2.1. Microscopic Characteristics
The quantitative analysis of samples led to the identification of honeys with a high quantity of pollen and fungal elements and others very poor in these elements. Pollen content varied between 1274 and 130,832 pollen grains per gram of honey, with a standard deviation of 25,585.
The qualitative pollen analysis identified 90 different pollen types in the samples.
Table 1 lists the most commonly occurring pollens. This table includes 16 pollen types from ten botanical families, along with their corresponding maximum values, the standard deviation, and the range and the number of honeys in which the various pollen types reached a determined percentage level.
Table 1.
Principal pollen types, their frequency classes and their representation in the samples.
Table 1.
Principal pollen types, their frequency classes and their representation in the samples.
Family | Pollen type | Frequency classes a | | |
---|
(%) | P (0–1%) | R (1–3%) | I (3–15%) | A (15–45%) | D >45% | Max. | St. Dv. |
---|
Fagaceae | Castanea | 100.0 | 0.0 | 7.0 | 9.3 | 48.8 | 34.9 | 87.9 | 21.55 |
Leguminosae | Cytisus t. | 100.0 | 18.6 | 25.6 | 52.3 | 2.3 | 1.2 | 48.6 | 6.81 |
Rosaceae | Rubus | 100.0 | 3.5 | 3.5 | 29.1 | 34.9 | 29.1 | 83.6 | 21.31 |
Ericaceae | Erica | 97.7 | 23.3 | 23.3 | 43.0 | 8.1 | 0.0 | 35.5 | 6.13 |
Leguminosae | Trifolium t. | 87.2 | 40.7 | 29.1 | 17.4 | 0.0 | 0.0 | 9.6 | 1.87 |
Myrtaceae | Eucalyptus | 86.0 | 22.1 | 18.6 | 20.9 | 12.8 | 11.6 | 81.0 | 22.81 |
Boraginaceae | Echium | 76.7 | 45.3 | 20.9 | 10.5 | 0.0 | 0.0 | 9.8 | 1.82 |
Fagaceae | Quercus | 66.3 | 52.3 | 10.5 | 2.3 | 1.2 | 0.0 | 27.8 | 3.03 |
Salicaceae | Salix | 59.3 | 43.0 | 9.3 | 7.0 | 0.0 | 0.0 | 10.9 | 1.55 |
Rosaceae | Crataegus monogyna t. | 40.7 | 33.7 | 5.8 | 1.2 | 0.0 | 0.0 | 3.6 | 0.61 |
Campanulaceae | Campanula t. | 39.5 | 33.7 | 4.7 | 1.2 | 0.0 | 0.0 | 3.9 | 0.59 |
Umbelliferae | Conium maculatum t. | 34.9 | 25.6 | 8.1 | 1.2 | 0.0 | 0.0 | 7.0 | 0.89 |
Rhamnaceae | Frangula alnus | 31.4 | 23.3 | 5.8 | 2.3 | 0.0 | 0.0 | 13.7 | 1.72 |
Leguminosae | Lotus t. | 20.9 | 17.4 | 2.3 | 1.2 | 0.0 | 0.0 | 3.7 | 0.45 |
Boraginaceae | Lithodora | 16.3 | 12.8 | 1.2 | 2.3 | 0.0 | 0.0 | 4.3 | 0.60 |
Boraginaceae | Myosotis | 11.6 | 10.5 | 0.0 | 1.2 | 0.0 | 0.0 | 5.6 | 0.61 |
| Others | 100.0 | 18.6 | 53.5 | 27.9 | 0.0 | 0.0 | 14.3 | 2.20 |
Castanea sativa, Cytisus type and Rubus were present in 100% of the samples, whereas Erica was present in 97.7% and Eucalyptus in 86%. These pollens were the principal types found in the honeys from north-western Spain. Echium, Quercus and Trifolium pollens were also frequently found. Pollens from Salix, Crataegus monogyna type, Campanula type, Conium maculatum types, Frangula alnus, Lotus type, Lithodora and Myosotis comprised more than 3% of pollen spectra and might also be important in honeys. Fifty pollen types were present only in a few honeys (less than 10%) and were included in other pollen type groups.
Regarding the botanical classification of honeys, 46 samples contained pollens from a variety of sources and were classified as polyfloral. Another 25 samples contained more than 45% Rubus pollens and were classified as blackberry monoflorals. Honeys containing more than 70% Castanea sativa pollen were classified as chestnut honeys (9 samples). The remaining 6 samples were Eucalyptus monofloral honeys with a percentage higher than 70%. Pollens from Erica plants were common, but none of the samples contained enough Erica pollen to be classified as heather monofloral.
