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DSC study of honey granulation stored at various temperatures
Food Research International, Vol. 30, No. 9, pp. 683±688, 1997 # 1998 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0963-9969(98)00030-1 0963-9969/97 $19.00+0.00
DSC study of honey granulation stored at various temperatures Cecilia Elena Lupano* Centro de InvestigacioÂn y Desarrollo en CriotecnologõÂa de Alimentos (CIDCA). Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP). Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). 47 y 116 (1900) La Plata, Argentina Non pasteurized, liquid and transparent honey containing very few and ®ne crystals was stored at 20, 10, 4 and ÿ20 C. The crystal growth was analysed by dierential scanning calorimetry (DSC) and by measuring the absorbance intensity at 660 nm over a 6 mth period. In honey stored at 20 C coarse crystals were formed with melting temperatures (Tm) between 45 and 65 C, whereas honey stored at ÿ20 C granulated as a ®nely-grained, fondant-like honey, with Tm between 25 and 45 C. In honeys stored at 10 and 4 C big and small crystals were produced having intermediate characteristics when compared with honeys stored at 20 and ÿ20 C. The rates of honey granulation were calculated from H values at the four temperatures. A linear relationship was found between the enthalpy of melting (H) and the absorbance at 660 nm. The activation energy for the melting of coarse crystals was also determined with DSC. # 1998 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: honey, dierential scanning calorimetry, honey granulation.
The phenomenon of honey granulation has long been acknowledged, but, although several works appeared in this subject (White Jr., 1978; Serra Bonhevi, 1989; Shinn and Wang, 1990; Assil et al., 1991), and DSC was utilized to study vitri®cation of glucose (Arvanitoyannis et al., 1993) or honey (Rubin et al., 1990), only very scarce information is available in the literature concerning the study of the crystal melting of honey by this technique. The objective of this work was to contribute to the understanding of the honey granulation process by using dierential scanning calorimetry. The granulation of honey consists of two processes: the formation of crystals and their gradual growth. It is well-known that the granulation rate and the crystal shape and size, depend on honey composition and storage temperature (Serra Bonhevi, 1989). Two opposite trends have been observed: when temperature decreases, the solubility of sugar decreases (Flink, 1983) thus favoring granulation, but at the same time viscosity increases (Johnson et al., 1975), retarding the mobility of molecules, thereby resulting in lower granulation rate. In the present study, the only variable is the storage temperature; dierent samples of the same honey were stored at 20, 10, 4 and ÿ20 C, in order to
INTRODUCTION When carbohydrates are heated in the solid state and/or in aqueous environments they undergo a series of interrelated physical transitions and chemical transformations. These transitions are manifested by changes in their physical properties including heat capacity, enthalpy and crystallinity. Dynamic thermal analysis methods, such as dierential scanning calorimetry (DSC), have proven powerful tools to probe the extent, rate and sequence of thermal events in both pure and complex carbohydrate systems (Biliaderis, 1990). Upon melting of a crystalline phase (®rst order transition) the thermodynamic functions show abrupt changes at the melting temperature (Tm) (Biliaderis, 1990; Rubin et al., 1990). Honey is a glucose supersaturated solution, and can granulate during storage. When granulated honey is heated, crystals melt and turn into a sugar solution. Changes produced in this process are usually analysed by DSC (Arvanitoyannis et al., 1993; Arvanitoyannis and Blanshard, 1994). *Fax: 54 21 25 4853; e-mail: [email protected] 683
684
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reproduce the room temperature and the temperatures of refrigerator and freezer. The rate of crystal growth and the type of crystals were analysed in each case by dierential scanning calorimetry, absorbance measurements at 660 nm and light microscopy. The kinetics of honey granulation was determined in honey stored at 4 C by a dynamic DSC method. MATERIALS AND METHODS Materials Honey utilized in this study was poly¯oral honey of the SE region of the province of Entre RõÂos (Argentina), provided by a beekeeper, harvested in January 1995. Honey was not pasteurized, and remained at room temperature (about 21 C) for three months prior to its storage at 20, 10, 4 and ÿ20 C. Single containers of honey were stored at each of the above temperatures for 6 mth and subsampled several times at room temperature during this period of time, reproducing the temperature variations that honey usually undergoes at home. Analytical Moisture was determined with an Abbe refractometer, calculating the moisture content by means of the Chataway table (FAO±WHO, 1969). Glucose, fructose and sucrose were determined by HPLC (Waters Associates, Milford, USA), using a column Accubond Amino SV (J & W Scienti®c) and a differential refractometer R 401 (Waters Associates, Milford, USA). The eluting solvent was acetonitrile: water 87:13. Light microscopy Honey stored at dierent temperatures was observed with a Leitz microscope (Wetzlar, Germany), at a magni®cation of 100. Dierential scanning calorimetry A Du Pont model 910 calorimeter calibrated with indium was used. Samples of 6±15 mg of honey were placed in weighed aluminum DSC hermetic pans, and an empty pan with four covers was used as reference. Sample and reference were heated between 0 and 100 C at a heating rate of 10 C minÿ1. The enthalpy of melting (H, Jgÿ1 honey) was computed from the endothermic peaks by means of the equation: ÿ1 ÿ1
