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Honey from Luso region (Portugal): Physicochemical characteristics and mineral contents
Microchemical Journal 93 (2009) 73–77
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Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
Honey from Luso region (Portugal): Physicochemical characteristics and mineral contents Luís R. Silva a, Romeu Videira b, Andreia P. Monteiro b, Patrícia Valentão a, Paula B. Andrade a,⁎ a b
REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal CI & DETS / Departamento de Ambiente, Escola Superior de Tecnologia do Instituto Politécnico de Viseu, Viseu, Portugal
a r t i c l e
i n f o
Article history: Received 29 April 2009 Accepted 3 May 2009 Available online 13 May 2009 Keywords: Luso honey Pollen analysis Physicochemical analysis Mineral content
a b s t r a c t This work was conducted to evaluate the quality of 38 honey samples from Luso region (Portugal), and to study the relation between Eucalyptus pollen and chemical properties of honey. Mean values obtained for physicochemical parameters were: pH 3.83; 16.65% moisture; 80.7 °Brix sugar; 0.35% ash; 419.6 μS cm− 1 electrical conductivity; 21.5 meq/kg free acidity; 9.6 meq/kg lactonic acidity; 31.2 meq/kg total acidity; 9.41 mg/kg HMF and 18.3° Gothe diastase activity. The mineral content was determined by atomic absorption spectrometry, and in the analysed samples, potassium was the major 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 by Apis mellifera bees from nectar of plants (nectar honey), from secretions of livings parts of plants or excretions of plant-sucking insects of the living part of plants (honeydew honey). This natural complex foodstuff is produced in almost every country and largely used as food source. Honey cannot be considered a complete food by human nutritional standards, but it offers potential as a dietary supplement [1]. For infants, senior citizens and invalids, honey can be a more easily digested and more palatable carbohydrate food than saccharose by itself. Honey mainly contains simple sugars or monosaccharides (of which fructose and glucose are the main components (65%)) and 18% of water, approximately [2]. Proteins, flavour and aroma, phenolic compounds (phenolic acids and flavonoids), free amino acids, organics acids, vitamins and minerals constitute minor components of honeys [2]. Honey commercially available varies greatly in quality all over the world. This is largely assessed on the basis of colour, flavour and density. Honey composition is influenced by the plant species, climate, environmental conditions and the contribution of the beekeeper [3,4]. In general, monofloral honeys are more expensive than multifloral ones [5]. In addition, some monofloral honeys are
⁎ Corresponding author. Tel.: +351 222078934; fax: +351 222003977. E-mail address: [email protected] (P.B. Andrade). 0026-265X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.microc.2009.05.005
more appreciated than others due to their organoleptic properties or their pharmacological attributes [6]. Honey has been reported to contain about 200 substances and is considered as an important part of traditional medicine [7]. It has been used in ethnomedicine since the early humans, and in more recent times its role in the treatment of burns, gastrointestinal disorders, asthma, infected wounds and skin ulcers have also been reported [8,9]. Several types of honey are produced in Portugal, where honey production is a traditional practice well implanted in several regions. Luso region is located in the centre of Portugal, being one of the most important region of honey production in this country, due to its edafoclimatic conditions and plants diversity, were Eucalyptus pollen predominates. The detailed characterization of the different honey type's existent in Portugal is important, once it will allow the establishment of technical specifications, avoiding occurrence of adulterations. Due to adulteration possibility, honey quality must be analytically controlled with the aim of guaranteeing its speculation. On the other hand, as consumers have been incrementing their interest in monofloral honeys in detriment of multifloral ones [10], pollen analysis is important for the commercial valorisation of honey. The work herein was conducted to investigate the quality of 38 different samples of honey proceeding from Luso region. For this purpose, pollen analysis was performed and physicochemical characteristics (pH, moisture, sugar, ash content, electrical conductivity, free, lactonic and total acidity, diastase activity and hydroxymethylfurfural) and mineral contents (K, Na, Ca and Mg) evaluated.
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2. Materials and methods 2.1. Sample collection Honey samples were collected in Luso province (centre region of Portugal). Sampling area covered the most important production zones (Table 1). Samples were stored at 0 °C until analysis, which occurred no longer than one month after extraction from the hives by beekeepers. 2.2. Pollen analysis The botanical origin of the samples was determined using techniques described before [11]. For floral identification, 5 g of diluted honey sample was centrifuged at 10,000 rpm for 15 min, to separate the pollens. Samples of separated pollen grains were spread with the help of a brush on a slide containing a drop of lactophenol. The slides were examined microscopically at 45×, using a bright-field microscope (Olympus, Tokyo). 2.3. Physicochemical characteristics Honey were analysed according to methods previously reported for pH, moisture, Brix, ash content, electrical conductivity, free, lactonic and total acidity, diastase activity, hydroxymethylfurfural determination [12]. Two replicate analyses were performed for each sample.