Further microscopic analysis identified additional elements in the sediments from samples.
Table 2 shows a descriptive analysis of principal fungal elements sorted by frequency.
Cladosporium conidia were the best represented elements (occurring in 97.7% of samples), with a mean of 325 spores per gram of honey. Spores from Myxomycetes and
Penicillium were also very abundant, with mean content of 135 and 151 spores/g, respectively. More than 40% of the samples contained these elements. The second-most common element was
Metschnikowia yeast, present in 79.1% of the honeys studied. These cells have appeared in high content in some samples, with a maximum value of 46,217 cells per gram of honey.
Table 2.
Descriptive analysis of microscopic elements per g of honey.
Table 2.
Descriptive analysis of microscopic elements per g of honey.
Fungal elements | Max. | Min. | Mean | St. Dv. | Rep. (%) |
---|
Cladosporium | 1,934 | 0 | 325 | 346 | 97.7 |
Metschnikowia a | 46,217 | 0 | 2,244 | 5,998 | 79.1 |
Leptosphaeria | 613 | 0 | 42 | 84 | 51.2 |
Myxomycete | 3,815 | 0 | 135 | 443 | 47.7 |
Penicillium | 1,594 | 0 | 151 | 324 | 41.9 |
Basidiospores | 679 | 0 | 20 | 78 | 32.6 |
Stemphylium | 251 | 0 | 19 | 46 | 32.6 |
Urediniospores | 454 | 0 | 25 | 67 | 30.2 |
Alternaria | 91 | 0 | 6 | 18 | 15.1 |
Bipolaris | 92 | 0 | 3 | 14 | 5.8 |
Torula | 241 | 0 | 4 | 27 | 4.7 |
Fusicladium | 56 | 0 | 1 | 8 | 4.7 |
Curvularia | 71 | 0 | 2 | 10 | 3.5 |
Botrytis | 51 | 0 | 1 | 6 | 2.3 |
Pleospora | 151 | 0 | 2 | 16 | 2.3 |
Fern spores | 1,126 | 0 | 20 | 135 | 2.3 |
Helminthosporium | 71 | 0 | 1 | 8 | 1.2 |
Sporidesmium | 19 | 0 | 0 | 2 | 1.2 |
Drechslera | 109 | 0 | 1 | 12 | 1.2 |
Unidentified | 761 | 0 | 22 | 86 | 27.9 |
HDE/P | 0.63 | 0 | 0.16 | 0.09 | 100 |
Fungal spores produced by plant pathogens, such as Leptosphaeria, Stemphylium, Urediniospores, Alternaria, Pleospora and Botrytis, were also counted. Of these, Leptosphaeria was the most common (more than 50% of samples contain its spores), with a mean of 42 spores/g and a maximum of 613 spores/g. The next most common spores found were Stemphylium and Urediniospores (present in more than 30% of samples), with means of 19 spores/g and 25 spores/g, respectively. Other elements from plant pathogens were uncommon (occurring in less than 15% of the samples), with mean values of 6, 2 and 1 spores/g for Alternaria, Pleospora and Botrytis, respectively.
Basidiospores, which include spores from Basidiomycota other than yeasts, were present in 32.6% of samples with a maximum of 679 spores/g. A high number of spores from ferns (Pterydophyta) were present in two samples, probably because the bees collected them as a protein source. In relation to the honeydew index, the highest value was 0.63 and the lowest was 0.00, with a standard deviation of 0.09.
2.3. Spearman’s Linear Rank Correlation between Microscopic and Physicochemical Variables
A linear rank correlation analysis was used to examine the relationships between the variables studied. We used a non-parametric method, in which a linear correlation coefficient was calculated using Spearman’s linear rank correlation analysis. In this procedure, the variables with the strongest relationship to the provenance of the honey were used.
Table 4 includes the value of the correlation coefficient and the significance (
P < 0.05,
P < 0.01,
P < 0.001) of its relationship with the variables obtained by microscopic analysis, that could be related to the source of the honey. The presence of
Metschnikowia cells had a positive correlation with
Eucalyptus and
Rubus pollen content (
P < 0.05) and a negative correlation with
Cytisus t. (
P < 0.01) and
Erica pollen content (
P < 0.001). The first taxon produced blossom honeys, and the other two were numerous in dark honeys.
Table 4.
Significant correlation coefficients for the microscopic analysis variables, based on Spearman’s test.
Table 4.
