1 Microchemical Journal 93 (2009) 7377

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Microchemical Journal
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Honey from Luso region (Portugal): Physicochemical characteristics and

mineral contents
Lus R. Silva a, Romeu Videira b, Andreia P. Monteiro b, Patrcia Valento a,Paula B. Andrade a
a
REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, Rua Anbal Cunha 164, 4050-047 Porto, Portugal
b
CI & DETS / Departamento de Ambiente, Escola Superior de Tecnologia do Instituto Politcnico de Viseu, Viseu, Portugal

ARTICLE INFO ABSTRACT

Article history: This work was conducted to evaluate the quality of 38 honey samples
Received 29 April 2009 from Luso region (Portugal), and to study the relation between
Accepted 3 May 2009 Eucalyptus pollen and chemical properties of honey. Mean values
Available online 13 May obtained for physicochemical parameters were: pH 3.83; 16.65%
2009 moisture; 80.7 Brix sugar; 0.35% ash; 419.6 S cm1 electrical
Keywords: conductivity; 21.5 meq/kg free acidity; 9.6 meq/kg lactonic acidity;
Luso honey 31.2 meq/kg total acidity; 9.41 mg/kg HMF and 18.3 Gothe diastase
Pollen analysis activity. The mineral content was determined by atomic absorption
Physicochemical analysis spectrometry, and in the analysed samples, potassium was the major
Mineral content element, being magnesium the minor one. Mean values obtained
were (mg/kg): Ca, 59.88; K, 1150.10; Mg, 35.57; Na, 261.43.
Among the overall determined parameters, only Mg, ash and
electrical conductivity were influenced by the presence of Eucalyptus
pollen in the honey samples: the values obtained for Mg, ash and
electrical conductivity in multifloral honey without Eucalyptus were
lower than those of either monofloral or multifloral honey with
Eucalyptus.
The results obtained for physicochemical characteristics of Luso
honey indicate a good quality level, adequate processing, good
maturity and freshness.
Published by Elsevier B.V.

1 Introduction

Honey is the natural sweet product produced Proteins, flavour and aroma, phenolic compounds
by Apis mellifera bees from nectar of plants (nectar (phenolic acids and flavonoids), free amino acids,
honey), from secretions of livings parts of plants or organics acids, vitamins and minerals constitute
excretions of plant-sucking insects of the living part minor components of honeys.2
of plants (honeydew honey). Honey cannot be Honey commercially available varies greatly
considered a complete food by human nutritional in quality all over the world. This is largely assessed
standards, but it offers potential as a dietary on the basis of colour, flavour and density. Honey
supplement.1 composition is influenced by the plant species,
For infants, senior citizens and invalids, honey climate, environmental conditions and the
can be a more easily digested and more palatable contribution of the beekeeper [3,4]. In general,
carbohydrate food than saccharose by itself. monofloral honeys are more expensive than
1 M.L. Gonzlez-Miret, A. Terrab, D. Hernanz, M.A. multifloral ones [5]. In addition, some monofloral
Fernndez-Recamales, F.J. Heredia, Multivariate
correlation between color and mineral composition of 2 E. Mendes, E. Brojo-Proena, I.M.P.L.V.O. Ferreira, M.A.
honeys and by their botanical origin, J. Agric. Food Chem. Ferreira, Quality evaluation of Portuguese honey,
53 (2005) 25742580. Carbohydr. Polym. 37 (1998) 219223.
 L.R. Silva et al. / Microchemical Journal 93 (2009) 7377 2

