5 (same units). The
`Saleki-Gerhardt,[45] sorption at 30 °C data showed a slope of 17.8 for sucrose and 22.0 for
`trehalose. Although moisture sorption is slightly temperature dependent, the difference
`between 25 and 30 °C may actually be less than the experimental error, and therefore data
`at these two temperatures are worth comparing. In general, given the problems in moisture
`measurement, our data agree well.
`As indicated previously in Fig. 5 the onset of Tg for dry sucrose and trehalose
`occurred at 61 and 107 °C, respectively. Based on previously reported data, these values
`are about 8 or 9 °C less than what was expected for those sugars (∼115 °C for trehalose
`and 70 °C for sucrose.) This could be due to the fact that even under the relatively rigorous
`drying conditions used, a small amount of moisture remained. Karl Fischer experiments
`confirmed a small but not negligible amount of moisture remained in the dry samples, par-
`ticularly the pure trehalose sample, which contained 0.36% water. Using data plotted in
`Fig. 3 as a guide, this moisture content could explain the slightly lower Tg value deter-
`mined from the DSC measurements. In this plot, a moisture content of roughly 0.5% cor-
`responds to a 10 °C drop in Tg.
`Fig. 7 shows the results of Tg determination of sample mixtures as a function of tre-
`halose-sucrose ratio for all runs done at each water activity. As expected the glass tra-
`nsition of the mixtures occurred at a temperature in between the Tgs of the pure
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`567
`
`Figure 7 Glass transition temperatures of amorphous, freeze-dried sucrose-trehalose mixtures at different water
`activities at 25°C.
`
`components, similar to what is found for carbohydrate-sugar mixtures.[46] In addition, the
`data appear to follow a roughly linear trend as a function of mixture composition at each
`water activity. At the highest humidity (aw = 0.36) samples with only 40% trehalose or
`less had Tgs lower than room temperature, and structural collapse was observed. The for-
`merly amorphous cake became a sticky residue with a reduced volume, as seen in Fig. 8.
`For the dry freeze-dried systems, Fig. 9 shows the influence of trehalose addition on
`sucrose crystallization during the DSC scan. The interesting feature of this data is that
`while the Tg of the mixture shifts only slightly with small amounts of trehalose added to
`the sucrose, the crystallization peaks are dramatically shifted to higher temperatures. Pure
`sucrose crystallizes with a peak at roughly 110 °C. With the addition of just 4% trehalose
`the peak shifts to about 135 °C and becomes less pronounced, while the 8% trehalose mix-
`ture had a peak at 150 °C. At 12% trehalose, the crystallization peak has already disap-
`peared, indicating some inhibitory effect of trehalose.
`A similar behavior was observed at a water activity of 0.15 (Fig. 10), but in this case
`some crystallization still occurs with 12% trehalose, and only at 16% is crystallization
`inhibited prior to melting. Given that sugar crystallization is accelerated at higher mois-
`ture, it is not surprising that more trehalose is required to inhibit it than for the dry sam-
`ples. It should be pointed out, however, that a sharp exothermic peak occurs at this water
`activity for pure trehalose, indicating that it recrystallized, while samples between 20%
`and 80% trehalose did not. This detail may be insightful, for it implies that perhaps
`
`
`
`568
`
`ROE AND LABUZA
`
`Figure 8 Freeze-dried trehalose-sucrose mixtures stored at 33% relative humidity for two weeks.
`
`Figure 9 DSC thermograms of the dry amorphous sucrose-trehalose mixtures, showing the influence of added
`trehalose on sucrose crystallization.
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`569
`
`Figure 10 DSC thermograms of the amorphous sucrose-trehalose mixtures, stored at 15% relative humidity,
`showing the influence of added trehalose on sucrose crystallization.
`
`crystallization is inhibited not only by the elevation of the glass transition temperature, but
`also by the mere fact that it is a mixture. Mazzobre et al.,[47] reported analogous data using
`mixtures of trehalose and lactose. The addition of trehalose, which has a dry Tg slightly
`higher than lactose (∼115 °C compared to ∼100 °C for lactose), caused little change in Tg
`at 33% RH, but greatly inhibited crystallization, as indicated by the shift in the exothermic
`crystallization peaks. These results further support the conclusion made above that a sugar
`added to another sugar may inhibit crystallization, regardless of the degree to which the
`glass transition is affected.
`As noted above, cotton candy samples were prepared with 0%, 25%, 50%, and
`100% trehalose, the balance sucrose, and stored in desiccators at 23%, 33%, and 54% rel-
`ative humidity. One concern in the preparation of the trehalose-sucrose cotton candy mix-
`tures was the possibility of a heterogeneous distribution of the two sugars. But based on
`the relative intensities of sucrose and trehalose peaks within individual cotton candy sam-
`ples, this did not occur. In the 50% mixture, the peaks corresponding to the pure compo-
`nent scans are present in equal proportion, and in the 25% trehalose mixture they are
`found in the corresponding proportion. This suggests that the sugar mixture is coming out
`of the cotton candy machine in the same proportion as the feed.