Table 1 Classification of honey samples. Sample identification
Honey type
H1 H2 H3 H4 H5 H6 H7 H8
Multifloral (Erica, Rubus, Castanea sativa) Multifloral (Erica, Rubus, Raphanus raphanistrium) Multifloral (Echium plantagineum, Cytisus scoparius, Rubus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rhamnus) Monofloral (Eucalyptus) Monofloral (Lavandula stoechas) Multifloral (Raphanus raphanistrium, Rubus, Echium plantagineum) Multifloral (Cytisus scoparius, Lavandula stoechas, Rubus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Echium plantagineum, Cytisus scoparius, Rubus) Multifloral (Erica, Eucalyptus, Rubus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Rubus, Erica, Eucalyptus) Multifloral (Eucalyptus, Trifolium hybridum, Rubus) Multifloral (Erica arborea, Eucalyptus, Castanea sativa) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rhamnus) Multifloral (Eucalyptus, Rubus, Echium plantagineum) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rubus) Monofloral (Cytisus scoparius) Monofloral (Erica)
H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38
2.3.1. pH The pH was measured by a pH-meter Consort C831 (USA), with a precision of ±0.002 pH units. The pH of the honey was measured in solution of 10 g honey in 75 ml of CO2 free distilled water. 2.3.2. Moisture content Moisture was determined by refractometry, using an Atago (Japan) model lT Abbe refractometer. All measurements were performed at 25 °C. 2.3.3. Sugar Sugar content was determined with a special refractometer with direct reading display, and the results were expressed as °Brix. 2.3.4. Ash Ash content was measured by calcination, overnight, in furnace at 550 °C, until constant mass. 2.3.5. Electrical conductivity Electrical conductivity of a honey solution at 20% (dry matter basis) in CO2-free deionised distilled water, was measured at 20 °C in a Consort C831 conductimeter, and the results were expressed as μS cm− 1. 2.3.6. Free, lactonic and total acidity Free, lactonic and total acidity were determined as follows, by titrimetric method: the addition of 0.05 M NaOH was stopped at pH 8.50 (free acidity), immediately a volume of 10 ml 0.05 M NaOH was added and, without delay, back-titrated with 0.05 M HCl to pH 8.30 (lactonic acidity). Total acidity results were obtained by adding free and lactone acidities. 2.3.7. Diastase activity Diastase activity was measured using a buffered soluble starch solution and honey, which was incubated in the thermostatic bath at 40 °C. Absorption was followed using a Perkin Elmer 25 UV/VIS spectrophotometer and a chronometer. Using regression (without using the data point at 0 min), lines were fitted to the absorption data and the diastase number was calculated from the time taken for the absorbance to reach 0.235. For samples of low diastase activity, the regression was made on the basis of the last three data points to improve the linear correlation. In samples of high diastase activity the time taken for the absorbance to reach 0.235 was determined with absorbance at 5 and 10, 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 550 °C, of approximately 5 g honey sample, until constant weight [13]. Five milliliters of nitric acid 0.1 M were added to the resultant ashes, and the mixture was stirred on a heating plate to almost complete dryness. Then, 10 ml of the same acid was added and the mixture was made up to 25 ml with distilled water. Calcium, potassium, sodium and magnesium were determined by atomic absorption spectrometry (Perkin Elmer AAnalyst 300), using an air/acetylene flame. Quantitative determination of the elements by atomic absorption spectrometry was carried out after calibrating the instrument, using Ca (1 to 5 mg/l), K (0.1 to 2 mg/l), Na (0.1 to 2 mg/l), Zn (0.05 to 1 mg/l) and Mg (1 to 10 mg/l)
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Table 2 Distribution data for physicochemical parameters in Luso (Portugal) honey samples. Sample
pH
Moisture (%)
°Brix (%)
Ash (%)
Electrical conductivity (μS cm− 1)
Free Acidity (meq/kg)
Lactonic acidity (meq/kg)
Total acidity (meq/kg)
HMF (mg/kg)
Diastase activity (° Gothe)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 Mean SD Minimum Maximum
3.88 4.31 4.34 3.74 4.12 4.23 4.25 3.78 3.83 3.90 4.70 4.16 4.30 4.32 3.82 4.07 4.01 4.11 3.55 3.99 3.57 3.46 3.60 3.46 3.60 3.71 3.60 3.60 3.54 3.79 3.52 3.54 3.64 3.45 3.81 3.94 3.91 4.26 3.88 0.32 3.45 4.70
17.04 15.35 15.83 14.82 15.27 15.65 13.52 13.98 14.49 15.71 14.98 14.67 14.30 17.04 15.70 15.60 15.00 14.51 18.90 18.00 16.80 19.00 19.20 19.10 18.00 19.00 17.00 16.90 17.30 16.90 19.40 16.70 16.20 17.60 17.60 18.20 19.70 17.80 16.65 1.71 13.52 19.70
80.6 80.6 80.6 81.2 80.2 80.4 82.2 80.8 81.0 80.4 80.6 80.4 82.0 80.6 79.8 79.6 81.6 81.0 80.4 81.0 82.0 80.4 79.6 79.8 81.0 79.8 82.0 81.4 80.2 81.2 79.0 82.0 81.2 81.0 81.0 80.0 79.0 80.3 80.7 0.8 79.0 82.2
0.39 0.26 0.25 0.34 0.22 0.35 0.09 0.14 0.34 0.35 0.32 0.35 0.35 0.17 0.36 0.41 0.32 0.27 0.39 0.53 0.38 0.35 0.43 0.41 0.35 0.42 0.36 0.37 0.37 0.31 0.34 0.39 0.39 0.34 0.46 0.46 0.37 0.49 0.35 0.09 0.09 0.53
473.5 318.2 301.3 412.0 263.2 418.2 114.7 168.8 415.1 420.3 385.2 417.4 420.1 206.2 436.3 497.4 391.5 323.3 477.5 636.5 462.2 422.0 517.5 496.0 429.5 506.0 437.0 443.5 446.0 373.5 411.0 474.5 478.0 410.0 555.5 553.0 441.0 594.5 419.6 106.9 114.7 636.5
33.8 26.1 22.9 29.7 25.1 26.1 16.4 12.9 38.1 17.1 24.0 20.0 18.2 19.9 25.0 26.5 12.6 10.7 24.5 19.5 19.5 22.0 27.5 20.5 17.0 22.5 19.0 18.0 18.5 10.5 18.0 20.0 20.0 19.0 17.0 35.0 30.0 15.5 21.5 6.4 10.5 38.1
8.3 7.0 10.5 9.5 6.2 4.2 4.7 4.5 9.0 11.5 5.2 5.3 6.1 9.3 14.3 10.4 9.7 10.7 11.0 8.5 15.0 11.5 11.5 8.5 8.5 15.0 10.0 10.5 12.0 6.5 8.0 15.0 12.5 12.5 8.0 16.5 11.5 5.0 9.6 3.2 4.2 16.5
42.0 33.1 33.4 39.2 31.2 30.3 21.1 17.4 47.1 28.6 29.2 25.3 24.4 29.2 39.2 36.9 22.2 21.4 35.5 28.0 34.5 33.5 39.0 29.0 25.5 37.5 29.0 28.5 30.5 17.0 26.0 35.0 32.5 31.5 25.0 51.5 41.5 20.5 31.2 7.8 17.0 51.5
1.75 5.90 11.75 3.11 2.45 6.95 5.35 4.35 14.60 6.35 7.22 5.82 2.54 15.54 8.50 8.36 5.09 9.08 10.00 6.38 9.31 15.05 9.10 12.25 15.80 8.10 5.09 8.84 17.23 14.70 25.45 10.26 4.58 32.75 13.85 2.94 6.56 4.63 9.41 6.35 1.75 32.75
23 14 4 6 10 13 5 5 27 16 8 19 16 3 17 15 15 18 27 22 26 29 25 33 21 24 23 23 27 19 7 21 38 11 20 25 28 14 18.3 8.6 3 38
SD: standard deviation.