H E A 60 Sx Sy w
where E=constant (mw/mV); A=area under the curve (cm2); 60 (s minÿ1); Sx=recorder chart speed ( C cmÿ1);
Sy=sensitivity of y axis (mV cmÿ1); w=weight of sample (mg); =heating rate ( C minÿ1). Each measure was performed at least three times. Although there exist various methodologies regarding the determination and calculation of activation energies (Ea) for melting (van Krevelen, 1990), Ozawa's (Ozawa, 1970) method was ®nally opted for determining the melting activation energy of coarse crystals. Samples of honey stored at 4 C were heated in the DSC at seven dierent rates ( ), between 2 and 21 C minÿ1. The peak temperature (Tm) was determined for each run. The temperature was calculated using indium as a reference for each heating rate applied. The Ea and the preexponential factor of the Arrhenius equation (Z) were calculated by plotting ÿln( /Tm2) vs 1/Tm, according to the equation: ln =Tm2 ln ZR=Ea ÿ Ea =RTm were =heating rate (K minÿ1); Tm=peak temperature (K); Z=pre-exponential factor of the Arrhenius equation (minÿ1); R=gas constant (cal molÿ1 Kÿ1). A least squares minimum linear regression was applied to 33 experimental points, selecting ÿln( /Tm2) as the independent variable (Pravisani et al., 1985). Absorbance of honey Honey was put into a cuvette with a path-length of 1 cm. The absorbance was measured at 660 nm with a spectrophotometer Beckman DU-650 (Beckman Instruments, Fullerton, California, USA). RESULTS AND DISCUSSION Characterization of honey Honey was liquid and homogeneous with few and very ®ne crystals before being stored at dierent temperatures. Glucose and fructose contents were 34.4 and 38.3%, respectively, and water percentage was 17.6%, w/w. Thus, the resulting glucose:water ratio was 1.95. Values of 1.7 or less are usually associated with nongranulating honeys; whereas values higher than 2.1 predict that the honey will rapidly granulate to a solid state unless preventive measures are taken (White, 1974). The honey used in this study was of an intermediate glucose:water ratio. The relation (glucose±water)/fructose was 0.44. These data correspond to a honey that will granulate more or less rapidly, according to the storage conditions. Thermograms of honey stored at dierent temperatures Figure 1 shows some thermograms of honey stored at 20, 10, 4 and ÿ20 C. At least one endothermic peak was observed with the maximum lying between 25 and 65 C.
DSC study of honey granulation stored at various temperatures The more granulated honey was the greater the recorded area. In several cases, two superimposed endothermic peaks were observed: one of them with a maximum between 25 and 45 C, and the other with a maximum between 45 and 65 C. These peaks correspond to the melting of crystals. The presence of two peaks indicates that there would be more than one type of crystals in these samples, with dierent melting temperatures. When honey was stored at 20 C the ®rst peak was practically absent, and the endothermic transitions were observed at temperatures higher than 45 C (Fig. 1(A)). Crystals with melting points between 25 and 35 C would not be present in this sample because the storage temperature was close to this temperature range. This honey granulated with coarse crystals, as was observed directly by optical microscopy (Fig. 2(A)). These results suggest that big-size crystals correspond to endothermic peaks with melting points higher than 45 C. The sample stored at 10 C showed two superimposed endothermic peaks: the ®rst with a maximum between 25 and 45 C, and the second with a maximum between 45 and 55 C (Fig. 1(B)), thus indicating that more than one type of crystals is present. The last peak was sharper
685
than the peak observed in the honey stored at 20 C, and became greater with storage time, whereas the ®rst peak became less pronounced. Honey stored at 10 C had a less sandy appearance than honey stored at 20 C, and granulated with crystals of various sizes (Fig. 2(B)). Honey stored at 4 C also showed two endothermic peaks, one of them with the maximum between 25 and 40 C and the other one with the maximum between 40 and 55 C. As was observed in sample stored at 10 C, the second peak became gradually greater at longer storage times, whereas the ®rst peak practically disappeared (Fig. 1(C)). These results indicate that the second peak corresponds to more stable crystals than the ®rst one. Peaks obtained from honey stored at 4 C were substantially sharper than peaks obtained from honey stored at other temperatures, thereby suggesting that crystals coming from the former sample are more homogeneous. This honey in particular granulated mainly with small crystals and only a low number of big-size crystals was observed (Fig. 2(C)). Honey stored at ÿ20 C showed very wide and not well de®ned peaks at short storage times, but after 70 days of storage at ÿ20 C, a peak with a maximum between 25 and 45 C was observed, being gradually enhanced at longer storage times. This honey granulated as a ®ne-grained, fondant-like honey, and very ®ne crystals were observed (Fig. 2(D)). DSC traces show that ®ne crystals correspond to the endothermic peaks with the maximum between 25 and 45 C. H of honey stored at dierent temperatures
Fig. 1. Dierential scanning calorimetry thermograms of honey stored at dierent temperatures: (A) 20 C; (B) 10 C; (C) 4 C; (D) ÿ20 C. Storage time: (a) 0 days; (b) 31±32 days; (c) 45 days; (d) 66±68 days; (e) 108±116 days; (f) 173 days.
