2.1. Mellisopalynogical analysis
Table 1 presents the pollen profile of the honeys used in the experiments. On the basis of mellisopalynogical analysis, 7 varietal honeys and 4 multifloral honeys were distinguished. Among the varietal honeys, the following were distinguished:
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Plum honey (P) – dominant pollen – Prunus type (46.98%);
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Willow honey (Sa) – dominant pollen – Salix sp. (70.25%);
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Rapeseed honey (Br) – dominant pollen – Brassicaceae type (81.70)%;
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Lime honey (Tc) – dominant pollen – Tilia sp. (28.99%);
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Phacelia honey (Ph) – dominant pollen – Phacelia thanacetifolia (65.62%);
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Honeydew honey (So) – dominant pollen – Solidago type (46.48%);
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Sunflower honey (He) – dominant pollen – Helianthus type (73.35%).
In the case of multifloral honeys, they were characterized:
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Multifloral-Br (MBr) – with a predominance of pollen from Brassicaceae type (33.01%), Aesculus hippocastanum (15.53%) and Poligonum bistorta (15.53%);
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Multifloral-Sa (MSa) - with a predominance of pollen from Salix sp. (21.55%) Solidago type (17.24%) and Tilia sp. (17.24%);
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Multifloral-AP (MAP) - with a predominance of pollen from Acer sp. (37.38%) and Prunus type (37.38%);
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Multifloral-P (MP) - with a predominance of pollen from Prunus type (29.47%), Brassicaceae type (15.79%) and Salix sp.(15.26%).
Table 1
Mellisopalynological analysis of different types of honey.
Nectar producing plant taxons | HONEY TYPE |
P | MAP | MP | MSa | Sa | MBr | Br | Tc | Ph | So | He |
Pollen grains |
No. | % | No. | % | No. | % | No. | % | No. | % | No. | % | No. | % | No. | % | No. | % | No. | % | No. | % |
Acer sp. | 1 | 0.47 | 200 | 37.38 | 6 | 3.16 | | | 1 | 0.21 | | | | | | | | | | | | |
Achillea millefolium | | | | | | | | | | | | | | | 1 | 0.21 | 1 | 0.26 | | | | |
Aesculus hippocastanum | 28 | 13.02 | 35 | 6.54 | | | 12 | 10.34 | 1 | 0.21 | 16 | 15.53 | | | 55 | 11.39 | 5 | 1.31 | | | | |
Anthriscus sp. | | | | | | | | | | | 1 | 0.97 | | | 5 | 1.04 | | | 5 | 10.87 | 1 | 0.24 |
Arctium sp. | | | | | | | | | | | | | | | | | | | | | 1 | 0.24 |
Aster type | | | | | | | | | | | 4 | 3.88 | | | | | 1 | 0.26 | | | | |
Brassicaceae type | 13 | 6.05 | | | 30 | 15.79 | | | | | 34 | 33.01 | 260 | 81.76 | 41 | 8.49 | 22 | 5.77 | | | 60 | 14.67 |
Centaurea cyanus | | | 25 | 4.67 | | | | | | | | | | | | | 5 | 1.31 | | | | |
Cirsium sp. | | | | | | | | | | | | | | | 2 | 0.41 | | | | | | |
Convolvulus arvensis | 1 | 0.47 | | | | | | | | | | | | | | | | | | | | |
Echium vulgare | | | | | | | | | | | 1 | 0.97 | | | | | | | | | | |
Fagopyrum esculentum | 3 | 1.40 | | | | | | | | | | | | | | | | | | | 1 | 0.24 |
Frangula alnus | | | | | | | | | 2 | 0.41 | 1 | 0.97 | | | | | | | | | 3 | 0.73 |
Helianthus type | 26 | 12.09 | | | | | | | | | 7 | 6.80 | | | 6 | 1.24 | | | 1 | 2.17 | 300 | 73.35 |