solutions dissolved in 0.1% lanthanum (La). La was utilized as a matrix modifier in order to overcome the chemical interferences in the air/ acetylene flame. All samples were analysed in triplicate.
The results obtained for the several physicochemical parameters determined are presented in Table 2. Honey pH is affected by the conditions during extraction and storage, which also influences texture, stability and shelf-life. pH is indeed a useful index of possible
2.5. Statistical analysis Data are represented as mean ± standard deviation. The results were statistically analysed by analysis of variance (ANOVA) methodology followed by Fisher's PLSD test. Differences were considered significant for p b 0.05. 3. Results and discussion Table 1 shows the floral origin of honey samples determined by microscopy pollen analyses. Data indicate that 63% of honey samples were monofloral and 37% were multifloral. Eucalyptus sp. was a predominant source used by honeybees in the Luso region, once Eucalyptus pollen was detected in 79% of the total analysed samples. Furthermore, 92% samples of monofloral honey were from Eucalyptus sp., 4% were from Erica sp. and 4% from Cytisus scoparius. Multifloral honeys contained several pollen types with a considerable percentage of pollen grains from Eucalyptus sp., Erica sp., Rubus sp., Lavandula stoechas, Castanea sativa and C. scoparius.
Fig. 1. Linear regression of ash content (% w/w) and conductivity (μS cm− 1).
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Table 3 Distribution data for cationic mineral content in Luso (Portugal) honey samples. Sample
Ca (mg/kg)
K (mg/kg)
Mg (mg/kg)
Na (mg/kg)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 Mean SD Minimum Maximum
10.80 6.24 17.70 85.33 15.07 82.33 13.38 48.84 134.35 68.40 19.90 71.65 58.65 16.49 16.27 54.80 106.91 79.17 80.47 49.40 122.45 107.25 62.68 106.10 85.45 37.01 91.65 104.37 50.20 56.02 53.33 75.13 68.55 52.55 44.15 15.96 71.41 35.14 59.88 33.97 6.24 134.35
1196.30 1013.50 436.56 1520.60 653.02 397.17 117.55 188.98 798.48 1016.20 958.25 670.00 809.81 1645.00 1097.50 2040.50 1320.20 1200.00 891.04 1564.60 1115.40 1305.70 1813.10 1906.70 718.75 1732.30 1274.20 928.76 850.00 977.69 906.38 1324.60 966.25 1303.70 1187.50 2590.60 1600.60 1667.00 1150.10 513.26 117.55 2590.60
36.23 34.97 20.66 34.30 28.27 25.04 12.61 10.63 46.66 28.69 47.87 36.91 35.47 22.07 41.13 44.66 25.25 25.10 26.36 70.00 35.86 27.64 39.74 42.88 25.25 45.13 30.69 30.70 48.84 37.00 40.92 39.46 42.05 38.19 35.49 70.41 31.97 36.78 35.57 12.21 10.62 70.41
272.25 209.94 244.02 667.39 253.76 151.62 95.029 100.38 225.92 233.00 534.72 224.50 228.59 90.224 629.27 727.78 181.91 147.35 154.00 464.01 271.53 195.41 244.66 266.88 168.75 242.21 184.20 138.63 256.50 183.10 236.71 175.20 430.50 203.25 169.49 455.79 101.22 174.45 261.43 158.31 90.22 727.79
SD: standard deviation.
