Characterisation of Moroccan Spurge (Euphorbia) honeys by their physicochemical characteristics, mineral contents and colour

2.1 Honey samples

27 Spurge honey samples (11 samples from E. officinarum subsp. echinus and 16 from E. regis-jubae) were collected directly from professional beekeepers during 2009 and 2010 in the Ifni Massif region (SW Morocco). All samples were immediately transferred to the laboratory, and kept stored unpasteurised at 4–5 °C. These samples were the same as analysed by Terrab et al. (2014).

2.2 Pollen analysis

The botanical origin of the honeys was stated using the techniques described by Maurizio (1979) and Erdtman (1960). 10 g of honey was dissolved in 20 mL of diluted sulphuric acid (5 g H₂SO₄/L) and centrifuged (10 min at 2500 rpm); the supernatant was decanted and the sediment washed twice with 10 ml distilled water and again centrifuged. The sediment was extended on a slide and dried at 70 °C, and then mounted with stained glycerine gelatine. Following Behm et al. (1996), at least 500 pollen grains were counted among four different slides for each honey sample. For the pollen identification, the general key to pollen types from Díez (1987) was used.

2.3 Physicochemical common parameters

Water content (moisture) was determined with an Erma refractometer reading at 20 °C, and using the Wedmore table (AOAC, 1990).

pH was measured in a pH-meter (Orion 420 A) from a solution containing 10 g honey in 75 mL of CO₂−free distilled water (AOAC, 1990).

The free, lactonic and total acidities were determined by the titrimetric method: the addition of 0.05 M NaOH was stopped at pH 8.50 (free acidity), then 10 mL 0.05 M NaOH was immediately added and back-titration with 0.05 M HCl to pH 8.30 was carried out without delay (lactone acidity). Total acidity results were obtained by adding free plus lactone acidities (AOAC, 1990).

Hydroxymethylfurfural (HMF) was determined by clarifying samples with Carrez reagents (I and II), adding sodium bisulphite, and the absorbance at 284 nm and 336 nm in a 1 cm quartz cuvette was measured in a spectrophotometer (Milton Roy UV–vis Spectronic 3000 Array) (AOAC, 1990).

Electrical conductivity of a honey solution at 20% (dry matter basis) in CO₂−free deionised distilled water was measured at 20 °C (AOAC, 1990) in a Crison Basic 30 conductimeter.

Ash percentage was determined by calcination in furnace at 550 °C, until attaining constant mass (AOAC, 1990).

2.4 Determination of mineral elements

Mineral elements were determined using an Agilent 7500 c with Octopole Reaction System inductively coupled plasma-mass spectrometer. The analytical parameters for ICP-MS were: Nebulizer, Babington; RF generator, 1500; Argon flow rate, 1.11 L min⁻¹; Nebulizer pump, 0.1 rps; reduction gas flow, H₂ at 3.5 mL min⁻¹ and He at 4 mL min⁻¹. The elements standards solutions were prepared by diluting stock solution (ICP standard CertiPUR) 1000 mg L⁻¹. The same procedure was applied to prepare a Rh solution (1 mg L⁻¹) which was chosen as the internal standard. All reagents were analytical grade. All dilutions were prepared with deionised water produced by a Milli Q water purification system (Millipore, Belford, USA).

Six mineral elements (Na, Mg, Al, K, Ca and Fe) were determined in each honey. Samples were prepared from exactly 0.5 g put into PTFE vessels and dissolving with 6 mL 67% HNO₃ (PlasmaPURE, SCP, Courtaboeuf, France). The digestion was carried out in microwave oven (Multiwave 3000, Anton Paar, Austria), setting the parameters 10 min 0 to 800 W, 10 min 800 W, and 15 min vent.

2.5 Colour parameters

Colour was assessed by tristimulus colorimetry based on the reflectance spectra, with the application of the equations proposed by the Commission Internationale de l’Éclairage (CIE, 2004). The reflectance spectra were measured directly on the honey against a white background, with a CAS-140B spectroradiometer (Instrument System, Munich, Germany) equipped with a Top 100 telescopic probe and a Tamron SP 23A zoom (Tamron USA, Inc., Commack, NY, USA), set to measure in the visible region (λ = 380–770 nm) at constant intervals (Δλ = 2 nm). Spectra were integrated using the CromaLab® software (Heredia et al., 2004), which takes into account the CIE recommendations. The uniform colour space CIE 1976-(L^∗a^∗b^∗) (CIELAB) was considered. As is required by the weighted-ordinate method (Δλ = constant), the visible reflectance spectra of the samples were weighted according to the characteristic factor of the selected visual reference conditions: 10° visual field observer and the CIE standard illuminant D65, which correspond to natural daylight (Wyszecki and Stiles, 1982). The CIELAB colour parameters L^∗, a^∗, b^∗, $C_{\text{ab}}^{\ast }$ and h_ab were determined. L^∗ represents the perceived lightness, a^∗ and b^∗ indicate the change in hue from red to green and from yellow to blue, respectively. Chroma ( $C_{\text{ab}}^{\ast }$ ) represents the amount of colour and is measured according to the distance to the origin of coordinates. It is the attribute that allows determining the difference in comparison to a grey colour with the same lightness for each hue, so it is considered to be the quantitative attribute of colourfulness. Hue (h_ab) is the attribute according to which colours have been traditionally defined as reddish, greenish, etc. This attribute is related to the differences in absorbance at different wavelengths and is considered the qualitative attribute of colour.