`Table 2 summarizes the time to crystallize for all samples. Representative X-ray dif-
`fraction patterns are shown in Figs. 11–15. No crystallinity was observed in any sample at
`
`
`
`570
`
`ROE AND LABUZA
`
`Table 2 Time to crystallize for sucrose-trehalose cotton candy samples stored at different relative humdities
`
`Storage
`Humidity
`
`23%
`33%
`54%
`
`100% sucrose
`
`>30 days
`11 days
`<48 hours
`
`75% sucrose
`25% trehalose
`
`50% sucrose
`50% trehalose
`
`>30 days
`>30 days
`<120 hours
`
`>30 days
`>30 days
`~120 hours
`
`100% trehalose
`
`>30 days
`>30 days
`~20 hours
`
`Figure 11 X-ray diffraction pattern of pure amorphous sucrose cotton candy after storage at 33% relative
`humidity and 25°C for 11 days, showing crystalline peaks.
`
`Figure 12 X-ray diffraction pattern of pure amorphous trehalose cotton candy after storage at 54% relative
`humidity and 25°C for 120 hours, showing crystalline peaks.
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`571
`
`Figure 13 X-ray diffraction pattern of an amorphous 50% sucrose-50% trehalose cotton candy mixture, after
`storage at 54% relative humidity and 25°C for 120 hours, showing crystalline peaks.
`
`Figure 14 X-ray diffraction pattern of an amorphous 75% sucrose-25% trehalose cotton candy mixture, after
`storage at 54% relative humidity and 25°C for 120 hours, showing crystalline peaks.
`
`23% RH, even after 30 days of storage, nor did the fluffy structure collapse. At 33% RH
`pure sucrose samples collapsed and crystallized within 11 days. XRD crystalline peaks for
`sucrose did not appear in the 25% trehalose samples at this humidity after 26 days,
`although some collapse of the structure occurred. At 54% RH, crystallization occurred in
`all samples, but notably faster in the pure sucrose sample.
`In all samples that underwent crystallization a collapse in the cotton candy structure
`occurred beforehand. This phenomenon was most clearly observed at 54% RH. In this
`case, after 18 hours, no samples exhibited crystallinity, but the 100% sucrose cotton candy
`had almost fully collapsed while the 100% trehalose cotton candy remained intact.
`“Collapse” entails an increased density of the sample and a corresponding decrease in
`
`
`
`572
`
`ROE AND LABUZA
`
`Figure 15 X-ray diffraction pattern of an amorphous 75% sucrose-25% trehalose cotton candy mixture, after
`storage at 23% relative humidity and 25°C for 30 days, showing retention of amorphous halo.
`
`volume. The collapsed structure is sticky until crystallization occurs, when it becomes
`brittle and dry. The implication of this observation is that crystallization cannot be the
`cause of collapse, but more likely the result since the pickup of water reduces local viscos-
`ity and induces faster diffusion.[3] These results show that the addition of trehalose to
`sucrose in cotton candy will both slow the collapse of the fluffy structure and delay crys-
`tallization. This is true even with only 25% trehalose.
`
`CONCLUSIONS
`
`The differential scanning calorimetry studies indicated that more than 20% dry
`weight fraction of trehalose is necessary to significantly inhibit the crystallization of
`pure amorphous sucrose. The results indicate that the effect is not just an increase in
`Tg, but may be due to diffusion resistance in the matrix. Due to the high price of treh-
`alose further economic analysis is required to determine if its use as an additive would
`be a practical shelf-life enhancer. Cotton candy samples with 25% or more of added
`trehalose stored at various humidities showed significantly slower crystallization than
`pure sucrose samples, as indicated by X-ray diffraction as well as visual observation
`of collapse. This fact is promising since, ultimately, it is texture that determines cot-
`ton candy shelf-life, not Tg. The next step will be to determine if this same phenome-
`non occurs in more complex, high-sucrose food systems, such as soft cookies, with
`the addition of trehalose.
`
`REFERENCES
`
`1. Adhikari, B.; Howes, T.; Bhandari, B.R.; Truong, V. Stickiness in foods: a review of mechanisms
`and test methods. International Journal of Food Properties 2001, 4 (1), 1–33.
`2. Panda, F.; Labuza, T.P. X-ray diffraction evaluation of sucrose crystallization in soft cookies.
`Journal of Food Science 2004, in press.
