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Color changes during storage of honeys in relation to their composition and initial color
Food Research International 32 (1999) 185±191
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Color changes during storage of honeys in relation to their composition and initial color Adriana Pereyra Gonzales a, Leila Burin b, MarõÂa del Pilar Buera b,*,1 Departamento de QuiÂmica OrgaÂnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina b Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina
a
Received 11 March 1999; accepted 22 April 1999
Abstract The causes of darkening in honey have been attributed to Maillard reaction, fructose caramelization and reactions of polyphenols, however, no systematic studies exist on this subject. The in¯uence of composition and initial color on the rate of darkening of several Argentine honeys submitted to storage at 37 C during 90 days was evaluated through spectrocolorimetric measurements. The most suitable color functions to evaluate darkening of honeys [lightness (Lab*), browning index (BI), metric chroma (Cab*), metric hue (Hab*) and 1/Z] increased linearly as storage time increased, after an initial induction period of very low browning development. The slope of the linear browning development zone with time was an index of browning rate, and it was analyzed in relation to the initial color and the composition of honeys (moisture content, total nitrogen, total lipids and polyunsaturated fatty acids, fructose and glucose content). Of the analyzed variables, the initial color was the parameter which better de®ned the rate of darkening of honeys. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. Keywords: Honey; Color; Darkening
1. Introduction The color of honey is one of the factors determining its price on the world market, and also its acceptability by the consumers. Light honeys are usually mild in ¯avor and of a higher commercial value than dark colored honeys (Wootton, Edwards, Faraji-Haremi & Johnson, 1976; Wootton, Edwards & Faraji-Haremi, 1976; White, 1978). Argentina is the third world producer of honey, which represents 62,000 ton per year (Nimo, 1998). During shipping to far countries and/or during storage, darkening of honey may occur, and parallel changes in its organoleptic properties have detrimental eects on its quality, masking its original aroma, which promotes loss of competitiveness in the world market (Milum, 1939; Aubert & Gonnet, 1983). * Corresponding author. Tel.: +54-11-4576-3397; fax: +56-114576-3366. E-mail address: [email protected] (M.P. Buera) 1 Member of Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas de la RepuÂblica, Argentina.
The rate of darkening has been related to the composition of honey and of the storage temperature (White, 1978; Gupta, Kaushik & Joshi, 1992). Of the compositional factors, the ratio of glucose to fructose, nitrogen content, free aminoacids, moisture content have been cited as possible factors determining the rate of darkening (Lynn, Englis & Milum, 1936; Schade, Marsh & Eckert, 1958). Lynn et al., (1936) indicated that the main causes of darkening in honey could be: (a) reaction aminoacidaldol (Maillard reaction); (b) combination of tannates and other oxydated polyphenols with ferrum salts; (c) instability of fructose (caramelization reaction). However, there is still controversy over the relative in¯uence of these factors on the darkening of honey. While Ramsay and Milum (1933) stated that the Maillard reaction was the main cause of darkening, Lynn et al. (1936) indicated that it was only a secondary factor. Milum (1948) observed that darkening during storage depended on the initial color of the honey. Despite the known incidence of the commercial value of the color of honeys, and of the occurrence of darkening
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00075-7
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A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
during storage, literature related to investigate the main causes of darkening is scarce. Wootton, Edwards, FarajiHaremi and Johnson (1976) and Wootton, Edwards and Faraji-Haremi (1976) analyzed the changes in chemical composition of six Australian honeys and reported that the amount of sugars and free aminoacids in them were not related to the extent of darkening after storage of the honeys at 50 C. Schade et al. (1958) indicated that the rate of honey darkening may increase as increasing the moisture content increases. The content and type of natural polyphenols (such as ¯avonoids) in¯uence the color of fresh honeys (Chandler, Fenwick, Orlova & Reynolds, 1974), and their degradation reactions may also cause color changes during storage. The objective of this present work was to analyze the rate of darkening of several Argentine honeys of multi¯oral origin as a function of their main components (glucose, fructose, total nitrogen, moisture content and lipids), and of their initial color, during storage at 37 C.
