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Color changes during storage of honeys in relation to theircomposition and initial color

Adriana Pereyra Gonzales a, Leila Burin b, Mara del Pilar Buera b,*,1

aDepartamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,

1428 Buenos Aires, ArgentinabDepartamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,

1428 Buenos Aires, Argentina

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 poly-

phenols, however, no systematic studies exist on this subject. The inuence of composition and initial color on the rate of darkening

of several Argentine honeys submitted to storage at 37C 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 dened the rate of

darkening of honeys. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Tech-

nology. 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 a-

vor 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).

The rate of darkening has been related to the composi-

tion 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 pos-

sible 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 aminoacid-

aldol (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

inuence of these factors on the darkening of honey.

While Ramsay and Milum (1933) stated that the Mail-

lard 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 rightsreserved.

P I I : S 0 9 6 3 - 9 9 6 9 ( 9 9 ) 0 0 0 7 5 - 7

Food Research International 32 (1999) 185191

www.elsevier.com/locate/foodres

* Corresponding author. Tel.: +54-11-4576-3397; fax: +56-11-

4576-3366.

E-mail address: [email protected] (M.P. Buera)1 Member of Consejo Nacional de Investigaciones Cientcas y

Te cnicas de la Repu blica, Argentina.


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during storage, literature related to investigate the main

causes of darkening is scarce. Wootton, Edwards, Faraji-

Haremi 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 thehoneys at 50C. Schade et al. (1958) indicated that the rate

of honey darkening may increase as increasing the moist-

ure content increases. The content and type of natural

polyphenols (such as avonoids) inuence 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 37C.

2. Materials and methods

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 gen-

uiness and known history.

Composition of honeys had been determined in a pre-vious work, as described by Bertoni, Pereyra Gonzales and

Catta neo, (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 & Catta neo, (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 dev-

elopment of browning pigments (Buera, Petriella &

Lozano, 1985; Buera & Resnik, 1989) were calculated for

illuminant C, and the 2 angle observer, through the tris-

timulus 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):

Luminosity: L*abL*ab =116 (Y/Yn)

1/316

a* =500 [(X/Xn)1/3(Y/Yn)1/3]

b* =200[(Y/Yn)1/3(Z/Zn)1/3]

Metric chroma: C*ab

C*ab =(a*2+b*2)1/2

Color dierence CIE 1976: E*abE*ab =[(L*ab)

2+(a*)2+(b*)2]1/2

Metric hue dierence: H*abH*ab=[(E*ab)2(L*ab)2(C*ab)2]

1/2

Metric saturation: suv

suv

=13[(uHuHn)2+(vHvH

n)2]1/2

uH =4X/(X+15 Y+3 Z)

vH

=9Y/(X+15 Y+3 Z)

where: uHn 0X2009; vH

n 0X461; Xn 98X041; Yn

100X00 y Zn 118X103, 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:

f 100 x 0X31 a0X172Y whee x

omtioodinte Xa X Y Z

Before placing the samples in the cuvette for color

measurement, they were placed at 50C 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 pre-

liminary 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 condence level of 95%.

2.3. Storage

The samples were distributed in aliquots in hermeti-

cally 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.

186 A.P. Gonzales et al. / Food Research International 32 (1999) 185191


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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 chro-

matic coordinates. Most of the samples are located on a

line to which corresponds a dominant wavelength of 575nm, as observed for samples of yellow to light-brown

colors obtained through non-enzymic browning reac-

tions (Buera et al., 1985), and also observed by Aubert

and Gonnet (1983) from spectrophotometric measure-

ments of honeys. The darker honeys were located in the

descending part of the curve, and showed a displace-

ment of the dominant wavelengh to the red zone of the

chromaticity diagram. Fig. 1(b) shows the CIE chro-

maticity coordinates of the honeys, after 90 days of

storage at 37C. It can be seen that a displacement of

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, den-

ing a picture of the chromaticity (yellow-red) of the

samples, which is not possible by the visual classica-

tion of honey by the ocial Pfund method from light to

dark (Aubert & Gonnet, 1983).During storage at 37C 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 pro-

ducts 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 sam-

ples, covering the whole range of color changes andbrowning 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 coe-

cient. 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 selec-

ted color functions (the rate coecients for the metric

hue development is reported in Table 1). The induction

period for browning development at 37C, calculated as

the value of the abscise for which the ordinate was zero,

was between 10 and 30 days, and the values were alsoincluded in Table 1.