microbial growth, since most bacteria grow in a neutral and mildly alkaline environment, while yeasts and moulds are capable of developing in an acidic environment (pH = 4.0–4.5) and do not
grow well in alkaline media [14]. The pH values of the analysed honey samples ranged from 3.45 to 4.70 (mean value = 3.88). These values are in accordance with acceptable range for honey [15] and similar to those obtained with others Portuguese honeys [5]. Percent moisture in the analysed honeys ranged from 13.53 to 19.70 (mean value = 16.65). The water content of honey depends on various factors, like the harvesting season, the degree of maturity reached in the hive and climatic factors. The maximum amount of water contained by honey is regulated for safety against fermentation. All the samples contained less than 20% water, the maximum amount allowed by international and Portuguese legislations [16]. Moisture and sugar content are strictly correlated and anomalous values of Brix degrees (directly related with sugar content) may be a reliable index of adulteration [13,14]. The analysed samples presented Brix degrees ranging from 79.0 to 82.2 (average = 80.7), which are similar to those from others Portuguese honey samples [5]. Ash content is a parameter used for the determination of the botanical origin (floral, mix or honeydew) [17]. The results found (0.09–0.53%) are within the limit allowed for floral honeys (0.6%), indicating clearness of honey samples and possibly lack of adulterations with molasses [1]. The electrical conductivity of honey is closely related to the concentration of mineral salts, organic acids and proteins. This parameter shows great variability according to the floral origin and it is important for the differentiation of honeys of different floral origins [18]. The results obtained for the honey samples under study varied between 114.7 and 636.5 μS cm− 1 (average = 419.6 μS cm− 1). These values are below the maximum limit indicated by Portuguese legislation for nectar honey (800 μS cm− 1). The increase in ash content of the honey samples from Luso region was accompanied by the increase of electrical conductivity, as previously reported by others [19,20]. This linear relationship is characterised by a correlation coefficient R equal to 0.99 (Fig. 1). Honey acidity is due to the presence of organic acids, mainly gluconic acid, in equilibrium with their corresponding lactones or internal esters, and to inorganic ions, such as phosphate, sulphate and chloride [13,21]. The lactonic acidity is considered as the acidity reserve when the honey becomes alkaline, while the total acidity is the sum of free and lactonic acidities [18]. Free acidity was within the limits of Portuguese and European legislations (below 50 meq/kg), indicating the absence of undesirable fermentation. Lactonic acidity ranged were from 4.2 to 16.5 meq/kg (average = 9.6 meq/kg). Total acidity varied between 17.0 and 51.5 meq/kg, with a mean value of 31.2 meq/kg. The results obtained for acidity were in agreement with data reported for other Portuguese honeys [1,5] as well as for samples
Fig. 2. Means of ash, electrical conductivity and magnesium values for three different honey groups, considering floral origin, particularly multifloral without Eucalyptus, monofloral Eucalyptus and multifloral with Eucalyptus.
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from other geographical locations [13,18,22–24]. The variation of total acidity has been attributed to harvest season [25]. HMF content is widely recognized as parameter of freshness for honey samples. Several factors influence the formation of HMF, such as storage conditions (e.g. temperature) and floral sources [18,26]. It is well known that honey heating results in the formation of HMF, which is produced during acid-catalysed dehydratation of hexoses, such as fructose and glucose [27]. The amounts found fell within the European legislation, corresponding to a high degree of freshness, which was in agreement with the information provided by the producers: all samples presented HMF levels below 40 mg/kg of honey, ranging from 1.75 to 32.75 mg/kg (average = 9.41 mg/kg). Diastase activity is a parameter used to determine if honey has been extensively heated during processing, because the enzyme is susceptible to heating and storage factors. The herein honey samples exhibited very different values, ranging between 3 and 38° Gothe, and only six samples presented an inappropriate diastase activity (Table 2), with values below 8° Gothe [16], suggesting inadequate storage or processing. The mineral content is an important index of possible environmental pollution and a potential indicator of geographical origin of honey [3]. The results of the cationic metals determined in Luso honey samples are summarized in Table 3. Potassium was quantitatively the most important mineral, since, in mean, it accounted for 76% of the total mineral, with an average content of 1150.1 ppm. Studies from other geographical locations also revealed potassium to be the most abundant element [17,19,20,28]. Sodium, calcium and magnesium were present in moderate amounts in the honey samples, with average contents of 261.43, 59.88 and 35.57 ppm, respectively. Thus, sodium accounted for 17%, calcium for 4% and magnesium represented 3% of total quantified minerals. The mineral contents are similar to those described previously [20,29]. To evaluate the possible relationship between floral origin and chemical contents of honey, the samples were segregated in three different brands considering the floral pollen analysis, namely: monofloral Eucalyptus honey, multifloral honey with Eucalyptus and multifloral honey without Eucalyptus. Significant differences among the honey brands were performed by variance analysis (ANOVA), followed by Fisher's PLSD test. Fig. 2 plots the mean and standard deviation of the chemical parameters that show significant differences between monofloral Eucalyptus honey and multifloral honey with or without Eucalyptus. Data of Fig. 2 revealed that the values found for Mg, ash and electrical conductivity in multifloral honey without Eucalyptus are lower than those of monofloral or multifloral honey with Eucalyptus. Thus, Mg, ash and electrical conductivity are chemical parameters that can be used to discriminate the monofloral Eucalyptus honey samples from the multifloral, independently to have or not Eucalyptus pollen. Additionally, the results obtained for the other parameters analysed in the present work are important to characterise the properties the honey samples from the Luso Portugal region, but not to be used to differentiate the floral origin. 4. Conclusions Honeys from Luso region present a good level of quality, once 32 of the 38 analysed samples are in agreement with the European honey directive [30] and Portuguese legislation [16], indicating adequate processing, good maturity and freshness. Six samples did not fit within European and Portuguese standards relative to the diastase activity, reflecting inadequate sample manufacture and/or storage. Potassium is the most abundant of the determined elements. Magnesium and ash contents and electrical conductivity may be used to discriminate the Eucalyptus monofloral from the multifloral honey samples, independently to have or not Eucalyptus pollen, suggesting that mineral content is highly dependent on the type of flower used by bees.