Luminosity: L*ab L*ab =116 (Y/Yn)1/3ÿ16 a* =500 [(X/Xn)1/3ÿ(Y/Yn)1/3] b* =200[(Y/Yn)1/3ÿ(Z/Zn)1/3]
2. Materials and methods
where: u0n 0:2009; v0n 0:461; Xn 98:041; Yn 100:00 y Zn 118:103, are the values calculated for illuminant C and at the 2 angle observer. : dierence between the values corresponding to the sample at time t and t=0. Browning index (BR) (Buera et al., 1985) was also calculated as:
2.1. Samples Sixteen ¯oral honeys from dierent geographic plain regions of temperate climate from Argentina, two from woody temperate areas and one from a tropical region, were analyzed. The samples were provided by ocial institutions or private producers with guarantee of genuiness and known history. Composition of honeys had been determined in a previous work, as described by Bertoni, Pereyra Gonzales and CattaÂneo, (1994). Moisture content was determined by AOAC (1980) method 31.111. Glucose and fructose were evaluated as described by Ugarte and Karman (1945). Total nitrogen by AOAC 2.24 (1950) method in semimicro scale. Total lipids were analyzed using the Folch, Lee and Sloane Stanley, (1957) method. Fatty acid composition as described in Pereyra, Gonzales, Bertoni, Gros & CattaÂneo, (1994). 2.2. Color measurements Measurements were done in a Hunterlab 5100 (Hunter Associates Laboratory Fairfax, VA) spectrocolorimeter, with white background in Plexiglass 5.3 cm diameterer sample holders, with a sample thickness of 4 mm. The illumination mode illuminated the aperture area (4.7 cm). The color functions, which in previous experiences have been proved to be adequated to follow the development of browning pigments (Buera, Petriella & Lozano, 1985; Buera & Resnik, 1989) were calculated for illuminant C, and the 2 angle observer, through the tristimulus values X, Y, Z, taking as standard values those of the white background (X=79.01; Y=83.96; Z=86.76). The following equations were employed (Lozano, 1977):
Metric chroma: C*ab C*ab =(a*2+b*2)1/2 Color dierence CIE 1976: E*ab E*ab =[(L*ab)2+(a*)2+(b*)2]1/2 Metric hue dierence: H*ab H*ab=[(E*ab)2ÿ(L*ab)2ÿ(C*ab)2]1/2 Metric saturation: suv suv =13[(u0 ÿu0 n)2+(v0 ÿv0 n)2]1/2 0 u =4X/(X+15 Y+3 Z) v0 =9Y/(X+15 Y+3 Z)
BR 100 x ÿ 0:31=0:172; where x cromatic coordinate X= X Y Z Before placing the samples in the cuvette for color measurement, they were placed at 50 C for 30 min in a water bath to decrease their viscosity and to allow the measurement of the volume with a syringe, carefully avoiding air bubbles. It was demonstrated, in a preliminary study, that this initial treatment of the samples did not alter their color. Illumination was done from the bottom side of the samples. The standard white plate was placed over the cuvette which contained the sample. The standard deviation of the color measurements, estimated from 20 determinations of the same sample was 2.5% and 5 measurements of each sample were necessary to obtain an error lower than 3% at a con®dence level of 95%. 2.3. Storage The samples were distributed in aliquots in hermetically closed containers of high density polyethylene. They were stored in forced circulation ovens maintained at a constant temperature 37(1) C during adequate periods of time, after which colorimetric determinations were done, over a total period of 90 days.
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
3. Results and discussion Fig. 1(a) shows the initial color of honeys in the CIE chromaticity diagram, in which the tristimulus values are limited to a plane, determined by the x and y chromatic coordinates. Most of the samples are located on a line to which corresponds a dominant wavelength of 575 nm, as observed for samples of yellow to light-brown colors obtained through non-enzymic browning reactions (Buera et al., 1985), and also observed by Aubert and Gonnet (1983) from spectrophotometric measurements of honeys. The darker honeys were located in the descending part of the curve, and showed a displacement of the dominant wavelengh to the red zone of the chromaticity diagram. Fig. 1(b) shows the CIE chromaticity coordinates of the honeys, after 90 days of storage at 37 C. It can be seen that a displacement of
Fig. 1. Scheme of the CIE chromaticity diagrams showing the initial (a) and the ®nal (b) color of honeys.
187
the dominant wavelengh occurred for all the honeys, to the red zone of the diagram. The CIE tristimulus values allowed an objective approach to color grading, de®ning a picture of the chromaticity (yellow-red) of the samples, which is not possible by the visual classi®cation of honey by the ocial Pfund method from light to dark (Aubert & Gonnet, 1983). During storage at 37 C the darkening of the samples could be followed by any of the color functions which were previously related to browning development: suv, Hab*, IB and 1/Z (Buera et al., 1985; Buera & Resnik, 1989). As shown in Fig. 2 for the color function Hab* after an initial induction period, in which no browning occurred, it followed a period of linear increase of color with time. This behavior was observed in many products subjected to non-enzymatic browning (Song & Chichester, 1966; Labuza & Saltmarch, 1980). The samples included in Fig. 1 were chosen in such a way to obtain a representative picture of all the analyzed samples, covering the whole range of color changes and browning rates observed. The slope of the linear part of the curves was calculated, and considered as an index of the rate of browning, as a pseudo zero-order rate coecient. Table 1 shows the correlation coecients resulting from the linear correlation between the color functions and storage time data, obtained by least squares. The rate of darkening could be followed by any of the selected color functions (the rate coecients for the metric hue development is reported in Table 1). The induction period for browning development at 37 C, calculated as the value of the abscise for which the ordinate was zero, was between 10 and 30 days, and the values were also included in Table 1.
Fig. 2. Evolution of the color function Hab*, with storage time at 37 C for some honeys, representative of the whole range of color changes.