Impateins sp. | | | | | | | | | | | | | | | | | | | 5 | 10.87 | | |
Lilium sp. | | | | | 5 | 2.63 | 1 | 0.86 | | | | | | | | | | | | | | |
Lotus corniculatus | | | | | | | | | 3 | 0.62 | | | | | | | | | | | | |
Mallus type | 7 | 3.26 | | | 15 | 7.89 | | | 1 | 0.21 | | | | | 8 | 1.66 | | | | | | |
Melilotus sp. | | | | | | | | | | | | | | | | | 50 | 13.12 | | | | |
Phacelia thanacetifolia | | | | | 5 | 2.63 | 18 | 15.52 | | | 2 | 1.94 | | | 3 | 0.62 | 250 | 65.62 | | | 2 | 0.49 |
Poligonum bistorta | | | 5 | 0.93 | 4 | 2.11 | | | | | 16 | 15.53 | | | 130 | 26.92 | | | | | | |
Prunus type | 101 | 46.98 | 200 | 37.38 | 56 | 29.47 | | | 130 | 26.86 | | | 20 | 6.29 | 23 | 4.76 | 3 | 0.79 | | | 7 | 1.71 |
Robinia pseudoacacia | | | | | | | | | | | 1 | 0.97 | | | 7 | 1.45 | | | | | | |
Rubus sp. | | | | | 15 | 7.89 | 10 | 8.62 | | | 1 | 0.97 | 20 | 6.29 | 25 | 5.18 | 4 | 1.05 | 15 | 32.61 | 6 | 1.47 |
Salix sp. | 31 | 14.42 | | | 29 | 15.26 | 25 | 21.55 | 340 | 70.25 | | | 18 | 5.66 | 2 | 0.41 | | | | | 13 | 3.18 |
Sedum sp. | | | | | | | | | | | | | | | 2 | 0.41 | | | | | | |
Solidago type | 1 | 0.47 | | | | | 20 | 17.24 | | | | | | | 23 | 4.76 | 40 | 10.50 | 20 | 43.48 | 6 | 1.47 |
Trifolim repens | 2 | 0.93 | | | | | | | | | | | | | | | | | | | | |
Trifolium pratense | | | | | | | 10 | 8.62 | 1 | 0.21 | 4 | 3.88 | | | 10 | 2.07 | | | | | | |
Taraxacum officinale | 1 | 0.47 | 20 | 3.74 | | | | | | | | | | | | | | | | | 4 | 0.98 |
Tilia sp. | | | 50 | 9.35 | 20 | 10.53 | 20 | 17.24 | 4 | 0.83 | 15 | 14.56 | | | 140 | 28.99 | | | | | | |
Viola type | | | | | | | | | 1 | 0.21 | | | | | | | | | | | | |
Others | | | | | | | | | | | | | | | | | | | | | 5 | 1.22 |
SUM/AMOUNT | 215 | 95.98 | 535 | 95.54 | 190 | 59.38 | 116 | 43.61 | 484 | 98.57 | 103 | 89.74 | 318 | 93.53 | 483 | 76.86 | 381 | 98.45 | 46 | 38.2 | 409 | 97.15 | |
Not nectarative plant taxons | | | | | | | | | | | | | | | | | | | | | | |
Artemisia sp. | 2 | 22.22 | | | | | 60 | 40 | | | 4 | 28.57 | | | 110 | 74.32 | | | | | | |
Bellis perennis | 1 | 11.11 | | | 5 | 2.63 | | | | | | | | | | | | | | | | |
Betula pendula | 1 | 11.11 | | | | | | | | | | | | | 1 | 0.68 | | | | | | |
Chenopodiaceae type | 2 | 22.22 | | | | | | | | | 2 | 14.29 | | | 2 | 1.35 | | | | | 6 | 50 |
Filipendula sp. | | | 20 | 80% | 130 | 100 | 90 | 60 | | | | | 20 | 90.91 | | | | | 50 | 66.67 | | |
Fragaria sp. | | | | | | | | | | | | | | | 1 | 0.68 | | | | | | |
Pinus sp. | | | 5 | 20 | | | | | 2 | 28.57 | | | 2 | 9.09 | | | 1 | 16.67 | | | | |
Plantago sp. | | | | | | | | | | | 4 | 28.57 | | | 17 | 11.49 | | | | | | |
Poaceae type | 2 | 22.22 | | | | | | | 5 | 71.43 | 4 | 28.57 | | | 12 | 8.11 | | | 25 | 33. 33 | | |
Rumex sp. | | | | | | | | | | | | | | | 5 | 3.38 | | | | | | |
Quercus sp. | 1 | 11.11 | | | | | | | | | | | | | | | | | | | 6 | 50 |
Verbascum sp. | | | | | | | | | | | | | | | | | 5 | 83.33 | | | | |
SUM/AMOUNT | 9 | 4.02 | 25 | 4.46 | 130 | 40.63 | 150 | 56.39 | 7 | 1.43 | 14 | 10.26 | 22 | 6.47 | 148 | 23.14 | 6 | 1.55 | 75 | 66.96 | 12 | 2.85 |
2.2. Physico-chemical properties of honey
Table 2 shows the physico-chemical properties of 11 tested honeys (average ± standard deviation). The water content in the tested honey samples was within the range of 14.67 ± 0.47–18.00%.