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Acknowledgment This work was supported by Programa Apicola 2006. Luís R. Silva is indebted to Eng. Nelson Miranda and to Eng. Andreia Chasqueira, from Associação de Apicultores do Litoral Centro (Luso), for supplying samples. References [1] E. Mendes, E. Brojo-Proença, I.M.P.L.V.O. Ferreira, M.A. Ferreira, Quality evaluation of Portuguese honey, Carbohydr. Polym. 37 (1998) 219–223. [2] M.L. González-Miret, A. Terrab, D. Hernanz, M.A. Fernández-Recamales, F.J. Heredia, Multivariate correlation between color and mineral composition of honeys and by their botanical origin, J. Agric. Food Chem. 53 (2005) 2574–2580. [3] E. Anklam, A review of the analytical methods to determine the geographical and botanical origin of honey, Food Chem. 63 (1998) 549–562. [4] L. Azeredo, M.A.A. Azeredo, S.R. de Souza, V.M.L. Dutra, Protein contents and physicochemical properties in honey samples of Apis mellifera of different floral origins, Food Chem. 80 (2003) 249–254. [5] P.B. Andrade, M.T. Amaral, P. 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Contents lists available at ScienceDirect
Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
Honey from Luso region (Portugal): Physicochemical characteristics and mineral contents Luís R. Silva a, Romeu Videira b, Andreia P. Monteiro b, Patrícia Valentão a, Paula B. Andrade a,⁎ a b
REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal CI & DETS / Departamento de Ambiente, Escola Superior de Tecnologia do Instituto Politécnico de Viseu, Viseu, Portugal
a r t i c l e
i n f o
Article history: Received 29 April 2009 Accepted 3 May 2009 Available online 13 May 2009 Keywords: Luso honey Pollen analysis Physicochemical analysis Mineral content
a b s t r a c t This work was conducted to evaluate the quality of 38 honey samples from Luso region (Portugal), and to study the relation between Eucalyptus pollen and chemical properties of honey. Mean values obtained for physicochemical parameters were: pH 3.83; 16.65% moisture; 80.7 °Brix sugar; 0.35% ash; 419.6 μS cm− 1 electrical conductivity; 21.5 meq/kg free acidity; 9.6 meq/kg lactonic acidity; 31.2 meq/kg total acidity; 9.41 mg/kg HMF and 18.3° Gothe diastase activity. The mineral content was determined by atomic absorption spectrometry, and in the analysed samples, potassium was the major 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 by Apis mellifera bees from nectar of plants (nectar honey), from secretions of livings parts of plants or excretions of plant-sucking insects of the living part of plants (honeydew honey). This natural complex foodstuff is produced in almost every country and largely used as food source. Honey cannot be considered a complete food by human nutritional standards, but it offers potential as a dietary supplement [1]. For infants, senior citizens and invalids, honey can be a more easily digested and more palatable carbohydrate food than saccharose by itself. Honey mainly contains simple sugars or monosaccharides (of which fructose and glucose are the main components (65%)) and 18% of water, approximately [2]. Proteins, flavour and aroma, phenolic compounds (phenolic acids and flavonoids), free amino acids, organics acids, vitamins and minerals constitute minor components of honeys [2]. Honey commercially available varies greatly in quality all over the world. This is largely assessed on the basis of colour, flavour and density. Honey composition is influenced by the plant species, climate, environmental conditions and the contribution of the beekeeper [3,4]. In general, monofloral honeys are more expensive than multifloral ones [5]. In addition, some monofloral honeys are
⁎ Corresponding author. Tel.: +351 222078934; fax: +351 222003977. E-mail address: [email protected] (P.B. Andrade). 0026-265X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.microc.2009.05.005
more appreciated than others due to their organoleptic properties or their pharmacological attributes [6]. Honey has been reported to contain about 200 substances and is considered as an important part of traditional medicine [7]. It has been used in ethnomedicine since the early humans, and in more recent times its role in the treatment of burns, gastrointestinal disorders, asthma, infected wounds and skin ulcers have also been reported [8,9]. Several types of honey are produced in Portugal, where honey production is a traditional practice well implanted in several regions. Luso region is located in the centre of Portugal, being one of the most important region of honey production in this country, due to its edafoclimatic conditions and plants diversity, were Eucalyptus pollen predominates. The detailed characterization of the different honey type's existent in Portugal is important, once it will allow the establishment of technical specifications, avoiding occurrence of adulterations. Due to adulteration possibility, honey quality must be analytically controlled with the aim of guaranteeing its speculation. On the other hand, as consumers have been incrementing their interest in monofloral honeys in detriment of multifloral ones [10], pollen analysis is important for the commercial valorisation of honey. The work herein was conducted to investigate the quality of 38 different samples of honey proceeding from Luso region. For this purpose, pollen analysis was performed and physicochemical characteristics (pH, moisture, sugar, ash content, electrical conductivity, free, lactonic and total acidity, diastase activity and hydroxymethylfurfural) and mineral contents (K, Na, Ca and Mg) evaluated.
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2. Materials and methods 2.1. Sample collection Honey samples were collected in Luso province (centre region of Portugal). Sampling area covered the most important production zones (Table 1). Samples were stored at 0 °C until analysis, which occurred no longer than one month after extraction from the hives by beekeepers. 2.2. Pollen analysis The botanical origin of the samples was determined using techniques described before [11]. For floral identification, 5 g of diluted honey sample was centrifuged at 10,000 rpm for 15 min, to separate the pollens. Samples of separated pollen grains were spread with the help of a brush on a slide containing a drop of lactophenol. The slides were examined microscopically at 45×, using a bright-field microscope (Olympus, Tokyo). 2.3. Physicochemical characteristics Honey were analysed according to methods previously reported for pH, moisture, Brix, ash content, electrical conductivity, free, lactonic and total acidity, diastase activity, hydroxymethylfurfural determination [12]. Two replicate analyses were performed for each sample.
