International Food Research Journal 25(3): 1044-1050 (June 2018)

Journal homepage: http://www.ifrj.upm.edu.my

Effect of storage on hydroxymethylfurfural (HMF) and color of some

Algerian honey
*
Mouhoubi-Tafinine, Z., Ouchemoukh S., Bachir bey M., Louaileche, H.
and Tamendjari, A.

Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la vie, Université de

Bejaia, 06000, Algérie

Article history Abstract

Received: 25 January 2017 In this study, the quality evaluation of six honey samples was carried out by the analysis
Received in revised form: of some physico-chemical parameters. All the samples showed water content within limits
12 April 2017 (20%), except for the sample H01. This may be the result of a premature harvest. Values of ash
Accepted: 17 July 2017
content, Electrical conductivity and pH prove that the samples were most likely of floral origin.
However, the samples H01, H02 and H04 may be elaborated from honeydew, because of their
high ash content and electrical conductivity. The very high level of sucrose of H06 can be due
Keywords to an adulteration by an addition of sucrose. The total acidity of all the samples was within
limits, indicating absence of undesirable fermentation. The highest HMF level and the lowest
Honey invertase activity of H06, suggesting that this sample has undergone a heat treatment. In the
Algerian other hand, the impact of storage at different temperature (4, 20, and 35°C) on HMF and color
Storage was also investigated. Storage at 4 and 20°C had no considerable effect on these parameters.
HMF
However, storage at 35°C caused an increase of HMF and the results exceed largely the allowed
Color
limit (40 mg/Kg). In the same time, the color of the samples is accentuated because of Maillard
reaction.
© All Rights Reserved

Introduction the quality of some Algerian honeys by the analysis of

some physico-chemical parameter and to investigate
Honey is a natural substance which is used as the effect of storage at 4°C, 20°C and 35°C, during 9
a medicine since ancient time. It is made by Apis months, on HMF formation and honey samples color.
mellifera bees from nectar of blossoms (floral
honey) or from secretions of living parts of plants Material and Methods
or excretions of plant sucking insects (honeydew
honey) (Sanz et al., 2004; Belay et al. 2013). Honey Honey samples
is essentially a concentrated aqueous solution of The present study was carried out on six honey
inverted sugar, but it also contains a very complex samples; samples H01, H02, H03 and H04 were
mixture of other saccharides, enzymes, amino and harvested between June and November 2012 and
organic acids, polyphenols, carotenoids, Maillard collected directly from beekeepers in different
reaction products, vitamins, and minerals (Blasa et regions of Bejaïa, Algeria. Samples H05 and H06
al., 2006). Honey types differ from one country to were purchased from a local market in the same year
another and in different regions in the same country (2012). The samples were stored in glass bottles.
due to the floral origin, soil composition and other
factors consequently (Alqarni et al., 2012). Honey Physico-chemical analyses
quality can undergo some changes with duration Moisture, ash, proline contents, and electrical
and temperature of storage, which leads essentially conductivity were determined by the methods of
to loss of enzymatic activities and formation of Bogdanov et al. (1999). Moisture was evaluated by
hydroxymethylfurfural (HMF), a cyclic aldehyde refractometry using Abbe-typ refractometer (RF 490,
that is produced by degradation of sugars (Gidamis Euromexholland). Ash content was determined by
et al., 2004). The presence of excessive amounts heating 5 g of honey at 625°C in a muffle furnace.
of HMF in honey has been considered as evidence Proline content was determined by the measuring
of overheating and implies loss of freshness (Serra the absorbance at 510 nm of the resulting product
Bonvehi et al., 2000). between proline and ninhydrin in an acidic medium.
The purposes of the present study is to estimate Electrical conductivity was measured in a 20%

*Corresponding author.
Email: [email protected]
1045 Mouhoubi-Tafinine et al./IFRJ 25(3): 1044-1050