honeys are more appreciated than others due to their covered the most important production zones (Table
organoleptic properties or their pharmacological 1). Samples were stored at 0 C until analysis, which
attributes [6]. occurred no longer than one month after extraction
Honey has been reported to contain about 200 from the hives by beekeepers.
substances and is considered as an important part of 2.2. Pollen analysis
traditional medicine [7]. It has been used in
ethnomedicine since the early humans, and in more The botanical origin of the samples was
recent times its role in the treatment of burns, determined using techniques described before [11].
gastrointestinal disorders, asthma, infected wounds For floral identification, 5 g of diluted honey sample
and skin ulcers have also been reported [8,9]. was centrifuged at 10,000 rpm for 15 min, to
separate the pollens. Samples of separated pollen
Several types of honey are produced in
grains were spread with the help of a brush on a
Portugal, where honey production is a traditional
slide containing a drop of lactophenol.
practice well implanted in several regions.
The slides were examined microscopically at
Luso region is located in the centre of
45, using a bright-field microscope (Olympus,
Portugal, being one of the most important region of
Tokyo).
honey production in this country, due to its
edafoclimatic conditions and plants diversity, were 2.3. Physicochemical characteristics
Eucalyptus pollen predominates. The detailed
characterization of the different honey type's existent Honey were analysed according to methods
in Portugal is important, once it will allow the previously reported for pH, moisture, Brix, ash
establishment of technical specifications, avoiding content, electrical conductivity, free, lactonic and
occurrence of adulterations. Due to adulteration total acidity, diastase activity, hydroxymethylfurfural
possibility, honey quality must be analytically determination [12]. Two replicate analyses were
controlled with the aim of guaranteeing its performed for each sample.
speculation. 2.3.1. pH
On the other hand, as consumers have been
incrementing their interest in monofloral honeys in The pH was measured by a pH-meter Consort
detriment of multifloral ones [10], pollen analysis is C831 (USA), with a precision of 0.002 pH units.
important for the commercial valorisation of honey. The pH of the honey was measured in solution of 10
g honey in 75 ml of CO2 free distilled water.
The work herein was conducted to investigate
the quality of 38 different samples of honey 2.3.2. Moisture content
proceeding from Luso region. For this purpose,
Moisturewas determined by refractometry,
pollen analysis was performed and physicochemical
using an Atago (Japan) model lT Abbe refractometer.
characteristics (pH, moisture, sugar, ash content,
All measurements were performed at 25 C.
electrical conductivity, free, lactonic and total
acidity, diastase activity and hydroxymethylfurfural) 2.3.3. Sugar
and mineral contents (K, Na, Ca and Mg) evaluated.
Sugar content was determined with a special
2. Materials and methods refractometer with direct reading display, and the
results were expressed as Brix.
2.1. Sample collection
2.3.4. Ash
Honey samples were collected in Luso
province (centre region of Portugal). Sampling area Ash content was measured by calcination,
overnight, in furnace at 550 C, until constant mass.
3 Microchemical Journal 93 (2009) 7377

Table 2
Distribution data for physicochemical parameters in Luso (Portugal) honey samples.

Sample pH Moisture (%) Brix (%) Ash (%) Electrical conductivity Free Acidity HMF

(meq/kg) (meq/kg) (mg/kg)

H1 3.88 17.04 80.6 0.39 473.5 33.8 5.2 8.3 42.0

H2 4.31 15.35 80.6 0.26 318.2 26.1 7.0 33.1 5.90

H3 4.34 15.83 80.6 0.25 301.3 22.9 10.5 33.4 11.75

H4 3.74 14.82 81.2 0.34 412.0 29.7 9.5 39.2 3.11

H5 4.12 15.27 80.2 0.22 263.2 25.1 6.2 31.2 2.45

H6 4.23 15.65 80.4 0.35 418.2 26.1 4.2 30.3 6.95

H7 4.25 13.52 82.2 0.09 114.7 16.4 4.7 21.1 5.35

H8 3.78 13.98 80.8 0.14 168.8 12.9 4.5 17.4 4.35

H9 3.83 14.49 81.0 0.34 415.1 38.1 9.0 47.1 14.60

H10 3.90 15.71 80.4 0.35 420.3 17.1 11.5 28.6 6.35

H11 4.70 14.98 80.6 0.32 385.2 24.0 5.2 29.2 7.22
 L.R. Silva et al. / Microchemical Journal 93 (2009) 7377 4