Another analyzed parameter was the electrical conductivity, which makes it possible to distinguish between nectar and honeydew honeys. The electrical conductivity was within the range of 0.25 (sunflower He) − 0.91 mS·cm− 1 (honeydew So). The obtained results were within the values applicable for nectar honeys, i.e. 0.2 ÷ 0.8 mS·cm− 1, and for honeydew, i.e. above 0.8 mS·cm− 1.
The phenolic compounds present in honey come from honeydew or pollen. It was observed that the content of phenolic compounds in dark honeys, e.g. honeydew (So) 808.05 ± 7.20 µg GAEs/g, is higher than the content of these compounds in light honeys, e.g. multifloral (MBr) 404.74 ± 9.12 µg GAEs/g and sunflower (He) 431.27 ± 5.45 µg GAEs/g. Rapeseed honey (Br) 378.27 ± 7.3 µg GAEs/g was characterized by the lowest content of phenolic compounds.
Among the tested honeys, the following honeys had the highest protein content above 100 mg/ml: multifloral-AP (116.80 ± 0.57 mg/ml), plum P (112.40 ± 2.47 mg/ml), willow Sa (107.60 ± 0.57 mg/ml) and multifloral-P (103.60 ± 0.57 mg/ml). The honeys: sunflower He (41.20 ± 2.47 mg/ml) and rapeseed Br (49.20 ± 3.40 mg/ml) had the lowest protein content below 50 mg/ml.
Table 2
Physico-chemical characteristics of tested honey types.
| Physico-chemical parameters of honey types (average ± SD, N = 3) |
Honey type | Water content (%) | pH | Electrical conductivity (mS/cm) | Total phenol (µg GAEs/g) | Proteins (mg/ml) |
P | 16.33 ± 0.47 | 4.43 ± 0.005 | 0.29 ± 0.008 | 670.20 ± 18.96 | 112.40 ± 2.47 |
MAP | 18.00 ± 0.00 | 4.62 ± 0.012 | 0.35 ± 0.009 | 776.54 ± 9.12 | 116.80 ± 0.57 |
MP | 14.67 ± 0.47 | 4.79 ± 0.009 | 0.29 ± 0.005 | 700.47 ± 18.18 | 103.60 ± 0.57 |
MSa | 15.67 ± 0.47 | 4.68 ± 0.017 | 0.46 ± 0.014 | 606.54 ± 27.66 | 96.80 ± 2.27 |
Sa | 15.00 ± 0.00 | 4.96 ± 0.029 | 0.29 ± 0.005 | 667.14 ± 4.79 | 107.60 ± 0.57 |
MBr | 18.00 ± 0.00 | 4.67 ± 0.014 | 0.36 ± 0.012 | 404.74 ± 9.12 | 95.20 ± 2.27 |
Br | 17.67 ± 0.47 | 4.22 ± 0.012 | 0.27 ± 0.005 | 378.27 ± 7.3 | 49.20 ± 3.40 |
Tc | 18.00 ± 0.00 | 4.08 ± 0.017 | 0.42 ± 0.012 | 624.40 ± 15.43 | 85.60 ± 3.00 |
Ph | 17.67 ± 0.47 | 4.62 ± 0.005 | 0.4 ± 0.005 | 524.40 ± 18.58 | 90.80 ± 0.57 |
So | 16.33 ± 0.47 | 4.85 ± 0.009 | 0.91 ± 0.008 | 808.05 ± 7.20 | 85.20 ± 0.00 |
He | 15.67 ± 0.47 | 4.35 ± 0.012 | 0.25 ± 0.005 | 431.27 ± 5.45 | 41.20 ± 2.47 |
The Pfund scale includes 7 classes of honey colours. Among the tested honeys, the following colours were distinguished: extra light amber (18.18%), light amber (45.45%) and amber (36.36%) (Table 3).