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Table 1 Correlation coecients (r2) for the linear regression between some of the color functions and storage time data at 37 C, obtained by least squaresa Honey
suv
BR
Lab
1/Z
Cab
Hab
k(Hab units/day)
I (days)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
0.9391 0.9674 0.9755 0.8623 0.9622 0.9105 0.9472 0.9673 0.9113 0.9675 0.9766 0.9632 0.9858 0.9589 0.9819 0.9295 0.7674 0.9362 0.9876
0.9434 0.9657 0.9354 0.8132 0.9245 0.9372 0.9472 0.9267 0.9139 0.9325 0.9123 0.8954 0.9543 0.9216 0.9325 0.9325 0.9432 0.8997 0.9354
0.9542 0.9391 0.9261 0.8817 0.9303 0.9811 0.8931 0.9815 0.9873 0.9625 0.9946 0.9600 0.9428 0.9604 0.9979 0.9778 0.8060 0.9837 0.9546
0.8868 0.9311 0.8341 0.7918 0.9539 0.9523 0.9748 0.9485 0.8460 0.9210 0.9056 0.9350 0.9431 0.9963 0.9353 0.9853 0.7977 0.8960 0.8990
0.9321 0.9625 0.8461 0.8490 0.9402 0.8381 0.9561 0.9554 0.8821 0.9623 0.9569 0.9614 0.9979 0.7214 0.9671 0.9427 0.8707 0.9189 0.9143
0.9316 0.9419 0.8547 0.8889 0.9193 0.9555 0.9654 0.8759 0.8885 0.9373 0.9690 0.9644 0.9769 0.9386 0.9881 0.9768 0.7214 0.9110 0.9089
0.104 0.0795 0.121 0.121 0.765 0.1397 0.0380 0.0992 0.126 0.0292 0.161 0.090 0.139 0.3722 0.124 0.149 0.5337 0.173 0.21
27.6 25.6 0 31.9 30.6 19.2 25.9 30.2 28.0 11.9 15.7 21.6 20.3 21.2 0 3.6 36.9 15.3 12.6
a The slope of the linear part of the curves (k) and the induction period (I) for browning development were calculated for the Hab* color function. I was calculated as the X-intercept.
To investigate the variable which most in¯uenced the rate of darkening, the slopes of the linear part of the curves were analyzed in relation to the main components of the honeys, and also to their initial color. Fig. 3 shows the dependence of the calculated browning rates (k) with some of the analyzed variables. Of the analyzed variables, the rate of darkening of honeys had no dependence with the compositional variables, and the initial color (IC) seemed to be the best parameter to de®ne browning rate. Metric saturation (suv), a color function de®ned above (see Materials and Methods section) is related to the purity of color and was selected as an index of the initial color value of honeys. suv has been found to be an adequate function to follow color changes in transparent products yellow to brown colorations (Petriella, Resnik, Lozano & Chirife, 1985; Buera et al., 1985). Fig. 3(a) shows that the rate of browning did not have any correlation with the concentration of the main sugar components of honey (glucose and fructose). At the acidic pH value of the honey (3.8±4.5), fructose is more reactive than glucose towards browning development (Buera, Resnik & Petriella, 1992), but as both sugars are in a large extent and the variability between samples was small, the concentration of fructose was not a limiting factor for browning development. Amino compounds play an important role in interactions involving reducing sugars. Wootton, Edwards, Faraji-Haremi and Johnson (1976) and Wootton, Edwards and Faraji-Haremi, (1976) indicated that although total nitrogen content was relatively unaected by storage at 50 C, the total free aminoacid content decreased, but the changes in free aminoacids were not related to the extent of darkening. Due to the low content of free aminoacids in honey, it
could be expected that, if the amino-sugar condensation was an important cause of honey darkening, nitrogen concentration must be considered a limiting factor and the rate of browning should be related to the initial nitrogen content. However, the correlation between browning rate and nitrogen content was poor (Fig. 3b). These results indicated that, contrary to the suggestions of Ramsay and Milum (1933) and in agreement with Lynn et al. (1936) the amino-sugar condensation, and the total nitrogen content had only a secondary eect on the darkening of honeys. The instability of fructose may be one important cause of discoloration, but the high concentration of this sugar and the narrow range of variability between dierent samples of honeys did not allow correlation of the rate of darkening to fructose concentration. Although during extended storage, lipids may generate carbonilic compounds through oxidation and promote browning (Taoukis & Labuza, 1996), the rate of darkening was neither aected by the total lipid content (Fig. 3c), nor by the individual concentrations of the unsaturated fatty acids linoleic (18:2) or linolenic (18:3) [the correlation coecients (r2) were lower than 0.2, not shown]. Schade et al. (1958) indicated that the rate of honey darkening may increase as the moisture content increases, but as shown in Fig. 3d, the water content was not a variable which in¯uenced clearly the browning rate. Wootton, Edwards, Faraji-Haremi and Johnson, (1976) reported that none of the changes observed during their study on storage of Australian honeys (color, acidity and total nitrogen content) were related to the initial moisture content, in spite of the known importance of moisture levels in browning reactions. When the rate of darkening was plotted as a func-
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
189
Fig. 3. Dependence of the calculated browning rates (k, in Hab* units/day) with some of the analyzed variables: (a) glucose and fructose content (in g/100 g of honey); (b) total nitrogen (N, in g/100 g of honey); (c) total lipids (in g/100 g of honey); (d) moisture content (in g H2O/100 g of honey).
tion of initial color (IC) of honeys, a better correlation than that obtained for the compositional variables was found (Fig. 4). The rate coecients, and also the induction periods were analyzed in relation to the compositional variables and initial color by stepwise regression analysis of data (Statistix for windows analytical software was employed). The best subset regression models that contained all the potential predictor variables were ®rst evaluated. To obtain a more complete picture of the dependence, some transformations in the variables were applied, such as squares or logarithms, and relationships between variables, which could account for interactions between factors, were also analyzed. None of the transformations proved, gave better correlation than the single variable ``initial color'' (IC). The results con®rmed that the initial color was the variable which most in¯uenced the browning rate, as observed in Fig. 3. The equation obtained through linear correlation by stepwise regression combined the variable IC and IC2 as follows:
Fig. 4. Correlation between the calculated browning rates (k, in Hab* units/day) and initial color (suv, metric saturation) obtained by stepwise linear regression of data.
k 0:1894 ÿ 0:3849 IC 0:3177 IC2 The curve predicted for this equation is shown in Fig. 4 as a dotted line. The square correlation coecient between observed and predicted values (r2 ) was 0.8018 for a p<0.06.