(w/v) solution of honey in deionized water with were determined by the same softwar.
low electrical conductivity using Leibohld model
conductimeter. Results and Discussion
Reducing sugars were determined by the
titrimetric method using Fehling reagent (Journal Physico-chemical analyses
Officiel Français, 1977). Table 1 shows the results expressed as mean
Protein content was determined by the method of (±SD) obtained from the physicochemical analysis of
Azeredo et al. (2003). A volume of 0.1 ml of honey honey samples. Moisture is regarded as an important
solution (50 % w/v) was added to 5 ml of Coomassie parameter of quality used to determine the maturity
Brillant Blue. After 2 min of incubation, the quantity degree of honey and to estimate its shelf life (De
of proteins was estimated at 595 nm in relation to Rodriguez et al., 2004). Values obtained for this
bovine serum albumin standard curve. parameter ranged between 17.28 and 21.34%. They
pH, acidity, invertase activity and HMF content are higher than those obtained by Ibrahim Khalil
were determined according to the Harmonised et al. (2012) (11.59-14.13%). One sample (H01)
Methods of the International Honey Commission presented water content over the maximum allowed
(Bogdanov, 1999). pH was assessed in a 10% (w/v) by the Codex Alimentarius (20%); this can be the
solution of honey in distilled water by mean of pH consequence of a premature harvest of honey. Values
meter (Crison micro pH 2000, Germany). Free, of ash content (0.137- 0.564%) fell within the limit
lactonic and total acidities were determined by the allowed for floral honeys (0.6%); these results were
titrimetric method. The addition of 0.05 M NaOH similar to those found by Mendes et al., (1998) who
was stopped at pH 8.50 (free acidity); immediately, investigated Portuguese floral honeys (0.1-0.5%).
the remained solution of NaOH was added and, Ash content depends on the material collected by
without delay, a back-titration was realised with bees during foraging on the flora. The soil type, in
0.05 M H2SO4 (lactonic acidity). Total acidity which the original nectar-bearing plant was located,
was obtained by adding free and lactone acidities. also influences the quantity of minerals present in
Invertase activity was determined basing on the honeys (De Rodriguez et al., 2004; Felsner et al.,
spectrophotometric measurement of 4-nitrophenol, 2004).
which is formed by the reaction of honey invertase Proline content serves as an additional
with 4-nitrophenyl-D-glucopyranoside, used as a determinant of quality and in some cases also as a
substrate. Results were expressed in Unit per Kg. criterion for estimating the maturity of honey and as
Hydroxymethylfurfural (HMF) was quantified after an indicator for detecting sugar adulteration (Meda
clarifying the samples with Carrez reagents (I and II) et al., 2005). The studied honey samples have good
and the addition of sodium bisulphate; absorbance proline levels (551.88–890 mg/Kg); they were higher
was determined at 284 nm and 336 nm (UV-Vis 1601 than the limit proposed by Bogdanov et al. (1999)
spectrophoto-meter, Shimadzu). (>180 mg/kg), indicating the maturity of the honeys
Color was estimated by measuring the absorbance and absence of adulteration.
of honey solutions at 420 nm according to Bath and Electrical conductivity (EC) shows a great
Singh (1999), after diluting 1.25 g of honey to 5 ml variability according to the floral origin. Floral
with hot distilled water and filtration. honeys should have conductivity values below than
0.8 mS/cm, while honeydew should have values over
Storage 0.8 mS/cm (Codex Alimentarius, 2001). EC values of
Each honey sample (500 g) was divided into 3 the tested honeys ranged from 0.417 to 1.412 mS/cm;
aliquots in hermetically closed glass containers. The H01, H02 and H04, which have the highest values
first aliquot was stored at 4°C, the second at 20 °C of ash content, have also EC values higher than 0.8
and the third at 35°C; the samples were stored during mS/cm, suggesting that these honeys were elaborated
9 months. from honeydew. Our results are superior to those
obtained by Khalil et al. (2012) (0,419-0,809 mS/
Statistical analysis cm).
Statistical analysis of the data was carried out with In this study, we confirmed the existence of a
STATISTICA 5.5 Fr. Analysis of variance (ANOVA) linear relationship (r = 0. 93, P <0.01) between ash
followed by LSD test (Least Significant Difference) content and electrical conductivity of honeys obtained
was performed to estimate the statistically significant by Člechovská and Vorlová (2001), Downey et al.
differences between honey samples for each (2005) and Ouchemoukh et al. (2007). Sugar content
parameter. In the other hand, statistical correlations present values varying from 62.75 to 76% (reducing
 Mouhoubi-Tafinine et al./IFRJ 25(3): 1044-1050 1046