Figure 3 shows the H values obtained when honey stored at various temperatures was heated in the DSC apparatus, vs storage time. It was observed that H rapidly increased with storage time in samples stored at 4 and 10 C, whereas the increase was much slower in samples stored at 20 C and even more at ÿ20 C. These results are in agreement with a previous publication acccording to which honey granulation is accelerated between 13 and 15.5 C, whereas granulation is retarded at freezer temperatures (White, 1974). It is well known that the viscosity of liquids decreases as temperature increases (Johnson et al., 1975). Thus, the viscosity of honey stored at 20 C is expected to be lower than that of honey at 10, 4 or ÿ20 C, and the higher mobility of molecules favors the crystal growth. However, the solubility of sugar increases with temperature (Flink, 1983) and, as can be seen from Fig. 1, the ®rst peak of melting begins to appear at 25 C. This result can explain the slow granulation rate observed at 20 C. This sample contains only crystals solubilized at temperatures higher than 45 C, as previously discussed (Figs 1(A) and 2(A)). On the other hand, honey stored at ÿ20 C would present a higher viscosity and a reduced molecular mobility thus leading to slower crystal growth.
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Fig. 2. Light microscopy of honey stored at dierent temperatures: (a) 20 C; (b) 10 C; (c) 4 C; (d) ÿ20 C.
Determination of the rate constants for honey granulation Bearing in mind that H values constitute an expression of honey granulation, and assuming that kinetics of order one with respect to time is predominant, the rate constants for honey granulation at dierent tempera-
tures were calculated from the slopes of the straight lines obtained from plotting ln(Hf-H) vs time, where Hf is the average of the maximum H of honey stored at 4 and 10 C. The obtained values and the correlation coecients are shown in Table 1. As shown from Fig. 3, rate constants were found to be very similar at 10 and 4 C, and higher than the respective constants at 20 and ÿ20 C. Although the granulation rate calculated by this method appears to be practically the same at 4 and 10 C, and at 20 and ÿ20 C as well, the crystal size was shown to be greatly dependent on the storage temperature. It should be taken into account that these values correspond to a unique honey sample stored at the above mentioned conditions, but results indicate that it is possible to estimate the rate constants for honey granulation by this method. Absorbance at 660 nm of honey stored at dierent temperatures
Fig. 3. Enthalpies of crystal melting of honey stored at dierent temperatures, as a function of the storage time. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C. Each data point is the average of at least three measures. The bars show standard deviation.
Turbidity of honey increases with granulation, and an intensity increase in the absorbance at 660 nm is considered a valid measure of determining the granulation extent. The same wavelength was previously employed for analysing turbidity in gels (Shimada and Cheftel, 1988; Lupano, 1994). Figure 4 shows the absorbance at 660 nm of honey stored at 20, 10, 4 and ÿ20 C as a function of storage time. The absorbance increased very
DSC study of honey granulation stored at various temperatures
687
Table 1. Rate constants for honey granulation at dierent storage temperatures Storage temperature coecient ( C) 20 10 4 ÿ20
Rate constant (daysÿ1)
Correlation
0.0046 0.0235 0.0237 0.0031
ÿ0.92 ÿ0.89 ÿ0.98 ÿ0.96
Fig. 5. Enthalpies of crystal melting of honey stored at dierent temperatures vs the absorbance of honey at 660 nm. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C.
whereas the H of honey stored at ÿ20 C to ®ne crystals. It is possible that ®ne crystals have lower H values than coarse ones, and turbidity is rather produced by ®ne crystals, the surface of which is exposed to a greater extent, thereby resulting in higher turbidity values than those produced by coarse crystals endowed with a smaller exposed surface. Activation energy (Ea) for the crystal melting The Ea for the melting of coarse crystals was calculated by the method of Ozawa, using honey stored at 4 C. Fig. 4. Absorbance at 660 nm of honey stored at dierent temperatures, as a function of the storage time. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C.
rapidly in samples stored at 10 and 4 C, and more slowly in samples stored at 20 and ÿ20 C similarly to the increase in H values (Fig. 3). Correlation between H and absorbance at 660 nm A linear relationship was shown to exist between H and the absorbance at 660 nm at all storage temperatures (Fig. 5). Honey stored at 20, 10 and 4 C, and the lower values of honey stored at ÿ20 C can be represented by the same straight line, with a correlation coecient r=0.96. Honey stored at ÿ20 C gave lower H values than honey stored at the other temperatures in the absorbance range of 1.6 to 3.0 (Fig. 5). These data had a linear correlation coecient r=0.98. In this sample the peak of lower melting point became progressively greater in the DSC traces, whereas the second peak disappeared at longer storage times. The ®rst peak is thought to correspond to small crystals whereas the second peak to coarse ones, as previously reported. Honeys stored at 20, 10 and 4 C mainly showed the second peak. Therefore, the H values of these samples should correspond to the presence of coarse crystals,
Fig. 6. Calculation of the activation energy (Ea) for the melting of coarse crystals in honey using dierential scanning calorimetry. =heating rate (K minÿ1), Tm=peak temperature (K).