Table 3
Honey type | Sample result (Absorbance) (average ± SD, N = 3) | Color |
P | 0,83 ± 0,01 | Light Amber |
MAP | 2,94 ± 0,01 | Amber |
MP | 1,36 ± 0 | Light Amber |
MSa | 1,05 ± 0,01 | Light Amber |
Sa | 0,42 ± 0,01 | Extra Light Amber |
MBr | 0,74 ± 0,01 | Light Amber |
Br | 1,76 ± 0,01 | Amber |
Tc | 0,97 ± 0,01 | Light Amber |
Ph | 1,85 ± 1,12 | Amber |
So | 2,23 ± 0,01 | Amber |
He | 0,36 ± 0,01 | Extra Light Amber |
The Principal Component Analysis (PCA) of the physico-chemical properties of tested honeys synthetically showed their differentiation and dependence on honey types. The eigenvalues of the first two axes were 2.47 and 1.13. The first axis explained over 49% and second axis over 22% of the variability of the analyzed data/physico-chemical properties of studied honeys, and all four axes over 98%. This proves the major role of the axis 1 and 2 in ordering the variables and determining the factor responsible for the distribution of the honey types in ordination diagram (Fig. 1). All the variables analysed, except for proteins content for axis 2, were statistically significant at a level of p < 0.05. The ordination diagram showed two main trends of the variation in the physico-chemical properties of the tested honey (Fig. 1). The first one was related to the first axis and positively correlated with all variables tested, except the water content. The strongest correlation with this axis was shown by total phenolics, pH and proteins content. This axis determined the gradient of the content of the analysed properties in the honey types. Group I of the study honeys (right side of ordination diagram) represents an increasing content of total phenolics, pH, and proteins content, starting from the honeydew (So), multifloral (MAP), multifloral (MP), plum (P) and willow (Sa) honey. Group II (left side of PCA diagram) was negatively correlated with the first axis and characterized by high water content and lower content of the phenolics, proteins and pH values. This group of honeys includes rapeseed (Br), sunflower (He), lime (Tc), multifloral (MBr) and phacelia (Ph). The second axis of the PCA ordination diagram was strongly and positively correlated with electrical conductivity and water content, and second axis determined the gradient of replaced variables in the studied honeys (Group III). The water content increased from the multifloral (MP) (14.67%) and willow (Sa), multifloral (MSa) and sunflower (He) (located under the 2nd axis and negatively correlated with it) to multifloral (MAP), multifloral (MBr), and lime (Tc), positively correlated with the discussed axis, where the highest content of water was recorded. The highest conductivity honey are: honeydew (So), multifloral (MSa), lime (Tc) and phacelia (Ph) (Fig. 1).
The main components of honey are sugars. Simple sugars, i.e. glucose and fructose, were identified in the highest amount in all the tested honey samples (Table 4).
Table 4
Major sugar components in tested honey samples as determined by HPLC.