Significant correlation coefficients for the microscopic analysis variables, based on Spearman’s test.
| Castanea | Eucalyptus | Rubus | Cytisus t. | Erica |
---|
Metschnikowia | −0.048 | 0.240 * | 0.239 * | −0.317 ** | −0.544 *** |
HD spores | 0.051 | −0.390 *** | 0.264 * | 0.043 | −0.041 |
The group called HD spores included the levels of Alternaria, Leptosphaeria, Stemphylium, Botrytis, Pleospora and Urediniospores. These plant pathogen fungi constitute an important subgroup of the identified fungal elements. This group had a negative correlation with the Eucalyptus pollen content (P < 0.001) and a positive correlation with Rubus pollen (P < 0.05).
Metschnikowia cells had negative correlation coefficients with most of the physicochemical variables (
P < 0.001) other than pH (no significance) and humidity (positive correlation
P < 0.01) (
Table 5).
Table 5.
Significant correlation coefficients among the microscopic analysis and physicochemical parameter variables, based on Spearman’s test.
Table 5.
Significant correlation coefficients among the microscopic analysis and physicochemical parameter variables, based on Spearman’s test.
| Diastase | Invertase | pH | EC | Humidity | Colour | Total mineral |
---|
Metschnikowia | −0.329 ** | −0.353 *** | −0.179 | −0.663 *** | 0.298 ** | −0.662 *** | −0.592 *** |
HD spores | 0.376 *** | 0.441 *** | 0.468 *** | 0.476 *** | −0.240 * | 0.399 *** | 0.436 *** |
Castanea | 0.141 | 0.038 | 0.105 | 0.247 * | 0.165 | 0.195 | 0.156 |
Eucalyptus | −0.386 *** | −0.344 ** | −0.346 ** | −0.520 *** | 0.197 | −0.491 *** | −0.456 *** |
Rubus | 0.232 * | 0.207 | 0.167 | 0.057 | −0.179 | 0.053 | 0.043 |
Cytisus t. | 0.251 * | 0.163 | 0.050 | 0.285 ** | −0.069 | 0.347 ** | 0.231 * |
Erica | 0.161 | 0.122 | −0.237 ** | 0.310 ** | 0.111 | 0.437 *** | 0.231 ** |
The presence of other fungal elements in honeys correlated positively with all physicochemical variables except humidity. HD spores in particular had a positive correlation (P < 0.001) with all the physicochemical parameters except humidity, with which it was negatively correlated (P < 0.05).
Regarding the honeys’ pollen content, Eucalyptus pollen had similar correlations to those of nectar yeast. Pollens from Cytisus and Erica types had positive correlations (P < 0.01) with electrical conductivity, mineral content (P < 0.05, P < 0.01, respectively) and colour (P < 0.01, P < 0.001, respectively). Cytisus t. also had positive correlations (P < 0.05) with diastase content, and Erica had negative correlations with pH (P < 0.01). Castanea pollen and Rubus pollen had positive correlation with electrical conductivity (P <0.05) and diastase (P < 0.05), respectively.
2.4. Cluster Analysis with the Main Microscopic Variables
The cluster analysis was performed with the main variables obtained by microscopy as independent variables. These were
Metschnikowia cells, HD spores,
Castanea pollen,
Eucalyptus pollen,
Rubus pollen,
Cytisus pollen and
Erica pollen. The groups (honeydew honeys or blossom honeys) were established according to the characteristics of the samples, as previously pointed out. The cluster (
Figure 1) represents the groups established in the analysis. Sixty four samples belonged to the blossom honey group (polyfloral honeys,
Eucalyptus honeys,
Rubus honeys and
Castanea honeys) and 22 to the honeydew honey group (including blend honeys and honeydew samples).
Figure 1.
Cluster analysis of the studied samples.
Figure 1.
Cluster analysis of the studied samples.
2.5. T Test and Levene’s Test of the Homogeneity of the Variance
The groups established by the cluster analysis were compared using a t-test for the equality of the means. Levene’s test examined the homogeneity of the variances by performing a one-way ANOVA on the absolute deviation scores.