688
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This sample was primarily selected because of its sharp peaks in conjunction with their clearly de®ned Tm. The selected peaks correspond to coarse crystals. The value obtained for Ea was 72.3 Kcal molÿ1, with a correlation coecient of 0.91, and the preexponential factor of the Arrhenius equation (Z) was 4.141049 minÿ1 (Fig. 6). The estimated value of Ea is in satisfactory agreement with other Ea values (according to Ozawa method) for other events that usually occur during the heating of foods, such as protein denaturation (Wagner and AnÄoÂn, 1985; Lupano and AnÄoÂn, 1986) and starch gelatinization (Pravisani et al., 1985). CONCLUSIONS Dierential scanning calorimetry appears to be an appropriate tool to analyse the granulation of honey. The peak temperature in the thermograms seems to be closely related to the crystal size: melting temperatures between 25 and 45 C should correspond to ®negrained honey, whereas melting temperatures between 45 and 65 C to coarse-grained honey. A linear relationship between the enthalpy of melting and the absorbance at 660 nm was established. The Ea required for melting coarse crystals in honey was within the same magnitude order to that required for protein denaturation and starch gelatinization. ACKNOWLEDGEMENTS The author would thank to E.M.B. GoÂmez for supplied honey samples and for water determination, to R. Ferreira for HPLC analysis, and to A. Colavita for photograph copies. This investigation was supported by the Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). C.E.L. is a member of the Researcher Career of the Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). REFERENCES Arvanitoyannis, I., Blanshard, J. M. V., Ablett, S., Izzard, M. J. and Lillford, P. J. (1993) Calorimetric study of the glass transition occurring in aqueous glucose:fructose solutions. J. Sci. Food Agric. 63, 177±188.
Arvanitoyannis, I. and Blanshard, J. M. V. (1994) Rates of crystallization of dried lactose±sucrose mixtures. J. Food Sci. 59, 197±205. Assil, H. I., Sterling, R. and Sporns, P. (1991) Crystal control in processed liquid honey. J. Food Sci. 56, 1034±1037, 1041. Biliaderis, C. G. (1990) Thermal analysis of food carbohydrates. In Thermal Analysis of Foods, eds V. R. Harwalkar and C.-Y. Ma, pp. 168±220. Elsevier Applied Science, Cambridge. FAO±WHO (1969) Norma Regional Europea recomendada para la miel. Codex Alimentarius Commission. Flink, J. M. (1983) Structure and structure transitions in dried carbohydrate materials. In Physical Properties of Foods, eds M. Peleg and E. B. Bagley, pp. 473±521. Avi Publishing Company, Westport, Connecticut. Johnson, J. F., Martin, J. R. and Porter, R. S. (1975) Determination of viscosity of food systems. In Theory, Determination and Control of Physical Properties of Food Materials, Ed. ChoKyun Rha, pp. 25±38. D. Reidel Publishing Company, Dordrecht-Holland. Lupano, C. E. (1994) Eect of heat treatments in very acidic conditions on whey protein isolate properties. J. Dairy Sci. 77, 2191±2198. Lupano, C. E. and AnÄoÂn, M. C. (1986) Denaturation of wheat germ proteins during drying. Cereal Chem. 63, 259±262. Ozawa, T. (1970) Kinetic analysis of derivative curves in thermal analysis. J. Thermal Anal. 2, 301. Pravisani, C. I., Califano, A. N. and Calvelo, A. (1985) Kinetics of starch gelatinization in potato. J. Food Sci. 50, 657±660. Rubin, C. A., Wasylyk, J. M. and Baust, J. G. (1990) Investigation of vitri®cation by nuclear magnetic resonance and dierential scanning calorimetry in honey: a model carbohydrate system. J. Agric. Food Chem. 38, 1824±1827. Serra Bonhevi, J. (1989) Estudio de la validez de los õÂndices que predicen la cristalizacioÂn de la miel. Rev. AgroquõÂm. Tecnol. Aliment. 29, 47±62. Shimada, K. and Cheftel, J. C. (1988) Texture characteristics, protein solubility, and sulphydryl group/disul®de bond contents of heat-induced gels of whey protein isolate. J. Agric. Food Chem. 36, 1018±1025. Shinn, J. M. and Wang, S. L. (1990) Textural analysis of crystallized honey using response surface methodology. Can. Inst. Food Sci. Technol. J. 23, 178±182. van Krevelen (1990) Properties of polymers, 3rd edn. pp. 585± 623. Elsevier, Amsterdam. Wagner, J. R. and AnÄoÂn, M. C. (1985) Denaturation kinetics of myo®brillar proteins in bovine muscle. J. Food Sci. 50, 1547±1550, 1563. White, J. W. (1974) Beekeeping: honey and honey products. In Encyclopedia of Food Technology, eds A. H. Johnson and M. S. Peterson, Vol. 2, pp. 103±108. Westport: AVI. White, J. W., Jr. (1978) Honey. Adv. Food Res. 24, 339±343
(Received 5 December 1997; accepted 11 March 1998)
DSC study of honey granulation stored at various temperatures Cecilia Elena Lupano* Centro de InvestigacioÂn y Desarrollo en CriotecnologõÂa de Alimentos (CIDCA). Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP). Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). 47 y 116 (1900) La Plata, Argentina Non pasteurized, liquid and transparent honey containing very few and ®ne crystals was stored at 20, 10, 4 and ÿ20 C. The crystal growth was analysed by dierential scanning calorimetry (DSC) and by measuring the absorbance intensity at 660 nm over a 6 mth period. In honey stored at 20 C coarse crystals were formed with melting temperatures (Tm) between 45 and 65 C, whereas honey stored at ÿ20 C granulated as a ®nely-grained, fondant-like honey, with Tm between 25 and 45 C. In honeys stored at 10 and 4 C big and small crystals were produced having intermediate characteristics when compared with honeys stored at 20 and ÿ20 C. The rates of honey granulation were calculated from H values at the four temperatures. A linear relationship was found between the enthalpy of melting (H) and the absorbance at 660 nm. The activation energy for the melting of coarse crystals was also determined with DSC. # 1998 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: honey, dierential scanning calorimetry, honey granulation.