Sugars content (g/100 g), average ± SD, N = 3 | Fructose/Glucose (Ratio) |
Sugar type Honey type | Glucose | Fructose | Sucrose | Rhamnose | Erlose | Fucose |
P | 38.80 ± 0.42 | 39.28 ± 0.12 | 3.26 ± 0.07 | 1.18 ± 0.04 | 3.02 ± 0.10 | 0.00 ± 0.00 | 1.01 |
MAP | 44.77 ± 0.1 | 43.57 ± 0.28 | 2.42 ± 0.08 | 1.79 ± 0.07 | 0.66 ± 0.02 | 0.23 ± 0.01 | 0.97 |
MP | 38.80 ± 0.17 | 38.71 ± 0.23 | 2.79 ± 0.08 | 1.24 ± 0.07 | 2.00 ± 0.07 | 0.00 ± 0.00 | 1.00 |
MSa | 40.75 ± 0.21 | 42.74 ± 0.39 | 3.08 ± 0.09 | 1.58 ± 0.04 | 1.74 ± 0.10 | 0.00 ± 0.00 | 1.05 |
Sa | 37.51 ± 0.18 | 39.24 ± 0.15 | 3.46 ± 0.07 | 1.71 ± 0.03 | 2.92 ± 0.10 | 0.00 ± 0.00 | 1.05 |
MBr | 39.56 ± 0.17 | 42.1 ± 0.58 | 2.45 ± 0.07 | 1.01 ± 0.08 | 1.57 ± 0.06 | 0.00 ± 0.00 | 1.06 |
Br | 39.05 ± 0.35 | 38.53 ± 0.47 | 4.96 ± 0.08 | 1.46 ± 0.05 | 8.26 ± 0.31 | 0.00 ± 0.00 | 0.99 |
Tc | 45.10 ± 0.27 | 38.23 ± 0.28 | 1.31 ± 0.07 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.85 |
Ph | 52.53 ± 0.46 | 37.17 ± 0.34 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.71 |
So | 39.51 ± 0.35 | 40.36 ± 0.47 | 2.89 ± 0.02 | 1.20 ± 0.05 | 1.77 ± 0.13 | 0.00 ± 0.00 | 1.02 |
He | 37.27 ± 0.29 | 42.61 ± 0.2 | 2.91 ± 0.08 | 1.55 ± 0.02 | 0.69 ± 0.02 | 0.35 ± 0.02 | 1.14 |
The PCA ordination analysis shows relationships between the honey type and diversity of sugars and their content (Fig. 2). All analysed sugar types were statistically significant at a level of p < 0.05 for two first axis of ordination PCA diagram, except glucose for axis 2. The first axis explaining ca. 53% (eigenvalue 3.18), and second axis ca. 32% (eigenvalue 1.9) of the data variability. All four axes explain over 97% of data variability.
Axis 1 is positively correlated with all variables tested, except glucose content. The first axis determined the falling share of glucose in the honey types form right side of PCA diagram for sunflower (He), willow (Sa), multifloral (MP) and plum (P) to multifloral (MAP) in the middle, and lime (Tc), phacelia (Ph) in left side. In relation to the content of other sugars, axis 1 is positively correlated with them. The strongest correlation with sucrose and rhamnose is observed and discussed axis shows rising gradient of this sugars from rapeseed (Br), willow (Sa) and multifloral (MSa) to lime (Tc) and phacelia (Ph).
Axis 2 of the diagram is strongly, positively correlated with erlose content and strong negatively correlated with fructose and fucose content. The erlose content decrease form rapeseed Br (8.26 g/100 g), plum P (3.02 g/100 g) and willow Sa (2.92 g/100 g) by honeydew So, multifloral MSa, multifloral MBr and multifloral MAP honey where erlose content ranges from 1.77 g /100 g to 0.66 g /100 g appropriately to lime (Tc) and phacelia (Ph) honey in which no erlose was found. The high fucose content in sunflower (He) and multifloral (MAP) honey is positively correlated with fructose.