Table 1 Classification of honey samples. Sample identification
Honey type
H1 H2 H3 H4 H5 H6 H7 H8
Multifloral (Erica, Rubus, Castanea sativa) Multifloral (Erica, Rubus, Raphanus raphanistrium) Multifloral (Echium plantagineum, Cytisus scoparius, Rubus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rhamnus) Monofloral (Eucalyptus) Monofloral (Lavandula stoechas) Multifloral (Raphanus raphanistrium, Rubus, Echium plantagineum) Multifloral (Cytisus scoparius, Lavandula stoechas, Rubus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Echium plantagineum, Cytisus scoparius, Rubus) Multifloral (Erica, Eucalyptus, Rubus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Rubus, Erica, Eucalyptus) Multifloral (Eucalyptus, Trifolium hybridum, Rubus) Multifloral (Erica arborea, Eucalyptus, Castanea sativa) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rhamnus) Multifloral (Eucalyptus, Rubus, Echium plantagineum) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Monofloral (Eucalyptus) Multifloral (Eucalyptus, Erica, Rubus) Monofloral (Cytisus scoparius) Monofloral (Erica)
H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38
2.3.1. pH The pH was measured by a pH-meter Consort C831 (USA), with a precision of ±0.002 pH units. The pH of the honey was measured in solution of 10 g honey in 75 ml of CO2 free distilled water. 2.3.2. Moisture content Moisture was determined by refractometry, using an Atago (Japan) model lT Abbe refractometer. All measurements were performed at 25 °C. 2.3.3. Sugar Sugar content was determined with a special refractometer with direct reading display, and the results were expressed as °Brix. 2.3.4. Ash Ash content was measured by calcination, overnight, in furnace at 550 °C, until constant mass. 2.3.5. Electrical conductivity Electrical conductivity of a honey solution at 20% (dry matter basis) in CO2-free deionised distilled water, was measured at 20 °C in a Consort C831 conductimeter, and the results were expressed as μS cm− 1. 2.3.6. Free, lactonic and total acidity Free, lactonic and total acidity were determined as follows, by titrimetric method: the addition of 0.05 M NaOH was stopped at pH 8.50 (free acidity), immediately a volume of 10 ml 0.05 M NaOH was added and, without delay, back-titrated with 0.05 M HCl to pH 8.30 (lactonic acidity). Total acidity results were obtained by adding free and lactone acidities. 2.3.7. Diastase activity Diastase activity was measured using a buffered soluble starch solution and honey, which was incubated in the thermostatic bath at 40 °C. Absorption was followed using a Perkin Elmer 25 UV/VIS spectrophotometer and a chronometer. Using regression (without using the data point at 0 min), lines were fitted to the absorption data and the diastase number was calculated from the time taken for the absorbance to reach 0.235. For samples of low diastase activity, the regression was made on the basis of the last three data points to improve the linear correlation. In samples of high diastase activity the time taken for the absorbance to reach 0.235 was determined with absorbance at 5 and 10, 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 550 °C, of approximately 5 g honey sample, until constant weight [13]. Five milliliters of nitric acid 0.1 M were added to the resultant ashes, and the mixture was stirred on a heating plate to almost complete dryness. Then, 10 ml of the same acid was added and the mixture was made up to 25 ml with distilled water. Calcium, potassium, sodium and magnesium were determined by atomic absorption spectrometry (Perkin Elmer AAnalyst 300), using an air/acetylene flame. Quantitative determination of the elements by atomic absorption spectrometry was carried out after calibrating the instrument, using Ca (1 to 5 mg/l), K (0.1 to 2 mg/l), Na (0.1 to 2 mg/l), Zn (0.05 to 1 mg/l) and Mg (1 to 10 mg/l)
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Table 2 Distribution data for physicochemical parameters in Luso (Portugal) honey samples. Sample
pH
Moisture (%)
°Brix (%)
Ash (%)
Electrical conductivity (μS cm− 1)
Free Acidity (meq/kg)
Lactonic acidity (meq/kg)
Total acidity (meq/kg)
HMF (mg/kg)
Diastase activity (° Gothe)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 Mean SD Minimum Maximum
3.88 4.31 4.34 3.74 4.12 4.23 4.25 3.78 3.83 3.90 4.70 4.16 4.30 4.32 3.82 4.07 4.01 4.11 3.55 3.99 3.57 3.46 3.60 3.46 3.60 3.71 3.60 3.60 3.54 3.79 3.52 3.54 3.64 3.45 3.81 3.94 3.91 4.26 3.88 0.32 3.45 4.70
17.04 15.35 15.83 14.82 15.27 15.65 13.52 13.98 14.49 15.71 14.98 14.67 14.30 17.04 15.70 15.60 15.00 14.51 18.90 18.00 16.80 19.00 19.20 19.10 18.00 19.00 17.00 16.90 17.30 16.90 19.40 16.70 16.20 17.60 17.60 18.20 19.70 17.80 16.65 1.71 13.52 19.70