`
`
`
`CRYSTALLIZATION OF TREHALOSE-SUCROSE
`
`573
`
`3. Roos, Y.; Karel, M. Plasticizing effect of water on thermal behavior and crystallization of amor-
`phous food models. Journal of Food Science 1991, 34, 324–329.
`4. Roser, B. Trehalose, a new approach to premium dried foods. Trends in Food Science and
`Technology 1991, 2, 166–169.
`5. Addler, M.; Lee, G. Stability and surface activity of lactate dehydrogenase in spray-dried treha-
`lose. Journal of Pharmaceutical Sciences 1998, 88, 199–208.
`6. Aldous, B.J.; Auffret, A.D.; Franks, F. The crystallization of hydrates from amorphous carbohy-
`drates. Cryo-Lett 1995, 16, 181–186.
`7. Cardona, S.; Schebor, C.; Buera, M.P.; Karel, M.; Chirife, J. Thermal stability of invertase in
`reduced-moisture amorphous matrices in relation to glassy state and trehalose crystallization.
`Journal of Food Science 1997, 62, 105–112.
`8. Crowe, J.H.; Hoekstra, F.A.; Nguyen, K.H.N.; Crowe, L.M. Is vitrification involved depression
`of the phase transition temperature in dry phospholipids? Biochim. Biophys. Acta. 1996, 1280,
`187–196.
`9. Crowe, L.M.; Reid, D.S.; Crowe, J.H. Is trehalose special for preserving dry biomaterials? Bio-
`phys. J. 1996, 71, 2087–2093.
`10. Ding, S.-P.; Fan, J.; Green, J.L.; Lu, Q.; Sanchez, E.; Angell, C.A. Vitrification of trehalose by
`water loss from its crystalline hydrate. J. Therm. Anal. 1996, 47, 1391–1405.
`11. Green, J.L.; Angell, C.A. Phase relations and vitrification in saccharide-water solutions and the
`trehalose anomaly. J. Phys. Chem. 1989, 93, 2880–2882.
`12. Lammert, A.M.; Schmidt, S.J.; Day, G.A. Water activity and solubility of trehalose. Food
`Chemistry 1997, 61, 139–144.
`13. Mehl, P.M. Solubility and glass transition in the system α-d-trehalose/water. J. Therm. Anal.
`1997, 49, 817–822.
`14. Miller, D.P.; de Pablo, J.J. Calorimetric solution properties of simple saccharides and their sig-
`nificance for the stabilization of biological structure and function. J. Phys. Chem. B. 2000, 104,
`8876–8883.
`15. Miller, D.P.; de Pablo, J.J.; Corte, H. Viscosity and glass transition temperature of aqueous mix-
`tures of trehalose with borax and sodium chloride. J. Phys. Chem. B. 1999, 103, 10243–10249.
`16. Miller, D.P.; Anderson, R.E.; de Pablo, J.J. Stabilization of lactate dehydrogenase following
`freeze-thawing and vacuum-drying in the presence of trehalose and borate. Pharm. Res. 1998,
`15, 1215–1221.
`17. Miller, D.P.; de Pablo, J.J.; Corte, H. Thermophysical properties of trehalose and its concen-
`trated aqueous solutions. Pharm. Res. 1997, 14, 578–590.
`18. Nicolajsen, H; Hvidt, A. Phase behavior of the system trehalose-NaCl-water. Cryobiology 1994,
`31, 199–205.
`19. Roos, Y. Melting and glass transition of low molecular weight carbohydrates. Carbohydr. Res.
`1993, 238, 39–48.
`20. Saleki-Gerhardt, A.; Zografi, G. Non-isothermal and isothermal crystallization of sucrose from
`the amorphous state. Pharmaceutical Research 1994, 11, 1166–1173.
`21. Shafizadeh, F; Susott, R.A. Crystalline transitions of carbohydrates. J. Org. Chem. 1973, 38,
`3710–3715.
`22. Schebor, C.; Burin, L.; del, Pilar Buera M.; Chirife, J. Stability to hydrolysis and browning of
`trehalose, sucrose and raffinose in low-moisture systems in relation to their use as protectants of
`dry biomaterials. Lebensm.-Wiss. u.-Technol 1999, 32, 1–5.
`23. Sussich, F.; Urbani, R.; Princivalle, F.; Cesàro, A. Polymorphic amorphous and crystalline
`forms of trehalose. J. Am. Chem. Soc. 1998, 120, 7893–7899.
`24. Suzuki, T.; Okazaki, M. Thermal stabilizing effect of amorphous matrices of sugars on freeze-
`dried proteins. Bulletin of the Polish Academy of Sciences Technical Sciences 1999, 48, 415–427.
`25. Wang, G.M.; Haymet, A.D.J. Trehalose and other sugar solutions at low temperatures: modu-
`lated differential scanning calorimetry (MDSC). Journal of Physical Chemistry B 1998, 102
`(27), 5341–5347.