Table 1. Physico-chemical characteristics of the honey samples

Values with the same letter in each line do not differ significantly. The results are arranged in ascending order;
a<b<c<d<e<f. SSD : statististical significance of the différences ; ** : highly significant (P<0,01), *** :very
highly significant (P<0,001), ND : not determined

sugar) and 61.35 to 73.39% (total reducing sugar). lactones or esters, and inorganic ions, contribute to
The sugar composition depends highly on the type of the honey acidity. The variation in acidity among
flowers used by bees, as well as regional and climatic different honey types may be attributed to variation
conditions. Sucrose content for all the samples fell in these constituents (Bath and Singh, 1999; De
within the limits of the Codex Alimentarius (5%), Rodriguez et al., 2004).
except the sample H06, which present a very high Invertase activity of the honey samples varies
level of sucrose (13.54%); this can be due to an from 5.45 to 75.97 U/Kg. These results are higher
adulteration by an addition of sucrose to honey or than those obtained by Serrano et al. (2007). The
an early harvest of honey responsible for the no full samples H05 and H06 purchased from the market
transformation of sucrose into glucose and fructose. have the lowest invertase activity (12.39 and 11.46
The analysed samples showed that protein U/Kg, respectively); this can be the consequence of
contents ranged from 669.93 to 1635.48 µg/g; an eventual heating of these honeys. A significant
they are lower than those obtained in our previous correlation was observed between invertase activity
study (3700-9400 µg/g) (Ouchemoukh et al., 2007). and electrical conductivity (r = 0.84, P< 0.05). A
Variations of the protein content can be attributed to similar correlation was also observed by Tsigouri and
the floral origin. During extraction and storage of Passaloglou-Katrali (2000).
honey, pH is of great importance, as it influences the HMF measurement is used to evaluate the quality
texture, stability and shelf-life of honey (Bath and of honey; generally not present in fresh honey, its
Singh, 1999). Values recorded for this parameter in content increases during conditioning and storage.
this study ranged from 3.97 and 4.58. They indicate Then it can be useful to estimate the freshness of
that the honeys tested were most likely of floral honey (Gonnet, 1993; Gidamis et al., 2004; Zappala
origin, since honeydew honeys generally have higher et al., 2005) and since it is formed during acid
ash content than floral, resulting in honey with less hydrolysis of sucrose. The presence of high levels
active acidity and therefore a higher pH (Downey et of this compound suggests the possibility that honey
al., 2005). Bath and Singh (1999) reported that honey has been adulterated with invert syrup (Azeredo et
obtained during spring, is often more acidic than in al., 2003; Sanz et al., 2003). HMF was undetectable
autumn, when it usually contains more honeydew. in the sample H01 indicating its high degree of
Samples H01 and H04, which were collected in freshness. H02, H03, H04 and H05 have an HMF
autumn, have the highest pH values, 4.58 and 4.41, content ranged between 11.04 and 34.53 mg/kg.
respectively. These values were lower than the allowed maximum
Values of free acidity are ranged between 6.80 limit of 40 mg/kg as recommended by the Codex
and 12.50 meq/kg; lactonic acidity is ranged between Alimentarius (2001). However, the commercial
8.50 and 18.50 meq/kg, while total acidity varied from sample H06 has the highest HMF level (82 mg/kg),
16 to 30.50 meq/kg. Total acidity was within limits which confirm the heating of this honey. Our results
(below 50 meq/kg of honey), indicating absence of are different from those reported by Makhloufi et al.
undesirable fermentation. The presence of organic (2007) (0, 5-124 mg/kg)
acids, particularly gluconic acid in equilibrium with The color of honey is one of the factors
1047 Mouhoubi-Tafinine et al./IFRJ 25(3): 1044-1050

Température : 4°C; 20°C; 35°C

Figure 1. Effect of storage temperature and duration on HMF formation in the honey.