80.6 80.6 80.6 81.2 80.2 80.4 82.2 80.8 81.0 80.4 80.6 80.4 82.0 80.6 79.8 79.6 81.6 81.0 80.4 81.0 82.0 80.4 79.6 79.8 81.0 79.8 82.0 81.4 80.2 81.2 79.0 82.0 81.2 81.0 81.0 80.0 79.0 80.3 80.7 0.8 79.0 82.2
0.39 0.26 0.25 0.34 0.22 0.35 0.09 0.14 0.34 0.35 0.32 0.35 0.35 0.17 0.36 0.41 0.32 0.27 0.39 0.53 0.38 0.35 0.43 0.41 0.35 0.42 0.36 0.37 0.37 0.31 0.34 0.39 0.39 0.34 0.46 0.46 0.37 0.49 0.35 0.09 0.09 0.53
473.5 318.2 301.3 412.0 263.2 418.2 114.7 168.8 415.1 420.3 385.2 417.4 420.1 206.2 436.3 497.4 391.5 323.3 477.5 636.5 462.2 422.0 517.5 496.0 429.5 506.0 437.0 443.5 446.0 373.5 411.0 474.5 478.0 410.0 555.5 553.0 441.0 594.5 419.6 106.9 114.7 636.5
33.8 26.1 22.9 29.7 25.1 26.1 16.4 12.9 38.1 17.1 24.0 20.0 18.2 19.9 25.0 26.5 12.6 10.7 24.5 19.5 19.5 22.0 27.5 20.5 17.0 22.5 19.0 18.0 18.5 10.5 18.0 20.0 20.0 19.0 17.0 35.0 30.0 15.5 21.5 6.4 10.5 38.1
8.3 7.0 10.5 9.5 6.2 4.2 4.7 4.5 9.0 11.5 5.2 5.3 6.1 9.3 14.3 10.4 9.7 10.7 11.0 8.5 15.0 11.5 11.5 8.5 8.5 15.0 10.0 10.5 12.0 6.5 8.0 15.0 12.5 12.5 8.0 16.5 11.5 5.0 9.6 3.2 4.2 16.5
42.0 33.1 33.4 39.2 31.2 30.3 21.1 17.4 47.1 28.6 29.2 25.3 24.4 29.2 39.2 36.9 22.2 21.4 35.5 28.0 34.5 33.5 39.0 29.0 25.5 37.5 29.0 28.5 30.5 17.0 26.0 35.0 32.5 31.5 25.0 51.5 41.5 20.5 31.2 7.8 17.0 51.5
1.75 5.90 11.75 3.11 2.45 6.95 5.35 4.35 14.60 6.35 7.22 5.82 2.54 15.54 8.50 8.36 5.09 9.08 10.00 6.38 9.31 15.05 9.10 12.25 15.80 8.10 5.09 8.84 17.23 14.70 25.45 10.26 4.58 32.75 13.85 2.94 6.56 4.63 9.41 6.35 1.75 32.75
23 14 4 6 10 13 5 5 27 16 8 19 16 3 17 15 15 18 27 22 26 29 25 33 21 24 23 23 27 19 7 21 38 11 20 25 28 14 18.3 8.6 3 38
SD: standard deviation.
solutions dissolved in 0.1% lanthanum (La). La was utilized as a matrix modifier in order to overcome the chemical interferences in the air/ acetylene flame. All samples were analysed in triplicate.
The results obtained for the several physicochemical parameters determined are presented in Table 2. Honey pH is affected by the conditions during extraction and storage, which also influences texture, stability and shelf-life. pH is indeed a useful index of possible
2.5. Statistical analysis Data are represented as mean ± standard deviation. The results were statistically analysed by analysis of variance (ANOVA) methodology followed by Fisher's PLSD test. Differences were considered significant for p b 0.05. 3. Results and discussion Table 1 shows the floral origin of honey samples determined by microscopy pollen analyses. Data indicate that 63% of honey samples were monofloral and 37% were multifloral. Eucalyptus sp. was a predominant source used by honeybees in the Luso region, once Eucalyptus pollen was detected in 79% of the total analysed samples. Furthermore, 92% samples of monofloral honey were from Eucalyptus sp., 4% were from Erica sp. and 4% from Cytisus scoparius. Multifloral honeys contained several pollen types with a considerable percentage of pollen grains from Eucalyptus sp., Erica sp., Rubus sp., Lavandula stoechas, Castanea sativa and C. scoparius.
Fig. 1. Linear regression of ash content (% w/w) and conductivity (μS cm− 1).
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Table 3 Distribution data for cationic mineral content in Luso (Portugal) honey samples. Sample
Ca (mg/kg)
K (mg/kg)
Mg (mg/kg)
Na (mg/kg)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 Mean SD Minimum Maximum
10.80 6.24 17.70 85.33 15.07 82.33 13.38 48.84 134.35 68.40 19.90 71.65 58.65 16.49 16.27 54.80 106.91 79.17 80.47 49.40 122.45 107.25 62.68 106.10 85.45 37.01 91.65 104.37 50.20 56.02 53.33 75.13 68.55 52.55 44.15 15.96 71.41 35.14 59.88 33.97 6.24 134.35
1196.30 1013.50 436.56 1520.60 653.02 397.17 117.55 188.98 798.48 1016.20 958.25 670.00 809.81 1645.00 1097.50 2040.50 1320.20 1200.00 891.04 1564.60 1115.40 1305.70 1813.10 1906.70 718.75 1732.30 1274.20 928.76 850.00 977.69 906.38 1324.60 966.25 1303.70 1187.50 2590.60 1600.60 1667.00 1150.10 513.26 117.55 2590.60
36.23 34.97 20.66 34.30 28.27 25.04 12.61 10.63 46.66 28.69 47.87 36.91 35.47 22.07 41.13 44.66 25.25 25.10 26.36 70.00 35.86 27.64 39.74 42.88 25.25 45.13 30.69 30.70 48.84 37.00 40.92 39.46 42.05 38.19 35.49 70.41 31.97 36.78 35.57 12.21 10.62 70.41
272.25 209.94 244.02 667.39 253.76 151.62 95.029 100.38 225.92 233.00 534.72 224.50 228.59 90.224 629.27 727.78 181.91 147.35 154.00 464.01 271.53 195.41 244.66 266.88 168.75 242.21 184.20 138.63 256.50 183.10 236.71 175.20 430.50 203.25 169.49 455.79 101.22 174.45 261.43 158.31 90.22 727.79
SD: standard deviation.