2.6 Statistical analysis

Multivariant statistical treatments (Principal Component Analysis, PCA, and Stepwise Discriminant Analysis, SDA) were carried out using the software Statistica v 8.0 (Statsoft Inc., 2007).

3.1 Pollen analysis

The microscopical analysis of the sediment showed that Euphorbia honeys contained between 27% and 99% pollen of Euphorbia sp. For the E. officinarum subsp. echinus honey, the most characteristic accompanying species were Eryngium ilycifolium, Bellis sp., Capsella and Reseda sp. However, for the E. regis-jubae honeys, the most characteristic accompanying species were Crepis Anthemis, Bellis, Capsella, Coronilla viminalis, Lotus and Limonium. For more details see Terrab et al. (2014).

3.2 Physicochemical common parameters

Table 1 shows the means, standard deviations and ranges of the different physicochemical parameters data.

Water content, which is related to the climatic conditions, the season and the degree of maturity (White, 1975; Bogdanov et al., 1999), showed values between 16% and 20% in most of the samples (90%), although two samples exceeded the permitted limit established by the European Community Directive (EU Council, 2002). No differences were found between the two Euphorbia honey types (E. officinarum subsp. echinus, mean = 18.50 and E. regis-jubae, mean = 18.52). Terrab and Heredia (2004) found similar values of moisture in avocado honeys from S Spain and Canary Island, a region with quite similar climatic conditions to the Ifni Massif region. They also match with those values reported for honeys from another arid region, the Argentinean Patagonia (Aloisi, 2010; Aloisi et al., 2013).

The pH exerts great importance during the extraction and storage of honey since influences the texture, stability and shelf life. In this study, pH values ranged between 3.69 and 4.67, with mean values 4.02 and 3.79 for E. officinarum subsp. echinus honeys and E. regis-jubae honeys, respectively.

Acidity of honey is due to the presence of organic acids and inorganic ions such as gluconic acid with their lactones or esters, phosphate and chloride. Acid measurement appeared useful to assess honey fermentation, to authenticate unifloral honeys and to differentiate nectars from honeydew honeys (Nandaa et al., 2003). Concerning free acidity established by legislations, European Community regulation requires in general not more than 50 meq kg⁻¹ and not more than 80 meq kg⁻¹ for baker’s honey (EU Council, 2002). Table 1 shows values ranging from 16 to 80 meq kg⁻¹ being 85% of the samples below the EU limit of tolerance. These values were higher than those reported by Terrab et al. (2002) for four different unifloral Moroccan honeys. No differences were found regarding free acidity between E. officinarum subsp. echinus honeys (mean = 43.38) and E. regis-jubae honeys (mean = 40.36); nevertheless, the laconic acidity (considered as the acidity reverse when honey becomes alkaline) showed a slight difference between the two honey types.

The hydroxymethylfurfural (HMF) content is a freshness indicator, so it is an important criterion to evaluate storage time and heat damage (Ruoff and Bogdanov, 2004). The EU legislation (EU Council, 2002) establishes a maximum limit of 40 mg/kg for this parameter. Fresh honey does not contain, or contains only trace amounts of HMF (Bogdanov et al., 2004). From consumers’ point of view, low HMF values guarantee that the honey is practically unaltered (Escriche et al., 2008). In this study, Euphorbia honey samples showed HMF values ranged from 2.54 to 85.48 mg/kg for E. officinarum subsp. echinus honeys, and from 0.75 to 25.92 mg/kg for E. regis-jubae honeys. Two samples were found to surpass the limit permitted by the European Community regulations.

The ash content is generally used to determine the botanical origin (floral, mixed or honeydew) of honeys (White, 1978). In this case, this parameter showed typical floral honeys values, and ranged between 0.04 and 0.4 for E. officinarum subsp. echinus honeys, and from 0.03 to 0.24 for E. regis-jubae honeys. None of the samples surpassed 0.6%, which is the permitted limit for flower honeys (EU Council, 2002). The mean value of ash found in this study was slightly lower than that found in Eucalyptus (0.22%) and Apiaceae (0.18%) Moroccan honeys, but higher than Lythrum (0.10%) and Citrus (0.07%) Moroccan honeys (Terrab et al., 2003).

The electrical conductivity of honeys is closely related to the concentration of mineral salts, organic acids and proteins. This parameter varies greatly according to the floral origin, so it is considered one of the best differentiating parameters to classify honeys regarding their floral origin (Krauze and Zalewski, 1991; Mateo and Bosch-Reig, 1998; Terrab et al., 2002). In this study the electrical conductivity showed values between 227 and 874 μS cm⁻¹ for E. officinarum subsp. echinus honeys, and between 322 and 690 μS cm⁻¹ for E. regis-jubae honeys. Based on these results and other from literature, the electrical conductivity could be considered a reliable parameter to differentiate our samples from other unifloral honeys such as citrus (136–474 μS cm⁻¹), rosemary (89–250 μS cm⁻¹), heather (815–1092 μS cm⁻¹), or honeydew honey (1329–2400 μS cm⁻¹) (Mateo and Bosch-Reig, 1998; Terrab et al., 2003).