It was observed in model systems subjected to nonenzymatic browning reactions (Petriella et al., 1985) that the induction period is inversely proportional to the rate coecient for darkening. In the present study,
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however, the induction period did not have any correlation either with the initial color (r2=0.46), or with the analyzed compositional variables (the best correlation obtained from a stepwise regression analysis had as r2 of 0.49 for a p<0.05). Fig. 5 shows that the chromatic coordinate x for the samples before storage (xi) and after storage for 90 days at 37 C (xf) had a good linear correlation. A similar dependence was observed for the chromatic coordinate y and other color functions (not shown). These results con®rmed that the ®nal color was strongly related to the initial color of honeys. Milum (1948) had reported that the rate of darkening in honeys during any given period of heating or storage was related to the amount of previous discoloration during the processing of honeys at high temperatures or during storage. However, the honeys analyzed in the present study had not been subjected to high temperatures and they were stored in similar conditions after they were produced, before the storage experiment at 37 C. It is to be noted that the honey in which the rate of darkening was the highest, was one from a woody temperate region, and had also the darkest initial color. The ¯oral origin, which is a variable that strongly aects the initial color of honeys (Aubert & Gonnet, 1983), the polyphenolic components (not analyzed in present paper) and their deteriorative reactions, may be important factors in determining the stability of the color of honeys during storage. It is known that the polyphenolic components form brown complexes with amino acids and proteins (Labuza and Schmidl, 1986). Quinones produced by oxidation of polyphenols are reactive substances in some stages of the complex reactions leading to browning (Pokorny, 1980). Enzymatic activities in honeys (not subjected to thermal treatment), may aect polyphenols and are not discarded as browning promotors.
Fig. 5. Correlation between the chromatic coordinate x for the samples before storage (xi) and after storage during 90 days at 37 C (xf).
Acknowledgements The authors acknowledge ®nancial support from Universidad de Buenos Aires and CONICET.
References A.O.A.C. (1980). Ocial methods of analysis of the Association of Ocial Agricultural Chemists, 7th edition. A.O.A.C. (1980). Ocial methods of analysis of the Association of Ocial Agricultural Chemists, 13th edition. Aubert, S., & Gonnet, M. (1983). Measure de la couleur des miels. Apidologie, 14, 105±118. Bertoni, M. H., Pereyra Gonzales, A., & CattaÂneo, P. (1994). Estudios sobre mieles de abeja. II Sobre contenido en glucosa, fructosa, sus relaciones y nitroÂgeno total. Anales AsociacioÂn Quimica Argentina, 82, 231±236. Buera, M. P., Petriella, C., & Lozano, R. D. (1985). De®nition of colour in the non-enzymatic browning. Die Farbe, 33, 316±326. Buera, M. P., & Resnik, S. (1989). Colorimetric measurements in a turbid medium: hydrolyzed concentrated cheese whey. Die Farbe, 36, 201±213. Buera, M. P., Resnik, S. L., & Petriella, C. (1992). Relative browning rate of glucose and fructose. Lebensmittel Wissenschaft und Technologie, 25, 35±37. Chandler, B. V., Fenwick, D., Orlova, T., & Reynolds, T. (1974). Composition of Australian honeys. Technical Paper. Division Food. Research. Australia: CSIRO. Folch, J., Lee, M., & Sloane Stanley, G. H. (1957). Journal of Biological Chemistry, 226, 49±53. Gupta, J. K., Kaushik, R., & Joshi, V. K. (1992). In¯uence of dierent treatments, storage temperature end period on some physico-chemical characteristics and sensory qualitoes of Indian honey. Journal of Food Science and Technology-India, 29, 84±87. Labuza, T. P., & Saltmarch, M. (1980). The non-enzymatic browning reactions as aected by water in foods. In L. B. Rockland, & G. F. Steward, Properties of water related to food quality and stability (pp. 605±650). Academic Press: San Francisco. Labuza, T. P., & Schimdl, M. K. (1986). Advances in the control of browning reactions in foods. In O. R. Fennema, W. H. Chang, & C. Y. Lii, Role of chemistry in the quality of processed food (pp. 65±95). Westport, CT: Food and Nutrition Press. Lozano, R. D. (1977). Evaluation of dierent color-dierence formulae by means of an experiment on color scaling. Color Research and Application, 2, 13±18. Lynn, E. G., Englis, D. T., & Milum, V. G. (1936). Eect of processing and storage on composition and color of honey. Food Research, 1, 23±27. Milum, V. G. (1939). Honey discoloration and loss of delicate ¯avor in processing. American Bee Journal, 17, 390±392,416. Milum, V. G. (1948). Some factors aecting the color of honey. Journal of Economic Entomology, 41, 495±505. Nimo, M. (1998). Batiendo records. Alimentos Argentinos, March, 75. Pereyra Gonzales, A., Bertoni, M. H., Gros, E. G., & CattaÂneo, P. (1994). On the fatty acid composition of bee's honey lipid fraction. Saturated, unsaturated and dicarboxylic acids from C10 to C18.. Anales de la AsociacioÂn QuiÂmica Argentina, 82, 43±51. Pokorny, J. (1980). Eect of browning reactions on the formation of ¯avour substances. Die Nahrung, 24, 115±127. Petriella, C., Resnik, S., Lozano, R. D., & Chirife, J. (1985). Kinetics of deteriorative reactions in model food systems of high water activity: color changes due to non-enzymatic browning. Journal of Food Science, 50, 622±626.