Fig. 1. Scheme of the CIE chromaticity diagrams showing the initial

(a) and the nal (b) color of honeys.

Fig. 2. Evolution of the color functionHab*, with storage time at 37C

for some honeys, representative of the whole range of color changes.

A.P. Gonzales et al. / Food Research International 32 (1999) 185191 187


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To investigate the variable which most inuenced 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 vari-

ables, 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 dene browningrate. Metric saturation (s

uv), a color function dened

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 corre-

lation with the concentration of the main sugar compo-

nents of honey (glucose and fructose). At the acidic pH

value of the honey (3.84.5), 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 rela-

tively unaected by storage at 50C, the total free ami-

noacid 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 thetotal 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 con-

centration of this sugar and the narrow range of varia-

bility between dierent samples of honeys did not allow

correlation of the rate of darkening to fructose con-

centration. 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 inuenced 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 reac-

tions. When the rate of darkening was plotted as a func-

Table 1

Correlation coecients (r2) for the linear regression between some of the color functions and storage time data at 37C, obtained by least squaresa

Honey suv BR v 1/Z g r

k(r

units/day) I (days)

1 0.9391 0.9434 0.9542 0.8868 0.9321 0.9316 0.104 27.6

2 0.9674 0.9657 0.9391 0.9311 0.9625 0.9419 0.0795 25.6

3 0.9755 0.9354 0.9261 0.8341 0.8461 0.8547 0.121 0

4 0.8623 0.8132 0.8817 0.7918 0.8490 0.8889 0.121 31.95 0.9622 0.9245 0.9303 0.9539 0.9402 0.9193 0.765 30.6

6 0.9105 0.9372 0.9811 0.9523 0.8381 0.9555 0.1397 19.2

7 0.9472 0.9472 0.8931 0.9748 0.9561 0.9654 0.0380 25.9

8 0.9673 0.9267 0.9815 0.9485 0.9554 0.8759 0.0992 30.2

9 0.9113 0.9139 0.9873 0.8460 0.8821 0.8885 0.126 28.0

10 0.9675 0.9325 0.9625 0.9210 0.9623 0.9373 0.0292 11.9

11 0.9766 0.9123 0.9946 0.9056 0.9569 0.9690 0.161 15.7

12 0.9632 0.8954 0.9600 0.9350 0.9614 0.9644 0.090 21.6

13 0.9858 0.9543 0.9428 0.9431 0.9979 0.9769 0.139 20.3

14 0.9589 0.9216 0.9604 0.9963 0.7214 0.9386 0.3722 21.2

15 0.9819 0.9325 0.9979 0.9353 0.9671 0.9881 0.124 0

16 0.9295 0.9325 0.9778 0.9853 0.9427 0.9768 0.149 3.6

17 0.7674 0.9432 0.8060 0.7977 0.8707 0.7214 0.5337 36.9

18 0.9362 0.8997 0.9837 0.8960 0.9189 0.9110 0.173 15.3

19 0.9876 0.9354 0.9546 0.8990 0.9143 0.9089 0.21 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.

188 A.P. Gonzales et al. / Food Research International 32 (1999) 185191


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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 con-

tained 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 trans-

formations proved, gave better correlation than the sin-

gle variable ``initial color'' (IC). The results conrmed

that the initial color was the variable which most inu-

enced 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:

k 0X1894 0X3849 sg 0X3177 sg 2

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


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however, the induction period did not have any corre-

lation 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


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A.P. Gonzales et al. / Food Research International 32 (1999) 185191 191

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