microbial growth, since most bacteria grow in a neutral and mildly alkaline environment, while yeasts and moulds are capable of developing in an acidic environment (pH = 4.0–4.5) and do not
grow well in alkaline media [14]. The pH values of the analysed honey samples ranged from 3.45 to 4.70 (mean value = 3.88). These values are in accordance with acceptable range for honey [15] and similar to those obtained with others Portuguese honeys [5]. Percent moisture in the analysed honeys ranged from 13.53 to 19.70 (mean value = 16.65). The water content of honey depends on various factors, like the harvesting season, the degree of maturity reached in the hive and climatic factors. The maximum amount of water contained by honey is regulated for safety against fermentation. All the samples contained less than 20% water, the maximum amount allowed by international and Portuguese legislations [16]. Moisture and sugar content are strictly correlated and anomalous values of Brix degrees (directly related with sugar content) may be a reliable index of adulteration [13,14]. The analysed samples presented Brix degrees ranging from 79.0 to 82.2 (average = 80.7), which are similar to those from others Portuguese honey samples [5]. Ash content is a parameter used for the determination of the botanical origin (floral, mix or honeydew) [17]. The results found (0.09–0.53%) are within the limit allowed for floral honeys (0.6%), indicating clearness of honey samples and possibly lack of adulterations with molasses [1]. The electrical conductivity of honey is closely related to the concentration of mineral salts, organic acids and proteins. This parameter shows great variability according to the floral origin and it is important for the differentiation of honeys of different floral origins [18]. The results obtained for the honey samples under study varied between 114.7 and 636.5 μS cm− 1 (average = 419.6 μS cm− 1). These values are below the maximum limit indicated by Portuguese legislation for nectar honey (800 μS cm− 1). The increase in ash content of the honey samples from Luso region was accompanied by the increase of electrical conductivity, as previously reported by others [19,20]. This linear relationship is characterised by a correlation coefficient R equal to 0.99 (Fig. 1). Honey acidity is due to the presence of organic acids, mainly gluconic acid, in equilibrium with their corresponding lactones or internal esters, and to inorganic ions, such as phosphate, sulphate and chloride [13,21]. The lactonic acidity is considered as the acidity reserve when the honey becomes alkaline, while the total acidity is the sum of free and lactonic acidities [18]. Free acidity was within the limits of Portuguese and European legislations (below 50 meq/kg), indicating the absence of undesirable fermentation. Lactonic acidity ranged were from 4.2 to 16.5 meq/kg (average = 9.6 meq/kg). Total acidity varied between 17.0 and 51.5 meq/kg, with a mean value of 31.2 meq/kg. The results obtained for acidity were in agreement with data reported for other Portuguese honeys [1,5] as well as for samples
Fig. 2. Means of ash, electrical conductivity and magnesium values for three different honey groups, considering floral origin, particularly multifloral without Eucalyptus, monofloral Eucalyptus and multifloral with Eucalyptus.
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from other geographical locations [13,18,22–24]. The variation of total acidity has been attributed to harvest season [25]. HMF content is widely recognized as parameter of freshness for honey samples. Several factors influence the formation of HMF, such as storage conditions (e.g. temperature) and floral sources [18,26]. It is well known that honey heating results in the formation of HMF, which is produced during acid-catalysed dehydratation of hexoses, such as fructose and glucose [27]. The amounts found fell within the European legislation, corresponding to a high degree of freshness, which was in agreement with the information provided by the producers: all samples presented HMF levels below 40 mg/kg of honey, ranging from 1.75 to 32.75 mg/kg (average = 9.41 mg/kg). Diastase activity is a parameter used to determine if honey has been extensively heated during processing, because the enzyme is susceptible to heating and storage factors. The herein honey samples exhibited very different values, ranging between 3 and 38° Gothe, and only six samples presented an inappropriate diastase activity (Table 2), with values below 8° Gothe [16], suggesting inadequate storage or processing. The mineral content is an important index of possible environmental pollution and a potential indicator of geographical origin of honey [3]. The results of the cationic metals determined in Luso honey samples are summarized in Table 3. Potassium was quantitatively the most important mineral, since, in mean, it accounted for 76% of the total mineral, with an average content of 1150.1 ppm. Studies from other geographical locations also revealed potassium to be the most abundant element [17,19,20,28]. Sodium, calcium and magnesium were present in moderate amounts in the honey samples, with average contents of 261.43, 59.88 and 35.57 ppm, respectively. Thus, sodium accounted for 17%, calcium for 4% and magnesium represented 3% of total quantified minerals. The mineral contents are similar to those described previously [20,29]. To evaluate the possible relationship between floral origin and chemical contents of honey, the samples were segregated in three different brands considering the floral pollen analysis, namely: monofloral Eucalyptus honey, multifloral honey with Eucalyptus and multifloral honey without Eucalyptus. Significant differences among the honey brands were performed by variance analysis (ANOVA), followed by Fisher's PLSD test. Fig. 2 plots the mean and standard deviation of the chemical parameters that show significant differences between monofloral Eucalyptus honey and multifloral honey with or without Eucalyptus. Data of Fig. 2 revealed that the values found for Mg, ash and electrical conductivity in multifloral honey without Eucalyptus are lower than those of monofloral or multifloral honey with Eucalyptus. Thus, Mg, ash and electrical conductivity are chemical parameters that can be used to discriminate the monofloral Eucalyptus honey samples from the multifloral, independently to have or not Eucalyptus pollen. Additionally, the results obtained for the other parameters analysed in the present work are important to characterise the properties the honey samples from the Luso Portugal region, but not to be used to differentiate the floral origin. 4. Conclusions Honeys from Luso region present a good level of quality, once 32 of the 38 analysed samples are in agreement with the European honey directive [30] and Portuguese legislation [16], indicating adequate processing, good maturity and freshness. Six samples did not fit within European and Portuguese standards relative to the diastase activity, reflecting inadequate sample manufacture and/or storage. Potassium is the most abundant of the determined elements. Magnesium and ash contents and electrical conductivity may be used to discriminate the Eucalyptus monofloral from the multifloral honey samples, independently to have or not Eucalyptus pollen, suggesting that mineral content is highly dependent on the type of flower used by bees.
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