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191 Ramsey, R. J., & Milum, V. G. (1993). The discoloration of honey. American Bee Journal, 73, 305±306. Schade, J. W., Marsh, G. L., & Eckert, J. E. (1958). Diastase activity and hydroxymethylfurfural in honey and their in¯uence in detecting heat alteration. Food Research, 23, 446±463. Song, P. S., & Chichester, C. O. (1966). Kinetic behavior and mechanism of inhibition in the Maillard reaction. II. Mechanistics considerations in the reaction between D-glucose and glycine. Journal of Food Science, 31, 914±919. Taoukis, P., & Labuza, T. (1996). Summary: integrative concepts. In R. Owen, & E. D. Fennema, Food Chemistry (pp. 1013±1042). New York: Marcel Dekker Inc. (3rd ed.). Ugarte, L., & Karman, G. (1945). Determination of the ratio glucose/ levulose in Argentine honeys. Anales AsociacioÂn QuiÂmica Argentina, 33, 181.
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White, J. W. (1978). Honey. Advances in Food Research, 24, 287±371. White, J. W., Kushnir, I., & Subers, M. H. (1964). Eect of storage and processing temperatures on honey quality. Food Technology 555-558. Wootten, M., Edwards, R. A., Faraji-Haremi, R., & Johnson, A. T. (1976). Eect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 1. Colour, acidity and total nitrogen content. Journal of Apicultural Research, 15, 23± 28. Wootten, M., Edwards, R. A., & Faraji-Haremi, R. (1976). Eect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 2. Changes in sugar and free amino acid contents. Journal of Apicultural Research, 15, 29±34.
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Color changes during storage of honeys in relation to their composition and initial color Adriana Pereyra Gonzales a, Leila Burin b, MarõÂa del Pilar Buera b,*,1 Departamento de QuiÂmica OrgaÂnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina b Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina
a
Received 11 March 1999; accepted 22 April 1999
Abstract The causes of darkening in honey have been attributed to Maillard reaction, fructose caramelization and reactions of polyphenols, however, no systematic studies exist on this subject. The in¯uence of composition and initial color on the rate of darkening of several Argentine honeys submitted to storage at 37 C during 90 days was evaluated through spectrocolorimetric measurements. The most suitable color functions to evaluate darkening of honeys [lightness (Lab*), browning index (BI), metric chroma (Cab*), metric hue (Hab*) and 1/Z] increased linearly as storage time increased, after an initial induction period of very low browning development. The slope of the linear browning development zone with time was an index of browning rate, and it was analyzed in relation to the initial color and the composition of honeys (moisture content, total nitrogen, total lipids and polyunsaturated fatty acids, fructose and glucose content). Of the analyzed variables, the initial color was the parameter which better de®ned the rate of darkening of honeys. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. Keywords: Honey; Color; Darkening
1. Introduction The color of honey is one of the factors determining its price on the world market, and also its acceptability by the consumers. Light honeys are usually mild in ¯avor and of a higher commercial value than dark colored honeys (Wootton, Edwards, Faraji-Haremi & Johnson, 1976; Wootton, Edwards & Faraji-Haremi, 1976; White, 1978). Argentina is the third world producer of honey, which represents 62,000 ton per year (Nimo, 1998). During shipping to far countries and/or during storage, darkening of honey may occur, and parallel changes in its organoleptic properties have detrimental eects on its quality, masking its original aroma, which promotes loss of competitiveness in the world market (Milum, 1939; Aubert & Gonnet, 1983). * Corresponding author. Tel.: +54-11-4576-3397; fax: +56-114576-3366. E-mail address: [email protected] (M.P. Buera) 1 Member of Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas de la RepuÂblica, Argentina.