determining its price on the World market, and also

its acceptability by the consumer (Gonzales et al.,
1999). The results obtained for this parameter varied
from 0.663 to 1.443. Many studies (Alvarez-Suarez
et al., 2010 ; Terrab et al., 2004) reported that honey
color is an indicator of its mineral content, the higher
the mineral content, the darker would be the color
and vice versa. The light color of samples H05 and
H06 was coherent with their low ash content, but this
relation was not observed for the other samples. The
color of honey can also reflect the pigments content
(carotenoids, flavonoids, etc) (Amiot et al., 1989).
Figure 2. HMF formation as a function of storage duration
Effect of storage at 35°C
Several factors influence the formation of
HMF, such as temperature, time of heating, storage ambient temperature. During the whole storage
conditions (Cordella et al., 2005), and some period at 20°C, the sample H01 had no detectable
chemical properties of honey (pH, total acidity, HMF content, while an important increase of HMF
mineral content, quantity and type of reducing was noted in the sample H03 (17.87 to 38.98 mg/
sugars) which are related to the floral source of Kg), which was the most acidic sample (pH 3.97).
honey (Ramirez Cervantes et al., 2000; Gidamis et Bath and Singh (1999) reported that honeys with
al., 2004). According to Janzowski et al. (2000), at low pH value produce more HMF during storage.
high concentrations, HMF can be cytotoxic, causing The samples H02, H04, H05, and H06 presented a
irritation to eyes and upper respiratory tract. The slight increase of HMF during storage at 20°C. At
effects of storage duration on HMF formation at the highest storage temperature (35°C), a significant
different temperature are illustrated in Figure 1. increase of HMF content was noted. After 9 months,
Storage at 4°C had no considerable effect on HMF HMF content varied from 100.84 (H01) to 353.09
formation. mg/Kg (H05) and exceed largely the allowed limit
Cherchi et al. (1997) did not observe significant (40 mg/Kg). The highest level of HMF observed in
changes in HMF in three types of honey even after the sample H05 after storage, can be due to its high
a storage period of 24 months at refrigeration and initial HMF content (82 mg/Kg) and its low pH
 Mouhoubi-Tafinine et al./IFRJ 25(3): 1044-1050 1048

Température : 4°C; 20°C; 35°C

Figure 3. Effect of storage temperature and duration on the color of honey.

(4.11). Other factors such as high level of moisture

and simple sugars (glucose and fructose) in honey
can also offer favourable conditions for the HMF
production (Fallico et al., 2004). Linear regression
models for the HMF production as a function of
storage time at 35°C are reported in Figure 2.
The effects of storage duration on the color
of honey at different temperature are shown in the
Figure 3. Evolution of the samples color (at 20°C)
was not very considerable, exception for the sample
H02 which color value varied from 1.443 to 2.466.
Storage at 35°C caused a high increase of the samples
darkening, especially after 6 months of storage.
Gonzales et al. (1999) who investigate the effect of Figure 4. Color formation as a function of storage duration
storage at 37°C during 3 months on the color of some at 35°C
Argentinean honeys reported that the final color was
strongly related to the initial color of honeys. In this been probably heated. In addition to that, the sample
study, the most pronounced darkening was observed H06 presents a very high level of sucrose (13.54%),
in the sample H02, which was initially the darkest. A which can be due to an adulteration. Ash content, pH
linear regression for the color evolution as a function and electrical conductivity indicate that the tested
of storage duration at 35°C is reported in the Figure honeys were most likely of floral origin. The storage
4. at 35°C accentuates HMF production, leading to loss
of honey freshness. At the same time, the samples
Conclusion undergo a significant darkening due essentially to
Maillard reaction. However, storage at 4 and 20°C
The honeys investigated in this study are of didn’t affect HMF content and color of honey.
acceptable quality standards as most of the quality Storage temperature of honey should be carefully
parameters fall within the recommended limits. controlled (lower than 35°C) to preserve its chemical
However, a few exceptions were observed. In fact, and sensory quality.
moisture values indicate that all the samples have
a good degree of maturity, except the sample H01. Acknowledgement
Concerning the freshness of the samples, the high
level of HMF and the low invertase activity of the We want to thank the Algerian Ministry of High
two samples (H05 and H06) indicate that they have Education and Scientific Research for sponsoring this
1049 Mouhoubi-Tafinine et al./IFRJ 25(3): 1044-1050

work and Mr MOUHOUBI Mustapha for collecting 2001. 1-7.

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