Table 6 shows the descriptive statistical analysis of each group. Blossom honeys (N group) had a mean electrical conductivity of 0.540 ± 0.2 mS/cm, lower than that of the HD group (mean: 0.830 ± 0.2 mS/cm). Colour was clearer for nectar honeys (85 ± 20 mm pfund) than for honeydew honeys (124 ± 21 mm pfund). Mineral content was generally lower in blossom honeys, particularly for potassium content (N group mean: 120.4 ± 50.7 mg/100 g
vs. HD group mean: 192.1 ± 65.2 mg/100 g), as was phosphorus content (7.4 ± 3.2 mg/100 g
vs. 13.2 ± 8.2 mg/100 g). Even the enzymatic activity was high in the nectar samples. The subindex of the variables indicates the significance levels between the groups. Some parameters led to differentiation between the groups. These were enzymatic activity,
Metschnikowia cells and
Cytisus pollen type with
p < 0.05, electrical conductivity, humidity, colour, potassium, calcium, magnesium, phosphorus, total mineral content,
Eucalyptus pollen,
Erica pollen and HD spores content with
p < 0.01.
Table 6.
Descriptive statistical analysis of the groups established by cluster analysis.
Table 6.
Descriptive statistical analysis of the groups established by cluster analysis.
Parameters | N group (N = 64) | HD group (N = 22) |
---|
Max. | Min. | Mean | St. Dv. | Max. | Min. | Mean | St. Dv. |
---|
Pollen grain per gram | 90,232 | 1,632 | 24,659 | 23,006 | 130,832 | 1,274 | 26,071 | 32,561 |
Diastase content (Shade) b | 31.8 | 7.5 | 15.7 | 5.4 | 30.9 | 6.1 | 18.8 | 5.9 |
Invertase content (IN) b | 35.9 | 4.3 | 16.8 | 5.8 | 26.0 | 10.1 | 19.7 | 4.8 |
HMF (mg/100 g) | 1.6 | 0.0 | 0.4 | 0.3 | 0.9 | 0.0 | 0.3 | 0.3 |
pH | 5.0 | 3.5 | 4.2 | 0.3 | 4.9 | 3.5 | 4.3 | 0.4 |
EC (mS/cm) a | 0.920 | 0.224 | 0.540 | 0.2 | 1.168 | 0.482 | 0.830 | 0.2 |
Humidity (%) a | 19.8 | 16.0 | 17.5 | 0.7 | 17.8 | 15.5 | 16.7 | 0.6 |
Colour (mm pfund) a | 118 | 39 | 85 | 20 | 150 | 79 | 124 | 20.9 |
Potassium (mg/100 g) a | 239.0 | 32.8 | 120.4 | 50.7 | 312.0 | 65.5 | 192.1 | 65.2 |
Calcium (mg/100 g) a | 13.7 | 4.7 | 8.4 | 2.3 | 16.6 | 4.4 | 10.6 | 3.4 |
Iron (mg/100 g) | 1.1 | 0.03 | 0.3 | 0.2 | 0.7 | 0.1 | 0.3 | 0.1 |
Magnesium (mg/100 g) a | 18.7 | 1.6 | 5.8 | 4.4 | 29.2 | 1.4 | 13.5 | 9.8 |
Sodium (mg/100 g) | 26.7 | 1.2 | 5.7 | 4.9 | 17.8 | 0.9 | 5.3 | 4.7 |
Phosphorus (mg/100 g) a | 17.1 | 3.3 | 7.4 | 3.2 | 31.5 | 2.9 | 13.2 | 8.2 |
Zinc (mg/100 g) | 0.4 | 0.1 | 0.2 | 0.1 | 0.4 | 0.1 | 0.2 | 0.1 |
Copper (mg/100 g) | 3.7 | 0.02 | 0.4 | 0.8 | 0.7 | 0.1 | 0.3 | 0.5 |
Total mineral (mg/100 g) a | 280.3 | 47.9 | 148.6 | 55.9 | 387.4 | 95.2 | 235.4 | 80.8 |
Castanea | 86.2 | 1.0 | 36.2 | 20.9 | 87.9 | 2.5 | 41.5 | 23.3 |
Rubus | 83.6 | 0.3 | 29.7 | 21.6 | 65.8 | 7.1 | 31.5 | 20.8 |
Erica a | 15.3 | 0.0 | 3.7 | 3.9 | 35.5 | 1.3 | 8.9 | 9.2 |
Eucalyptus a | 81.0 | 0.0 | 17.9 | 4.6 | 14.2 | 0.0 | 2.7 | 4.3 |
Cytisus t. b | 31.4 | 0.1 | 4.0 | 25.2 | 26.8 | 0.3 | 7.7 | 10.6 |
Mestchnikowia (cells/g) b | 46217 | 17.1 | 3013 | 6796 | 41 | 0 | 4.7 | 286 |
HD spores (spores/g) a | 406 | 0 | 61 | 84 | 1159 | 5 | 193 | 11.5 |