2.3.5. Electrical conductivity

Electrical conductivity of a honey solution at 0.1 M were added to the resultant ashes, and the
20% (dry matter basis) in CO2-free deionised mixture was stirred on a heating plate to almost
distilledwater,wasmeasured at 20 C in a Consort complete dryness. Then, 10 ml of the same acid was
C831 conductimeter, and the results were expressed added and the mixture was made up to 25 ml with
as Scm1. distilled water. Calcium, potassium, sodium and
magnesium were determined by atomic absorption
2.3.6. Free, lactonic and total acidity spectrometry (Perkin Elmer AAnalyst 300), using an
Free, lactonic and total acidity were air/acetylene flame. Quantitative determination of
determined as follows, by titrimetric method: the the elements by atomic absorption spectrometry was
addition of 0.05 M NaOH was stopped at pH 8.50 carried out after calibrating the instrument, using Ca
(free acidity), immediately a volume of 10 ml 0.05 (1 to 5 mg/l), K (0.1 to 2 mg/l), Na (0.1 to 2 mg/l),
M NaOH was added and, without delay, back- Zn (0.05 to 1 mg/l) and solutions dissolved in 0.1%
titrated with 0.05 M HCl to pH 8.30 (lactonic lanthanum (La). Lawas utilized as a matrix modifier
acidity). Total acidity results were obtained by in order to overcome the chemical interferences in
adding free and lactone acidities. the air/ acetylene flame. All samples were analysed
in triplicate.
2.3.7. Diastase activity
2.5. Statistical analysis
Diastase activity was measured using a
buffered soluble starch solution and honey, which Data are represented as meanstandard
was incubated in the thermostatic bath at 40 C. deviation. The results were statistically analysed by
Absorption was followed using a Perkin Elmer 25 analysis of variance (ANOVA) methodology
UV/VIS spectrophotometer and a chronometer. followed by Fisher's PLSD test. Differences were
Using considered significant for pb0.05.
Tegression (without using the data point at 0 3. Results and discussion
min), lines were fitted to the absorption data and the
diastase number was calculated from the time taken Table 1 shows the floral origin of honey
for the absorbance to reach 0.235. For samples of samples determined by microscopy pollen analyses.
low diastase activity, the regression was made on the Data indicate that 63% of honey samples were
basis of the last three data points to improve the monofloral and 37% were multifloral. Eucalyptus
linear correlation. In samples of high diastase sp. was a predominant source used by honeybees in
activity the time taken for the absorbance to reach the Luso region, once Eucalyptus pollen was
0.235 was determined with absorbance at 5 and 10, detected in 79% of the total analysed samples.
or 5,15, and 20 min, depending on the activity.
Results were expressed (as Gothe degrees) as
ml of 1% starch hydrolysed by enzyme in 1 g of
honey, in 1 h.
2.3.8. Hydroxymethylfurfural content (HMF)
The Winkler method was used to determine
the HMF content of honey samples: 5 g of each
sample was treated with a clarifying agent (Carrez),
the volume was completed to 50 ml and the solution
was filtered. The absorbance of the filtered solution
was measured at 284 and 336 nm against an aliquot
treated with NaHSO3.
2.4. Determination of mineral elements
Ash values were obtained by calcination, at Ilustracin 1: Linear regression of ash
550 C, of approximately 5 g honey sample, until content (% w/w) andconductivity (S cm1).
constant weight [13]. Five milliliters of nitric acid
5 Microchemical Journal 93 (2009) 7377