The phenomenon of honey granulation has long been acknowledged, but, although several works appeared in this subject (White Jr., 1978; Serra Bonhevi, 1989; Shinn and Wang, 1990; Assil et al., 1991), and DSC was utilized to study vitri®cation of glucose (Arvanitoyannis et al., 1993) or honey (Rubin et al., 1990), only very scarce information is available in the literature concerning the study of the crystal melting of honey by this technique. The objective of this work was to contribute to the understanding of the honey granulation process by using dierential scanning calorimetry. The granulation of honey consists of two processes: the formation of crystals and their gradual growth. It is well-known that the granulation rate and the crystal shape and size, depend on honey composition and storage temperature (Serra Bonhevi, 1989). Two opposite trends have been observed: when temperature decreases, the solubility of sugar decreases (Flink, 1983) thus favoring granulation, but at the same time viscosity increases (Johnson et al., 1975), retarding the mobility of molecules, thereby resulting in lower granulation rate. In the present study, the only variable is the storage temperature; dierent samples of the same honey were stored at 20, 10, 4 and ÿ20 C, in order to
INTRODUCTION When carbohydrates are heated in the solid state and/or in aqueous environments they undergo a series of interrelated physical transitions and chemical transformations. These transitions are manifested by changes in their physical properties including heat capacity, enthalpy and crystallinity. Dynamic thermal analysis methods, such as dierential scanning calorimetry (DSC), have proven powerful tools to probe the extent, rate and sequence of thermal events in both pure and complex carbohydrate systems (Biliaderis, 1990). Upon melting of a crystalline phase (®rst order transition) the thermodynamic functions show abrupt changes at the melting temperature (Tm) (Biliaderis, 1990; Rubin et al., 1990). Honey is a glucose supersaturated solution, and can granulate during storage. When granulated honey is heated, crystals melt and turn into a sugar solution. Changes produced in this process are usually analysed by DSC (Arvanitoyannis et al., 1993; Arvanitoyannis and Blanshard, 1994). *Fax: 54 21 25 4853; e-mail: [email protected] 683
684
C. E. Lupano
reproduce the room temperature and the temperatures of refrigerator and freezer. The rate of crystal growth and the type of crystals were analysed in each case by dierential scanning calorimetry, absorbance measurements at 660 nm and light microscopy. The kinetics of honey granulation was determined in honey stored at 4 C by a dynamic DSC method. MATERIALS AND METHODS Materials Honey utilized in this study was poly¯oral honey of the SE region of the province of Entre RõÂos (Argentina), provided by a beekeeper, harvested in January 1995. Honey was not pasteurized, and remained at room temperature (about 21 C) for three months prior to its storage at 20, 10, 4 and ÿ20 C. Single containers of honey were stored at each of the above temperatures for 6 mth and subsampled several times at room temperature during this period of time, reproducing the temperature variations that honey usually undergoes at home. Analytical Moisture was determined with an Abbe refractometer, calculating the moisture content by means of the Chataway table (FAO±WHO, 1969). Glucose, fructose and sucrose were determined by HPLC (Waters Associates, Milford, USA), using a column Accubond Amino SV (J & W Scienti®c) and a differential refractometer R 401 (Waters Associates, Milford, USA). The eluting solvent was acetonitrile: water 87:13. Light microscopy Honey stored at dierent temperatures was observed with a Leitz microscope (Wetzlar, Germany), at a magni®cation of 100. Dierential scanning calorimetry A Du Pont model 910 calorimeter calibrated with indium was used. Samples of 6±15 mg of honey were placed in weighed aluminum DSC hermetic pans, and an empty pan with four covers was used as reference. Sample and reference were heated between 0 and 100 C at a heating rate of 10 C minÿ1. The enthalpy of melting (H, Jgÿ1 honey) was computed from the endothermic peaks by means of the equation: ÿ1 ÿ1
H E A 60 Sx Sy w
where E=constant (mw/mV); A=area under the curve (cm2); 60 (s minÿ1); Sx=recorder chart speed ( C cmÿ1);