The rate of darkening has been related to the composition of honey and of the storage temperature (White, 1978; Gupta, Kaushik & Joshi, 1992). Of the compositional factors, the ratio of glucose to fructose, nitrogen content, free aminoacids, moisture content have been cited as possible factors determining the rate of darkening (Lynn, Englis & Milum, 1936; Schade, Marsh & Eckert, 1958). Lynn et al., (1936) indicated that the main causes of darkening in honey could be: (a) reaction aminoacidaldol (Maillard reaction); (b) combination of tannates and other oxydated polyphenols with ferrum salts; (c) instability of fructose (caramelization reaction). However, there is still controversy over the relative in¯uence of these factors on the darkening of honey. While Ramsay and Milum (1933) stated that the Maillard reaction was the main cause of darkening, Lynn et al. (1936) indicated that it was only a secondary factor. Milum (1948) observed that darkening during storage depended on the initial color of the honey. Despite the known incidence of the commercial value of the color of honeys, and of the occurrence of darkening
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00075-7
186
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
during storage, literature related to investigate the main causes of darkening is scarce. Wootton, Edwards, FarajiHaremi and Johnson (1976) and Wootton, Edwards and Faraji-Haremi (1976) analyzed the changes in chemical composition of six Australian honeys and reported that the amount of sugars and free aminoacids in them were not related to the extent of darkening after storage of the honeys at 50 C. Schade et al. (1958) indicated that the rate of honey darkening may increase as increasing the moisture content increases. The content and type of natural polyphenols (such as ¯avonoids) in¯uence the color of fresh honeys (Chandler, Fenwick, Orlova & Reynolds, 1974), and their degradation reactions may also cause color changes during storage. The objective of this present work was to analyze the rate of darkening of several Argentine honeys of multi¯oral origin as a function of their main components (glucose, fructose, total nitrogen, moisture content and lipids), and of their initial color, during storage at 37 C.
Luminosity: L*ab L*ab =116 (Y/Yn)1/3ÿ16 a* =500 [(X/Xn)1/3ÿ(Y/Yn)1/3] b* =200[(Y/Yn)1/3ÿ(Z/Zn)1/3]
2. Materials and methods
where: u0n 0:2009; v0n 0:461; Xn 98:041; Yn 100:00 y Zn 118:103, are the values calculated for illuminant C and at the 2 angle observer. : dierence between the values corresponding to the sample at time t and t=0. Browning index (BR) (Buera et al., 1985) was also calculated as:
2.1. Samples Sixteen ¯oral honeys from dierent geographic plain regions of temperate climate from Argentina, two from woody temperate areas and one from a tropical region, were analyzed. The samples were provided by ocial institutions or private producers with guarantee of genuiness and known history. Composition of honeys had been determined in a previous work, as described by Bertoni, Pereyra Gonzales and CattaÂneo, (1994). Moisture content was determined by AOAC (1980) method 31.111. Glucose and fructose were evaluated as described by Ugarte and Karman (1945). Total nitrogen by AOAC 2.24 (1950) method in semimicro scale. Total lipids were analyzed using the Folch, Lee and Sloane Stanley, (1957) method. Fatty acid composition as described in Pereyra, Gonzales, Bertoni, Gros & CattaÂneo, (1994). 2.2. Color measurements Measurements were done in a Hunterlab 5100 (Hunter Associates Laboratory Fairfax, VA) spectrocolorimeter, with white background in Plexiglass 5.3 cm diameterer sample holders, with a sample thickness of 4 mm. The illumination mode illuminated the aperture area (4.7 cm). The color functions, which in previous experiences have been proved to be adequated to follow the development of browning pigments (Buera, Petriella & Lozano, 1985; Buera & Resnik, 1989) were calculated for illuminant C, and the 2 angle observer, through the tristimulus values X, Y, Z, taking as standard values those of the white background (X=79.01; Y=83.96; Z=86.76). The following equations were employed (Lozano, 1977):
Metric chroma: C*ab C*ab =(a*2+b*2)1/2 Color dierence CIE 1976: E*ab E*ab =[(L*ab)2+(a*)2+(b*)2]1/2 Metric hue dierence: H*ab H*ab=[(E*ab)2ÿ(L*ab)2ÿ(C*ab)2]1/2 Metric saturation: suv suv =13[(u0 ÿu0 n)2+(v0 ÿv0 n)2]1/2 0 u =4X/(X+15 Y+3 Z) v0 =9Y/(X+15 Y+3 Z)
BR 100 x ÿ 0:31=0:172; where x cromatic coordinate X= X Y Z Before placing the samples in the cuvette for color measurement, they were placed at 50 C for 30 min in a water bath to decrease their viscosity and to allow the measurement of the volume with a syringe, carefully avoiding air bubbles. It was demonstrated, in a preliminary study, that this initial treatment of the samples did not alter their color. Illumination was done from the bottom side of the samples. The standard white plate was placed over the cuvette which contained the sample. The standard deviation of the color measurements, estimated from 20 determinations of the same sample was 2.5% and 5 measurements of each sample were necessary to obtain an error lower than 3% at a con®dence level of 95%. 2.3. Storage The samples were distributed in aliquots in hermetically closed containers of high density polyethylene. They were stored in forced circulation ovens maintained at a constant temperature 37(1) C during adequate periods of time, after which colorimetric determinations were done, over a total period of 90 days.
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
3. Results and discussion Fig. 1(a) shows the initial color of honeys in the CIE chromaticity diagram, in which the tristimulus values are limited to a plane, determined by the x and y chromatic coordinates. Most of the samples are located on a line to which corresponds a dominant wavelength of 575 nm, as observed for samples of yellow to light-brown colors obtained through non-enzymic browning reactions (Buera et al., 1985), and also observed by Aubert and Gonnet (1983) from spectrophotometric measurements of honeys. The darker honeys were located in the descending part of the curve, and showed a displacement of the dominant wavelengh to the red zone of the chromaticity diagram. Fig. 1(b) shows the CIE chromaticity coordinates of the honeys, after 90 days of storage at 37 C. It can be seen that a displacement of
Fig. 1. Scheme of the CIE chromaticity diagrams showing the initial (a) and the ®nal (b) color of honeys.