The ratio of fructose to glucose was typical for honey. The more glucose a honey has, the faster it tends to crystallize. In honey, the ratio of fructose to glucose ideally should range from 0.9 to 1.35. A fructose to glucose ratio below 1.0 leads to faster honey crystallization whereas crystallization become slower when this ratio is more than 1.0 [18, 19, 20]. In the present study, the average ratio of fructose to glucose was around 1. However, two tested honeys (Tc and Ph) had ratio well below 1.0 (0.85 and 0.71, respectively) which indicates the greater chances for honey crystallization (Table 4).
2.3. Antimicrobial activity of honey
The antimicrobial activity of the honey samples expressed by inhibition of the growth of the tested bacteria around the wells on the agar medium was varied (Fig. 3). The Gram-positive bacteria B. circulans proved to be the most sensitive to the activity of the honeys. The inhibition zones of bacterial growth were observed in all concentrations (62.5–500 mg/ml) in 7 honey samples: plum (P), rapeseed (Br), lime (Tc) and multifloral (MBr, MAP, MP, MSa). In the case of 3 honeys: willow (Sa), phacelia (Ph) and sunflower (He) no activity against B. circulans waqs found at concentrations of 125 and 62.5 mg/ml. On the other hand, honeydew honey (So) didn`t inhibit bacterial growth only in the concentration of 62.5 mg/ml. Taking into account the antibacterial activity observed after the use of the lowest concentration of honeys (62.5 mg/ml), it should be stated that the most effective B. circulans were honeys: multifloral (MSa), plum (P) and rapeseed (Br) (zones of growth inhibition 15.22, 14.17, 13.55 mm respectively).
The analysis of the results of the significance of differences test showed that the factors influencing the antibacterial activity of honeys against B. circulans are the type of honey and its concentration. The highest activity, expressed by the size of the inhibition zone, was observed for rapeseed honey (Br), this result is significantly different from plum (P) and multifloral (MAP) honey, which show similar activity, and willow (Sa), phacelia (Ph) and sunflower (He ) (Figs. 4A, B). At a lower concentration (Fig. 4C) rapeseed (Br), lime (Tc), multifloral (MSa), plum (P) and multifloral (MBr) are less efficient, but retain their antibacterial properties, which significantly differs from willow honeys (Sa), phacelia (Ph) and sunflower (He) which show no activity. At the lowest concentration, rapeseed (Br), multifloral (MSa), plum (P) and smaller multifloral (MBr) honeys show high activity, which significantly differs from the others, which have lost their properties (Fig. 4D).
For all tested honey types, inhibition of bacterial growth in all tested microorganisms at the highest concentration of 500 mg/ml was visible (Figs. 4A, 5).
It should be noted that the tested honey varieties showed significantly lower activity against other Gram-positive bacteria used in the experiments, i.e. S. aureus. In this case, the zones of inhibition of bacterial growth were observed only after the application of 50% honey concentrations. The growth of S. aureus was most strongly inhibited by the honeys: multifloral MBr (8.42 mm) and multifloral MSa (9.97 mm) (Fig. 5).
Similarly, only at a concentration of 500 mg/ml, the tested honeys inhibited the growth of Gram-negative bacteria E. coli. The largest zones of growth inhibition (9.6–11.9 mm), and thus the highest activity, was recorded for the following honeys: multifloral MBR and MAP. The exception was sunflower honey (He), which showed no activity against this bacteria (Fig. 5).
At a lower concentration (250, 125, 62.5 mg/ml), all tested honeys did not cause a decrease in E. coli and Staphylococcus aureus growth. A broader effect was evident when testing was done with honey at lower concentrations in relation to B. circulans and A. niger (Figs. 4B, C, D; Figs. 9B, C, D). In addition, based on the results obtained by the diffusion method, it was found that P. aeruginosa bacteria, both standard and clinical strain, was the microorganism completely insensitive to the tested honey varieties was.
The analyzed honeys show inhibitory activity against E. coli and S. aureus only in the highest concentration (Figs. 6, 7). Honeys: multifloral MAP, MP, MBr have the highest activity against E. coli and statistically significantly differ in this respect from honeys: multifloral (MSa) and plum (P) (Fig. 6). However, the activity of honeys: multifloral (MSa) and (MBr) against S. aureus is statistically significantly different from the properties of multifloral honeys: MAP and MP and lime (Tc) (Fig. 7).