Sy=sensitivity of y axis (mV cmÿ1); w=weight of sample (mg); =heating rate ( C minÿ1). Each measure was performed at least three times. Although there exist various methodologies regarding the determination and calculation of activation energies (Ea) for melting (van Krevelen, 1990), Ozawa's (Ozawa, 1970) method was ®nally opted for determining the melting activation energy of coarse crystals. Samples of honey stored at 4 C were heated in the DSC at seven dierent rates ( ), between 2 and 21 C minÿ1. The peak temperature (Tm) was determined for each run. The temperature was calculated using indium as a reference for each heating rate applied. The Ea and the preexponential factor of the Arrhenius equation (Z) were calculated by plotting ÿln( /Tm2) vs 1/Tm, according to the equation: ln =Tm2 ln ZR=Ea ÿ Ea =RTm were =heating rate (K minÿ1); Tm=peak temperature (K); Z=pre-exponential factor of the Arrhenius equation (minÿ1); R=gas constant (cal molÿ1 Kÿ1). A least squares minimum linear regression was applied to 33 experimental points, selecting ÿln( /Tm2) as the independent variable (Pravisani et al., 1985). Absorbance of honey Honey was put into a cuvette with a path-length of 1 cm. The absorbance was measured at 660 nm with a spectrophotometer Beckman DU-650 (Beckman Instruments, Fullerton, California, USA). RESULTS AND DISCUSSION Characterization of honey Honey was liquid and homogeneous with few and very ®ne crystals before being stored at dierent temperatures. Glucose and fructose contents were 34.4 and 38.3%, respectively, and water percentage was 17.6%, w/w. Thus, the resulting glucose:water ratio was 1.95. Values of 1.7 or less are usually associated with nongranulating honeys; whereas values higher than 2.1 predict that the honey will rapidly granulate to a solid state unless preventive measures are taken (White, 1974). The honey used in this study was of an intermediate glucose:water ratio. The relation (glucose±water)/fructose was 0.44. These data correspond to a honey that will granulate more or less rapidly, according to the storage conditions. Thermograms of honey stored at dierent temperatures Figure 1 shows some thermograms of honey stored at 20, 10, 4 and ÿ20 C. At least one endothermic peak was observed with the maximum lying between 25 and 65 C.
DSC study of honey granulation stored at various temperatures The more granulated honey was the greater the recorded area. In several cases, two superimposed endothermic peaks were observed: one of them with a maximum between 25 and 45 C, and the other with a maximum between 45 and 65 C. These peaks correspond to the melting of crystals. The presence of two peaks indicates that there would be more than one type of crystals in these samples, with dierent melting temperatures. When honey was stored at 20 C the ®rst peak was practically absent, and the endothermic transitions were observed at temperatures higher than 45 C (Fig. 1(A)). Crystals with melting points between 25 and 35 C would not be present in this sample because the storage temperature was close to this temperature range. This honey granulated with coarse crystals, as was observed directly by optical microscopy (Fig. 2(A)). These results suggest that big-size crystals correspond to endothermic peaks with melting points higher than 45 C. The sample stored at 10 C showed two superimposed endothermic peaks: the ®rst with a maximum between 25 and 45 C, and the second with a maximum between 45 and 55 C (Fig. 1(B)), thus indicating that more than one type of crystals is present. The last peak was sharper
685
than the peak observed in the honey stored at 20 C, and became greater with storage time, whereas the ®rst peak became less pronounced. Honey stored at 10 C had a less sandy appearance than honey stored at 20 C, and granulated with crystals of various sizes (Fig. 2(B)). Honey stored at 4 C also showed two endothermic peaks, one of them with the maximum between 25 and 40 C and the other one with the maximum between 40 and 55 C. As was observed in sample stored at 10 C, the second peak became gradually greater at longer storage times, whereas the ®rst peak practically disappeared (Fig. 1(C)). These results indicate that the second peak corresponds to more stable crystals than the ®rst one. Peaks obtained from honey stored at 4 C were substantially sharper than peaks obtained from honey stored at other temperatures, thereby suggesting that crystals coming from the former sample are more homogeneous. This honey in particular granulated mainly with small crystals and only a low number of big-size crystals was observed (Fig. 2(C)). Honey stored at ÿ20 C showed very wide and not well de®ned peaks at short storage times, but after 70 days of storage at ÿ20 C, a peak with a maximum between 25 and 45 C was observed, being gradually enhanced at longer storage times. This honey granulated as a ®ne-grained, fondant-like honey, and very ®ne crystals were observed (Fig. 2(D)). DSC traces show that ®ne crystals correspond to the endothermic peaks with the maximum between 25 and 45 C. H of honey stored at dierent temperatures
Fig. 1. Dierential scanning calorimetry thermograms of honey stored at dierent temperatures: (A) 20 C; (B) 10 C; (C) 4 C; (D) ÿ20 C. Storage time: (a) 0 days; (b) 31±32 days; (c) 45 days; (d) 66±68 days; (e) 108±116 days; (f) 173 days.