187
the dominant wavelengh occurred for all the honeys, to the red zone of the diagram. The CIE tristimulus values allowed an objective approach to color grading, de®ning a picture of the chromaticity (yellow-red) of the samples, which is not possible by the visual classi®cation of honey by the ocial Pfund method from light to dark (Aubert & Gonnet, 1983). During storage at 37 C the darkening of the samples could be followed by any of the color functions which were previously related to browning development: suv, Hab*, IB and 1/Z (Buera et al., 1985; Buera & Resnik, 1989). As shown in Fig. 2 for the color function Hab* after an initial induction period, in which no browning occurred, it followed a period of linear increase of color with time. This behavior was observed in many products subjected to non-enzymatic browning (Song & Chichester, 1966; Labuza & Saltmarch, 1980). The samples included in Fig. 1 were chosen in such a way to obtain a representative picture of all the analyzed samples, covering the whole range of color changes and browning rates observed. The slope of the linear part of the curves was calculated, and considered as an index of the rate of browning, as a pseudo zero-order rate coecient. Table 1 shows the correlation coecients resulting from the linear correlation between the color functions and storage time data, obtained by least squares. The rate of darkening could be followed by any of the selected color functions (the rate coecients for the metric hue development is reported in Table 1). The induction period for browning development at 37 C, calculated as the value of the abscise for which the ordinate was zero, was between 10 and 30 days, and the values were also included in Table 1.
Fig. 2. Evolution of the color function Hab*, with storage time at 37 C for some honeys, representative of the whole range of color changes.
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Table 1 Correlation coecients (r2) for the linear regression between some of the color functions and storage time data at 37 C, obtained by least squaresa Honey
suv
BR
Lab
1/Z
Cab
Hab
k(Hab units/day)
I (days)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
0.9391 0.9674 0.9755 0.8623 0.9622 0.9105 0.9472 0.9673 0.9113 0.9675 0.9766 0.9632 0.9858 0.9589 0.9819 0.9295 0.7674 0.9362 0.9876
0.9434 0.9657 0.9354 0.8132 0.9245 0.9372 0.9472 0.9267 0.9139 0.9325 0.9123 0.8954 0.9543 0.9216 0.9325 0.9325 0.9432 0.8997 0.9354
0.9542 0.9391 0.9261 0.8817 0.9303 0.9811 0.8931 0.9815 0.9873 0.9625 0.9946 0.9600 0.9428 0.9604 0.9979 0.9778 0.8060 0.9837 0.9546
0.8868 0.9311 0.8341 0.7918 0.9539 0.9523 0.9748 0.9485 0.8460 0.9210 0.9056 0.9350 0.9431 0.9963 0.9353 0.9853 0.7977 0.8960 0.8990
0.9321 0.9625 0.8461 0.8490 0.9402 0.8381 0.9561 0.9554 0.8821 0.9623 0.9569 0.9614 0.9979 0.7214 0.9671 0.9427 0.8707 0.9189 0.9143
0.9316 0.9419 0.8547 0.8889 0.9193 0.9555 0.9654 0.8759 0.8885 0.9373 0.9690 0.9644 0.9769 0.9386 0.9881 0.9768 0.7214 0.9110 0.9089
0.104 0.0795 0.121 0.121 0.765 0.1397 0.0380 0.0992 0.126 0.0292 0.161 0.090 0.139 0.3722 0.124 0.149 0.5337 0.173 0.21
27.6 25.6 0 31.9 30.6 19.2 25.9 30.2 28.0 11.9 15.7 21.6 20.3 21.2 0 3.6 36.9 15.3 12.6
a The slope of the linear part of the curves (k) and the induction period (I) for browning development were calculated for the Hab* color function. I was calculated as the X-intercept.