2.4. Antifungal activity of honey
The antifungal activity of the honey samples used at concentrations ranging from 62.5 to 500 mg/ml was tested against A. niger, C. albicans and S. cerevisiae using the radial diffusion method. On the basis of the obtained results, it was found that C. albicans and S. cerevisiae showed resistance to the tested honey samples at all concentrations.
On the other hand, the tested honey varieties effectively inhibited the growth of A. niger (Fig. 8). The maximum antifungal activity was found in all honey samples at a concentration of 500 mg/ml in the range from 62 to 99.25 µg/ml based on the activity of amphotericin B (µg/ml). At this concentration, the most active honeys were: multifloral (MSa), plum (P) and honeydew (So), which differs significantly from multifloral (MBr) and phacelia (Ph) honeys (Fig. 9A). At a lower concentration (Fig. 9B) the properties of multifloral (MP), multifloral (MSa) and willow (Sa) honeys are comparable and significantly different from phacelia (Ph). At the next concentration, i.e. 125 mg/ml (Fig. 9C), multifloral (MSa) and honeydew (So) honeys retained antifungal properties, significantly different from rapeseed (Br) and phacelia (Ph). At the lowest concentration (Fig. 9D) willow (Sa) and multifloral (MBr) honeys are the most active, differing from plum (P) and rapeseed (Br), which show the lowest antifungal activity.
2.5. Catalase
All the honey samples with catalase addition had the same or similar growth inhibition zones compared to the control i.e. honey without catalase. The tested honeys remained active against B. circulans, E. coli and S. aureus, which proves that the activity was related to other factors and that hydrogen peroxide didn`t affect the antimicrobial activity of these honeys (Figs. 10–12).
2.6. Lysozyme activity of honey
In subsequent experiments, lysozyme activity was checked by applying the tested honey samples to plates containing M. lysodeikticus according to the procedure of Mohrig and Messner [10]. Lysozyme activity was found in all the tested honeys. The highest lysozyme activity corresponding to the activity of 447.26 ug/ml and 159.74 ug/ml EWL measured in multifloral honeys: MAP and MP. The other varietal honeys have low lysozyme activity. Comparable values were obtained for the following honeys: multifloral (MSa), willow (Sa), multifloral (MBr), sunflower (He) and plum (P), rapeseed (Br), lime (Tc), phacelia (Ph), honeydew (So) which are statistically significantly different from other samples (Fig. 13).
2.7. HPLC analysis of phenolic compounds in honey samples
The obtained results are presented in Table 5. The presence of caffeic and syringic acid in various amounts was found in all tested honeys. Some honeys have identified coumaric acid (in 45% of samples) and cinnamic acid (in 73% of samples). The highest content of caffeic acid was observed in honeys: phacelia (Ph) − 356.72 µg/g, multifloral Sa (MSa) and multifloral Br (MBr) − 318.9 µg/g, and cinnamic acid in willow honey (Sa) − 11.9 µg/g. The content of coumaric and syringic acid in the honey samples didn`t exceed 10 µg/g.
Table 5
Selected phenolic acids content.
Honey type | Phenolic acids (µg/g) |
Coumaric | Caffeic | Cinnamic | Syringic |
P | 8.2 | 7.2 | 0.15 | 3.96 |
MAP | – | 210.78 | – | 1.6 |
MP | – | 221.6 | 0.15 | 1.99 |
MSa | – | 318.9 | 0.15 | 2.6 |
Sa | 5.0 | 0.36 | 0.33 | 1.1 |
MBr | – | 318.9 | 0.3 | 2.7 |
Br | 4.4 | 3.0 | 1.6 | 4.7 |
Tc | – | 286.45 | 0.3 | 2.6 |
Ph | – | 356.72 | – | 3.2 |
So | 4.4 | 10.8 | 0.15 | 0.99 |
He | 0.65 | 0.7 | – | 2.7 |