3.3 Mineral elements

Mean contents of each mineral found in the 27 honeys expressed in mg/kg fresh weight are shown in Table 2. The potassium was quantitatively the most important mineral (66% of total minerals quantified), having an average content $\bar{x}=687\text{mg}/\text{kg}$ ; sodium and calcium were present in moderate amounts in the honeys (20% and 11% of total minerals, respectively), showing magnesium, aluminium and iron the lowest average contents (2%, 1.5% and less than 0.5% of total minerals, respectively). Sodium and magnesium showed average values that can help to differentiate between E. officinarum subsp. echinus (Na = 127 mg/kg; Mg = 35 mg/kg) and E. regis-jubae (Na = 264 mg/kg, Mg = 13.7 mg/kg) honeys.

3.4 Colour parameters

Table 2 shows the results obtained for the different colour parameters in the CIELAB colour space against white background. The lightness (L^∗) values ranged between 38.55 and 50.68 units for E. officinarum subsp. echinus honeys (dark honeys), and between 45.92 and 83.46 units for E. regis-jubae honeys (light honey). The chroma ( $C_{\text{ab}}^{\ast }$ ) showed significant differences between E. officinarum subsp. echinus (mean = 44.37 units) and E. regis-jubae honeys (mean = 74.86 units). The results showed greater differentiation regarding the hue (h_ab), the qualitative attribute of colour, between E. officinarum subsp. echinus (36.94–53.15°, amber honeys) and E. regis-jubae honeys (49.84–84.37°, yellowish honeys). This can be observed graphically in Figure 1 which shows the projection of the colour points corresponding to each honey sample on the (a^∗, b^∗)-plane. The samples were located in a wide range of hue angles and chroma values, being the E. officinarum subsp. echinus honey samples separated from E. regis-jubae honey samples, except for one sample of E. regis-jubae that was located within the E. officinarum subsp. echinus honey samples area.

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Figure 1

Distribution of the Moroccan Euphorbia honeys (E. officinarum subsp. echinus and E. regis-jubae) within the CIELAB colour space (a^∗b^∗-diagram).

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3.5 Statistical approach

To distinguish between the two Euphorbia honey types (E. officinarum subsp. echinus and E. regis-jubae honeys), Principal Component Analysis (PCA) and Stepwise Discriminant Analysis (SDA) statistical techniques were applied to the data obtained from the common physicochemical parameters, the mineral composition, and the colour characteristics analysed.

Table 3 shows the factor loading matrix obtained for the two factors and the variance explained by each one. The first principal component accounted for 41.43% of the variance, and the second for 14.49%, the former being strongly chemically correlated with Na, Mg, K, L^∗, a^∗, b^∗, C_ab, and h_ab, and the latter specifically with the free and the total acidity. The cumulative variance was about 56%. A scatter plot was obtained by correlating the factorial weights of features in the first factor against the factorial weights in the second factor. It can be seen from Figure 2 that Mg, a^∗, K, Fe and pH are the dominant parameters in the first factor, while free acidity, total acidity and moisture dominate the second factor. Figure 3 represents the graphic distribution of the 27 samples according to their factor scores, and shows that E. officinarum subsp. echinus honey samples are perfectly differentiated from the E. regis-jubae honey samples, except for the one sample which was intermingled with the E. regis-jubae honeys.

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Figure 2

Plot of factorial weights. First factor versus second factor from the Principal Component Analysis of 19 physicochemical, minerals and colorimetric parameters.

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Figure 3

Plot of the first factor versus second factor for the classification of the two Euphorbia honey types. E. officinarum subsp. echinus (samples 1–11) and E. regis-jubae (samples 12–27).

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With respect to the discriminant analysis, a forward iterative inclusion of variables was performed in order to choose the parameters with higher discriminant power. A tolerance of 0.01 eliminates the variables that provide superfluous information at a 99% level, along with those previously included in the model. The variables selected by stepwise discriminant analysis were, Na, Mg, L^∗, C_ab and h_ab, as well as the Wilks’ lambda, which indicates the contribution of each variable to the discrimination. As can be seen, the latter does not surpass 0.175 (see Table 4). Table 5 lists the cumulative proportion of total dispersion, and the standardised coefficient for the canonical variable. The higher the absolute value of a standardised coefficient, the more significant is the related selected variable in the canonical variable. h_ab appeared to be the variable that accounts for most of the discrimination between the two Euphorbia honey types (standardised coefficient = 0.770). Classification functions are linear combinations of the variables selected by the programme. The coefficients and constants for these functions (data not shown) were applied to the honey samples; their validity can be verified according to the agreement percentages of the cases in their corresponding group (Table 6). It is seen that all E. officinarum subsp. echinus honey samples were correctly classified into their a priori established honey types (100%), and also, except for one sample, the E. regis-jubae honey samples were correctly classified.