Figure 3 shows the H values obtained when honey stored at various temperatures was heated in the DSC apparatus, vs storage time. It was observed that H rapidly increased with storage time in samples stored at 4 and 10 C, whereas the increase was much slower in samples stored at 20 C and even more at ÿ20 C. These results are in agreement with a previous publication acccording to which honey granulation is accelerated between 13 and 15.5 C, whereas granulation is retarded at freezer temperatures (White, 1974). It is well known that the viscosity of liquids decreases as temperature increases (Johnson et al., 1975). Thus, the viscosity of honey stored at 20 C is expected to be lower than that of honey at 10, 4 or ÿ20 C, and the higher mobility of molecules favors the crystal growth. However, the solubility of sugar increases with temperature (Flink, 1983) and, as can be seen from Fig. 1, the ®rst peak of melting begins to appear at 25 C. This result can explain the slow granulation rate observed at 20 C. This sample contains only crystals solubilized at temperatures higher than 45 C, as previously discussed (Figs 1(A) and 2(A)). On the other hand, honey stored at ÿ20 C would present a higher viscosity and a reduced molecular mobility thus leading to slower crystal growth.
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Fig. 2. Light microscopy of honey stored at dierent temperatures: (a) 20 C; (b) 10 C; (c) 4 C; (d) ÿ20 C.
Determination of the rate constants for honey granulation Bearing in mind that H values constitute an expression of honey granulation, and assuming that kinetics of order one with respect to time is predominant, the rate constants for honey granulation at dierent tempera-
tures were calculated from the slopes of the straight lines obtained from plotting ln(Hf-H) vs time, where Hf is the average of the maximum H of honey stored at 4 and 10 C. The obtained values and the correlation coecients are shown in Table 1. As shown from Fig. 3, rate constants were found to be very similar at 10 and 4 C, and higher than the respective constants at 20 and ÿ20 C. Although the granulation rate calculated by this method appears to be practically the same at 4 and 10 C, and at 20 and ÿ20 C as well, the crystal size was shown to be greatly dependent on the storage temperature. It should be taken into account that these values correspond to a unique honey sample stored at the above mentioned conditions, but results indicate that it is possible to estimate the rate constants for honey granulation by this method. Absorbance at 660 nm of honey stored at dierent temperatures
Fig. 3. Enthalpies of crystal melting of honey stored at dierent temperatures, as a function of the storage time. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C. Each data point is the average of at least three measures. The bars show standard deviation.
Turbidity of honey increases with granulation, and an intensity increase in the absorbance at 660 nm is considered a valid measure of determining the granulation extent. The same wavelength was previously employed for analysing turbidity in gels (Shimada and Cheftel, 1988; Lupano, 1994). Figure 4 shows the absorbance at 660 nm of honey stored at 20, 10, 4 and ÿ20 C as a function of storage time. The absorbance increased very
DSC study of honey granulation stored at various temperatures
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Table 1. Rate constants for honey granulation at dierent storage temperatures Storage temperature coecient ( C) 20 10 4 ÿ20
Rate constant (daysÿ1)
Correlation
0.0046 0.0235 0.0237 0.0031
ÿ0.92 ÿ0.89 ÿ0.98 ÿ0.96
Fig. 5. Enthalpies of crystal melting of honey stored at dierent temperatures vs the absorbance of honey at 660 nm. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C.
whereas the H of honey stored at ÿ20 C to ®ne crystals. It is possible that ®ne crystals have lower H values than coarse ones, and turbidity is rather produced by ®ne crystals, the surface of which is exposed to a greater extent, thereby resulting in higher turbidity values than those produced by coarse crystals endowed with a smaller exposed surface. Activation energy (Ea) for the crystal melting The Ea for the melting of coarse crystals was calculated by the method of Ozawa, using honey stored at 4 C. Fig. 4. Absorbance at 660 nm of honey stored at dierent temperatures, as a function of the storage time. Storage temperatures: (*) 20 C; (*) 10 C; (~) 4 C; (!) ÿ20 C.
rapidly in samples stored at 10 and 4 C, and more slowly in samples stored at 20 and ÿ20 C similarly to the increase in H values (Fig. 3). Correlation between H and absorbance at 660 nm A linear relationship was shown to exist between H and the absorbance at 660 nm at all storage temperatures (Fig. 5). Honey stored at 20, 10 and 4 C, and the lower values of honey stored at ÿ20 C can be represented by the same straight line, with a correlation coecient r=0.96. Honey stored at ÿ20 C gave lower H values than honey stored at the other temperatures in the absorbance range of 1.6 to 3.0 (Fig. 5). These data had a linear correlation coecient r=0.98. In this sample the peak of lower melting point became progressively greater in the DSC traces, whereas the second peak disappeared at longer storage times. The ®rst peak is thought to correspond to small crystals whereas the second peak to coarse ones, as previously reported. Honeys stored at 20, 10 and 4 C mainly showed the second peak. Therefore, the H values of these samples should correspond to the presence of coarse crystals,
Fig. 6. Calculation of the activation energy (Ea) for the melting of coarse crystals in honey using dierential scanning calorimetry. =heating rate (K minÿ1), Tm=peak temperature (K).