To investigate the variable which most in¯uenced the rate of darkening, the slopes of the linear part of the curves were analyzed in relation to the main components of the honeys, and also to their initial color. Fig. 3 shows the dependence of the calculated browning rates (k) with some of the analyzed variables. Of the analyzed variables, the rate of darkening of honeys had no dependence with the compositional variables, and the initial color (IC) seemed to be the best parameter to de®ne browning rate. Metric saturation (suv), a color function de®ned above (see Materials and Methods section) is related to the purity of color and was selected as an index of the initial color value of honeys. suv has been found to be an adequate function to follow color changes in transparent products yellow to brown colorations (Petriella, Resnik, Lozano & Chirife, 1985; Buera et al., 1985). Fig. 3(a) shows that the rate of browning did not have any correlation with the concentration of the main sugar components of honey (glucose and fructose). At the acidic pH value of the honey (3.8±4.5), fructose is more reactive than glucose towards browning development (Buera, Resnik & Petriella, 1992), but as both sugars are in a large extent and the variability between samples was small, the concentration of fructose was not a limiting factor for browning development. Amino compounds play an important role in interactions involving reducing sugars. Wootton, Edwards, Faraji-Haremi and Johnson (1976) and Wootton, Edwards and Faraji-Haremi, (1976) indicated that although total nitrogen content was relatively unaected by storage at 50 C, the total free aminoacid content decreased, but the changes in free aminoacids were not related to the extent of darkening. Due to the low content of free aminoacids in honey, it
could be expected that, if the amino-sugar condensation was an important cause of honey darkening, nitrogen concentration must be considered a limiting factor and the rate of browning should be related to the initial nitrogen content. However, the correlation between browning rate and nitrogen content was poor (Fig. 3b). These results indicated that, contrary to the suggestions of Ramsay and Milum (1933) and in agreement with Lynn et al. (1936) the amino-sugar condensation, and the total nitrogen content had only a secondary eect on the darkening of honeys. The instability of fructose may be one important cause of discoloration, but the high concentration of this sugar and the narrow range of variability between dierent samples of honeys did not allow correlation of the rate of darkening to fructose concentration. Although during extended storage, lipids may generate carbonilic compounds through oxidation and promote browning (Taoukis & Labuza, 1996), the rate of darkening was neither aected by the total lipid content (Fig. 3c), nor by the individual concentrations of the unsaturated fatty acids linoleic (18:2) or linolenic (18:3) [the correlation coecients (r2) were lower than 0.2, not shown]. Schade et al. (1958) indicated that the rate of honey darkening may increase as the moisture content increases, but as shown in Fig. 3d, the water content was not a variable which in¯uenced clearly the browning rate. Wootton, Edwards, Faraji-Haremi and Johnson, (1976) reported that none of the changes observed during their study on storage of Australian honeys (color, acidity and total nitrogen content) were related to the initial moisture content, in spite of the known importance of moisture levels in browning reactions. When the rate of darkening was plotted as a func-
A.P. Gonzales et al. / Food Research International 32 (1999) 185±191
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Fig. 3. Dependence of the calculated browning rates (k, in Hab* units/day) with some of the analyzed variables: (a) glucose and fructose content (in g/100 g of honey); (b) total nitrogen (N, in g/100 g of honey); (c) total lipids (in g/100 g of honey); (d) moisture content (in g H2O/100 g of honey).
tion of initial color (IC) of honeys, a better correlation than that obtained for the compositional variables was found (Fig. 4). The rate coecients, and also the induction periods were analyzed in relation to the compositional variables and initial color by stepwise regression analysis of data (Statistix for windows analytical software was employed). The best subset regression models that contained all the potential predictor variables were ®rst evaluated. To obtain a more complete picture of the dependence, some transformations in the variables were applied, such as squares or logarithms, and relationships between variables, which could account for interactions between factors, were also analyzed. None of the transformations proved, gave better correlation than the single variable ``initial color'' (IC). The results con®rmed that the initial color was the variable which most in¯uenced the browning rate, as observed in Fig. 3. The equation obtained through linear correlation by stepwise regression combined the variable IC and IC2 as follows:
Fig. 4. Correlation between the calculated browning rates (k, in Hab* units/day) and initial color (suv, metric saturation) obtained by stepwise linear regression of data.
k 0:1894 ÿ 0:3849 IC 0:3177 IC2 The curve predicted for this equation is shown in Fig. 4 as a dotted line. The square correlation coecient between observed and predicted values (r2 ) was 0.8018 for a p<0.06.
It was observed in model systems subjected to nonenzymatic browning reactions (Petriella et al., 1985) that the induction period is inversely proportional to the rate coecient for darkening. In the present study,
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however, the induction period did not have any correlation either with the initial color (r2=0.46), or with the analyzed compositional variables (the best correlation obtained from a stepwise regression analysis had as r2 of 0.49 for a p<0.05). Fig. 5 shows that the chromatic coordinate x for the samples before storage (xi) and after storage for 90 days at 37 C (xf) had a good linear correlation. A similar dependence was observed for the chromatic coordinate y and other color functions (not shown). These results con®rmed that the ®nal color was strongly related to the initial color of honeys. Milum (1948) had reported that the rate of darkening in honeys during any given period of heating or storage was related to the amount of previous discoloration during the processing of honeys at high temperatures or during storage. However, the honeys analyzed in the present study had not been subjected to high temperatures and they were stored in similar conditions after they were produced, before the storage experiment at 37 C. It is to be noted that the honey in which the rate of darkening was the highest, was one from a woody temperate region, and had also the darkest initial color. The ¯oral origin, which is a variable that strongly aects the initial color of honeys (Aubert & Gonnet, 1983), the polyphenolic components (not analyzed in present paper) and their deteriorative reactions, may be important factors in determining the stability of the color of honeys during storage. It is known that the polyphenolic components form brown complexes with amino acids and proteins (Labuza and Schmidl, 1986). Quinones produced by oxidation of polyphenols are reactive substances in some stages of the complex reactions leading to browning (Pokorny, 1980). Enzymatic activities in honeys (not subjected to thermal treatment), may aect polyphenols and are not discarded as browning promotors.
Fig. 5. Correlation between the chromatic coordinate x for the samples before storage (xi) and after storage during 90 days at 37 C (xf).
Acknowledgements The authors acknowledge ®nancial support from Universidad de Buenos Aires and CONICET.
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