Furthermore, 92% samples of monofloral important for the differentiation of honeys of

honey were from Eucalyptus sp., 4% were from different floral origins [18]. The results obtained for
Erica sp. and 4% from Cytisus scoparius. Multifloral the honey samples under study varied between 114.7
honeys contained several pollen types with a and 636.5 S cm1 (average=419.6 S cm1).
considerable percentage of pollen grains from These values are below the maximum limit indicated
Eucalyptus sp., Erica sp., Rubus sp., Lavandula by Portuguese legislation for nectar honey (800 S
stoechas, Castanea sativa and C. scoparius. cm1).
The results obtained for the several The increase in ash content of the honey
physicochemical parameters determined are samples from Luso region was accompanied by the
presented in Table 2. Honey pH is affected by the increase of electrical conductivity, as previously
conditions during extraction and storage, which also reported by others [19,20]. This linear relationship is
influences texture, stability and shelf-life. pH is characterised by a correlation coefficient R equal to
indeed a useful index of possible microbial growth, 0.99 (Fig. 1).
since most bacteria grow in a neutral and mildly Honey acidity is due to the presence of organic
alkaline environment, while yeasts and moulds are acids, mainly gluconic acid, in equilibrium with their
capable of developing in an acidic environment corresponding lactones or internal esters, and to
(pH=4.04.5) and do not grow well in alkaline inorganic ions, such as phosphate, sulphate and
media [14]. chloride [13,21]. The lactonic acidity is considered
The pH values of the analysed honey samples as the acidity reserve when the honey becomes
ranged from 3.45 to 4.70 (mean value=3.88). These alkaline, while the total acidity is the sum of free and
values are in accordance with acceptable range for lactonic acidities [18]. Free acidity was within the
honey [15] and similar to those obtained with others limits of Portuguese and European legislations
Portuguese honeys [5]. (below 50 meq/kg), indicating the absence of
Percent moisture in the analysed honeys undesirable fermentation.
ranged from 13.53 to 19.70 (mean value=16.65). Lactonic acidity ranged were from 4.2 to 16.5
The water content of honey depends on various meq/kg (average=9.6 meq/kg). Total acidity varied
factors, like the harvesting season, the degree of between 17.0 and 51.5 meq/kg, with a mean value of
maturity reached in the hive and climatic factors. 31.2 meq/kg. The results obtained for acidity were in
The maximum amount of water contained by honey agreement with data reported for other Portuguese
is regulated for safety against fermentation. honeys [1,5] as well as for
All the samples contained less than 20% water, 4. Conclusions
the maximum amount allowed by international and
Portuguese legislations [16]. Honeys from Luso region present a good level
Moisture and sugar content are strictly of quality, once 32 of the 38 analysed samples are in
correlated and anomalous values of Brix degrees agreement with the European honey directive [30]
(directly related with sugar content) may be a and Portuguese legislation [16], indicating adequate
reliable index of adulteration [13,14]. processing, good maturity and freshness. Six
samples did not fit within European and Portuguese
The analysed samples presented Brix degrees standards relative to the diastase activity, reflecting
ranging from 79.0 to 82.2 (average=80.7), which are inadequate sample manufacture and/or storage.
similar to those from others Portuguese honey
samples [5]. Potassium is the most abundant of the
determined elements.
Ash content is a parameter used for the
determination of the botanical origin (floral, mix or Magnesium and ash contents and electrical
honeydew) [17]. The results found (0.090.53%) are conductivity may be used to discriminate the
within the limit allowed for floral honeys (0.6%), Eucalyptus monofloral from the multifloral honey
indicating clearness of honey samples and possibly samples, independently to have or not Eucalyptus
lack of adulterations with molasses [1]. pollen, suggesting that mineral content is highly
dependent on the type of flower used by bees.
The electrical conductivity of honey is closely
related to the concentration of mineral salts, organic Acknowledgment
acids and proteins. This parameter shows great
variability according to the floral origin and it is
 L.R. Silva et al. / Microchemical Journal 93 (2009) 7377 6