`
`
`
`574
`
`ROE AND LABUZA
`
`26. Chen, T.; Fowler, A.; Toner, M. Literature review: supplemented phase diagram of the treha-
`lose-water binary mixture. Cryobiology 2000, 40, 277–282.
`27. Ablett, S.; Izzard, M.J.; Lillford, P.J. Differential scanning calorimetry study of frozen sucrose
`and glycerol solutions. J. Chem. Soc. Faraday Trans. 1992, 88, 789–794.
`28. Blanshard, J.M.V.; Muhr, A.H.; Gough, A. Crystallization from concentrated sucrose solutions.
`Adv. Exp. Med. Biol. 1991, 302, 639–655.
`29. Blond, G.; Simatos, D.; Catte, M.; Dussap, C.G.; Gros, J.B. Modeling of the water-sucrose state
`diagram below 0 °C. Carbohydrate Research 1997, 298, 139–145.
`30. Finegold, L.; Franks, F.; Hatley, R.H.M. Glass/rubber transitions and heat capacities of binary
`sugar blends. J. Chem. Soc. Faraday Trans. 1989, 85, 2945–2951.
`31. Gayle, F.W.; Cocks, F.H.; Shepard, M.L. The H2O-NaCl-sucrose phase diagram and applica-
`tions in cryobiology. J. Appl. Chem. Biotechnol. 1977, 27, 599–607.
`32. Hancock, B.C.; Zografi, G. The relationship between glass transition temperature and the water
`content of amorphous pharmaceutical solids. Pharmaceutical Research 1994, 11, 471–477.
`33. Mackenzie, A.P. Non-equilibrium freezing behavior of aqueous systems. Phil. Trans. R. Soc.
`Lond. B. 1977, 278, 167–189.
`34. Orford, P.D.; Parker, R.; Ring, S.G. Aspects of the glass transition behavior of mixtures of car-
`bohydrates of low molecular weight. Carbohydr. Res. 1990, 196, 11–18.
`35. Roos, Y.; Karel, M. Amorphous state and delayed ice formation in sucrose solutions. Interna-
`tional Journal of Food Science and Technology 1991, 26, 553–566.
`36. Roos, Y.; Karel, M. Differential scanning calorimetry study of phase transitions affecting the
`quality of dehydrated materials. Biotechnol. Progress. 1990, 6, 159–163.
`37. Shamblin, S.L.; Zografi, G. The effect of absorbed water on the properties of amorphous mix-
`tures containing sucrose. Pharmaceutical Research 1999, 16, 1119–1124.
`38. Slade, L.; Levine, H. Non-equilibrium behavior of small carbohydrate-water systems. Pure &
`Appl. Chem 1988, 60, 1841–1864.
`39. Sun, W.Q.; Leopold, A.C.; Crowe, L.M.; Crowe, J.H. Stability of dry liposomesin sugar glasses.
`Biophys. J. 1996, 70, 1769–1776.
`40. Urbani, R.; Sussich, F.; Prejac, S.; Cesàro, A. Enthalpy relaxation and glass transition behaviour
`of sucrose by static and dynamic DSC. Thermochimica Acta 1997, 304/305, 359–367.
`41. Young, F.E.; Jones, F.T. Sucrose hydrates: the sucrose-water phase diagram. J. Phys. Colloid
`Chem. 1949, 53, 1334–1350.
`42. Iglesias, H.A.; Chirife, J.; Buera, M.P. Adsorption isotherm of amorphous trehalose. J. Sci.
`Food Agric. 1997, 75, 183–186.
`43. Makower, B.; Dye, W.B. Equilibrium moisture content and crystallization of amorphous
`sucrose and glucose. Agricultural and Food Chemistry 1956, 4, 72–77.
`44. Karmas, E. Measurement of moisture content. Cereal Foods World 1981, 26, 332.
`45. Saleki-Gerhardt, A. Role of Water in the Solid-State Properties of Crystalline and Amorphous
`Sugars. Ph.D. Thesis, University of Wisconsin-Madison, 1993.
`46. Biliaderis, C.G.; Lazaridou, A.; Mavropoulos, A.; Barbayiannis, N. Water plasticization effects on
`crystallization behavior of lactose in a co-lyophilized amorphous polysaccharide matrix and its rel-
`evance to the glass transition. International Journal of Food Properties 2002, 5 (2), 463–483.
`47. Mazzobre, M.F.; Soto, G.; Aguilera, J.M.; Buera, M.P. Crystallization kinetics of lactose in sys-
`tems co-lyofilized with trehalose. Analysis by differential scanning calorimetry. Food Research
`International 2001, 34, 903–911.
`
`