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This sample was primarily selected because of its sharp peaks in conjunction with their clearly de®ned Tm. The selected peaks correspond to coarse crystals. The value obtained for Ea was 72.3 Kcal molÿ1, with a correlation coecient of 0.91, and the preexponential factor of the Arrhenius equation (Z) was 4.141049 minÿ1 (Fig. 6). The estimated value of Ea is in satisfactory agreement with other Ea values (according to Ozawa method) for other events that usually occur during the heating of foods, such as protein denaturation (Wagner and AnÄoÂn, 1985; Lupano and AnÄoÂn, 1986) and starch gelatinization (Pravisani et al., 1985). CONCLUSIONS Dierential scanning calorimetry appears to be an appropriate tool to analyse the granulation of honey. The peak temperature in the thermograms seems to be closely related to the crystal size: melting temperatures between 25 and 45 C should correspond to ®negrained honey, whereas melting temperatures between 45 and 65 C to coarse-grained honey. A linear relationship between the enthalpy of melting and the absorbance at 660 nm was established. The Ea required for melting coarse crystals in honey was within the same magnitude order to that required for protein denaturation and starch gelatinization. ACKNOWLEDGEMENTS The author would thank to E.M.B. GoÂmez for supplied honey samples and for water determination, to R. Ferreira for HPLC analysis, and to A. Colavita for photograph copies. This investigation was supported by the Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). C.E.L. is a member of the Researcher Career of the Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas (CONICET). REFERENCES Arvanitoyannis, I., Blanshard, J. M. V., Ablett, S., Izzard, M. J. and Lillford, P. J. (1993) Calorimetric study of the glass transition occurring in aqueous glucose:fructose solutions. J. Sci. Food Agric. 63, 177±188.
Arvanitoyannis, I. and Blanshard, J. M. V. (1994) Rates of crystallization of dried lactose±sucrose mixtures. J. Food Sci. 59, 197±205. Assil, H. I., Sterling, R. and Sporns, P. (1991) Crystal control in processed liquid honey. J. Food Sci. 56, 1034±1037, 1041. Biliaderis, C. G. (1990) Thermal analysis of food carbohydrates. In Thermal Analysis of Foods, eds V. R. Harwalkar and C.-Y. Ma, pp. 168±220. Elsevier Applied Science, Cambridge. FAO±WHO (1969) Norma Regional Europea recomendada para la miel. Codex Alimentarius Commission. Flink, J. M. (1983) Structure and structure transitions in dried carbohydrate materials. In Physical Properties of Foods, eds M. Peleg and E. B. Bagley, pp. 473±521. Avi Publishing Company, Westport, Connecticut. Johnson, J. F., Martin, J. R. and Porter, R. S. (1975) Determination of viscosity of food systems. In Theory, Determination and Control of Physical Properties of Food Materials, Ed. ChoKyun Rha, pp. 25±38. D. Reidel Publishing Company, Dordrecht-Holland. Lupano, C. E. (1994) Eect of heat treatments in very acidic conditions on whey protein isolate properties. J. Dairy Sci. 77, 2191±2198. Lupano, C. E. and AnÄoÂn, M. C. (1986) Denaturation of wheat germ proteins during drying. Cereal Chem. 63, 259±262. Ozawa, T. (1970) Kinetic analysis of derivative curves in thermal analysis. J. Thermal Anal. 2, 301. Pravisani, C. I., Califano, A. N. and Calvelo, A. (1985) Kinetics of starch gelatinization in potato. J. Food Sci. 50, 657±660. Rubin, C. A., Wasylyk, J. M. and Baust, J. G. (1990) Investigation of vitri®cation by nuclear magnetic resonance and dierential scanning calorimetry in honey: a model carbohydrate system. J. Agric. Food Chem. 38, 1824±1827. Serra Bonhevi, J. (1989) Estudio de la validez de los õÂndices que predicen la cristalizacioÂn de la miel. Rev. AgroquõÂm. Tecnol. Aliment. 29, 47±62. Shimada, K. and Cheftel, J. C. (1988) Texture characteristics, protein solubility, and sulphydryl group/disul®de bond contents of heat-induced gels of whey protein isolate. J. Agric. Food Chem. 36, 1018±1025. Shinn, J. M. and Wang, S. L. (1990) Textural analysis of crystallized honey using response surface methodology. Can. Inst. Food Sci. Technol. J. 23, 178±182. van Krevelen (1990) Properties of polymers, 3rd edn. pp. 585± 623. Elsevier, Amsterdam. Wagner, J. R. and AnÄoÂn, M. C. (1985) Denaturation kinetics of myo®brillar proteins in bovine muscle. J. Food Sci. 50, 1547±1550, 1563. White, J. W. (1974) Beekeeping: honey and honey products. In Encyclopedia of Food Technology, eds A. H. Johnson and M. S. Peterson, Vol. 2, pp. 103±108. Westport: AVI. White, J. W., Jr. (1978) Honey. Adv. Food Res. 24, 339±343
(Received 5 December 1997; accepted 11 March 1998)