This work was supported by Programa Apicola [4] L. Azeredo, M.A.A. Azeredo, S.R. de
2006. Lus R. Silva is indebted to Eng. Nelson Souza, V.M.L. Dutra, Protein contents and
Miranda and to Eng. Andreia Chasqueira, from physicochemical properties in honey samples of
Associao de Apicultores do Litoral Centro (Luso), Apis mellifera of different floral origins, Food
for supplying samples. Chem. 80 (2003) 249254.
References [5] P.B. Andrade, M.T. Amaral, P. Isabel, J.
Carvalho, R. Seabra, A. Cunha, Physicochemical
[1] E. Mendes, E. Brojo-Proena, I.M.P.L.V.O. attributes and pollen spectrum of Portuguese heather
Ferreira, M.A. Ferreira, Quality evaluation of honeys, Food Chem. 66 (1999) 503510.
Portuguese honey, Carbohydr. Polym. 37 (1998) [6] R.C. Martins, V.V. Lopes, P. Valento,
219223. J.C.M.F. Carvalho, P. Isabel, M.T. Amaral, M.T.
[2] M.L. Gonzlez-Miret, A. Terrab, D. Batista, P.B. Andrade, Relevant principal component
Hernanz, M.A. Fernndez-Recamales, F.J. Heredia, analysis applied to the
Multivariate correlation between color and mineral characterisation of Portuguese heather honey,
composition of honeys and by their botanical origin, Nat. Prod. Res. 17 (2008) 15601582.
J. Agric. Food Chem. 53 (2005) 25742580.
[7] J.W. White, Composition of Honey, in: E.
[3] E. Anklam, A review of the analytical Crane (Ed.), Honey: A Comprehensive Survey,
methods to determine the geographical and botanical Heinemann, London, 1979, pp. 157158.
origin of honey, Food Chem. 63 (1998) 549562.

Ilustracin 3: Means of ash, electrical conductivity and magnesiumvalues for three different honey
groups, considering floral origin, particularly multifloral without
Eucalyptus,monofloralEucalyptus and multifloral with Eucalyptus
Ilustracin 2: Means of ash, electrical conductivity and magnesiumvalues for three different honey
groups, considering floral origin, particularly multifloral without
Eucalyptus,monofloralEucalyptus and multifloral with Eucalyptus
7 Microchemical Journal 93 (2009) 7377

[8] M. Al-Mamary, M. Al-Meeri, M. Al- and alternative medicine in children with asthma,
Habori, Antioxidant activities and total phenolics of Ann. Allergy Asthma Immunol. 90 (2003) 611615.
different types of honey, Nutr. Res. 22 (2002) 1041 [10] A. Terrab, A.G. Gonzlez, M.J. Dez, F.J.
1047. Heredia, Characterization of Moroccan uniforal
[9] F. Orhan, B.E. Sekerel, C.N. Kocabas, C. honeys using multivariate analysis, Eur. Food Res.
Sackesen, G. Adalioglu, A. Tuncer, Complementary Technol. 218 (2003) 8895.

Ilu
 L.R. Silva et al. / Microchemical Journal 93 (2009) 7377 8

Sumario
1 Introduction....................................................................................................................1

2. Materials and methods...................................................................................................2

2.1. Sample collection........................................................................................................2

2.2. Pollen analysis.............................................................................................................2

2.3. Physicochemical characteristics..................................................................................2

2.3.1. pH.............................................................................................................................2

2.3.2. Moisture content......................................................................................................2

2.3.3. Sugar........................................................................................................................2

2.3.4. Ash............................................................................................................................2

2.3.5. Electrical conductivity..............................................................................................4

2.3.6. Free, lactonic and total acidity.................................................................................4

2.3.7. Diastase activity.......................................................................................................4

2.3.8. Hydroxymethylfurfural content (HMF).....................................................................4

2.4. Determination of mineral elements.............................................................................4

2.5. Statistical analysis.......................................................................................................4

3. Results and discussion...................................................................................................4

4. Conclusions....................................................................................................................5

Acknowledgment................................................................................................................5

References..........................................................................................................................6