The chemical composition of pollen and honey primarily depends on the botanical and geographical origin of the species, as well as other factors – climatic conditions, soil type, plant species, etc. The present study was to knowledge the biochemical profile of pollen, staminate catkins, and honey samples of
Major honey components are carbohydrates (mainly monosaccharides, fructose, glucose, and sucrose) and water. In small amounts, honey also contains minerals, proteins, organic acids, volatile components, phenolics, vitamins, and pigments. Although present in small amounts, these components are very important for honey characterization and nutritive properties (
Due to ecological-geographical conditions very important are not only differences in some compounds (biogenic elements, heavy metals), which can increase/decrease the quality of pollen or honey but also evaluation of pollen sizes (
The chemical composition of honey primarily depends on the botanical origin of species or cultivars. However, climatic conditions and/or geographical origin can also affect the chemical composition even within the same honey type (
The aim of the present study was to knowledge the biochemical profile of pollen, staminate catkins, and honey samples of
We examined the biological material of
Middle phase of blooming
Inflorescences of staminate catkins
Ethanol (Centralchem s.r.o., Bratislava, Slovakia, p.a.).
Acetonitrile (Fisher Chemical, Loughborough, UK, HPLC grade).
Petroleum ether (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, Sigma Grade, ≥99%).
Ninhydrin (Ingos, Czech Republic), nitric acid (Analytika Praha Ltd, Czech Republic).
Hydrochloric acid (Analytika Praha Ltd, Czech Republic).
Methyl cellosolve (Ingos, Czech Republic).
Filter with 0.45 μm pore size (Labicom, Czech Republic).
Tin chloride (SnCl2) (Centralchem s.r.o., Bratislava, Slovakia, p.a.).
HPLC system with an ELSD detector (Agilent Technologies 1260 Infinity, Santa Clara, CA, USA).
HPLC system with ninhydrin and a VIS detector (Model AAA-400 amino acid analyzer, Ingos, Czech Republic).
UV-VIS spectrophotometer (UV Jenway Model 6405, UV/VIS, England).
ICP-OES system (Ultima 2, Horiba Scientific, France).
ES column (Zorbax SB-C18, 4.6x25.0 mm, 5 μm particle size, Agilent, Santa Clara, CA, USA).
Microwave oven (Milestone 1200, Milestone, Italy). Vertical shake table (GFL, Germany).
Сentrifuge (EBA 21, Hettich, Germany). Cation exchanger (LG ANB sodium cycle, Laboratory of Spolchemie).
Total dry matter, ash, and protein content were determined according to the EN method (
For the determination of saccharides, 1 g of sample was extracted with 10 mL of extraction solution (ultrapure water and ethanol mixed in ration 4:1) in a 50 mL centrifugation tube placed on a vertical shake table (GFL, Germany). After 1 h of extraction, samples were centrifuged for 4 min at 6000 rpm in a centrifuge (EBA 21, Hettich, Germany); the supernatant was filtered using a filter with 0.45 μm pore size (Labicom, Czech Republic) and filled up to 50 mL in a volumetric flask with ultrapure water. An Agilent Infinity 1260 liquid chromatography (Agilent Technologies, USA) equipped with an ELSD detector was used for the determination of saccharides. A Prevail Carbohydrates ES column (250/4.6 mm) was used as a stationary phase and acetonitrile (VWR) mixed with water in a 75:25 volume ratio was used as the mobile phase.
Total carotenoid content expressed as beta-carotene was analyzed at a wavelength of 445 nm spectrophotometrically (VIS spectrophotometer UV Jenway Model 6405 UV/VIS). Sample (1 g) was disrupted with sea sand and extracted with acetone until complete discoloration. Petroleum-ether was added and then water, in purpose to the separation of phases. After the separation, the petroleum ether-carotenoid phase was obtained and the absorbance was measured (
Sample for elemental analysis was prepared using the wet ashing method in a microwave oven (Milestone 1200, Milestone, Italy). A total of 0.25 g sample matrix was decomposed in a mixture of nitric acid (6 mL) (Analytika Praha Ltd, Czech Republic) and hydrochloric acid (2 mL) (Analytika Praha Ltd, Czech Republic).
After the decomposition sample was filtered using a filter with 0.45 μm pore size and filled up to 25 mL in a volumetric flask with ultrapure water. Elemental analysis was performed using ICP-OES (Ultima 2, Horiba Scientific, France) according to the procedure described by
Amino acids were determined by ion-exchange liquid chromatography (Model AAA-400 amino acid analyzer, Ingos, Czech Republic) using post-column derivatization with ninhydrin and a VIS detector. A glass column (inner diameter 3.7 mm, length 350 mm) was filled manually with a strong cation exchanger in the LG ANB sodium cycle (Laboratory of Spolchemie) with average particles size 12 μM and 8% porosity. The column was tempered within the range of 35 to 95 °C. The elution of the studied amino acids took place at a column temperature set to 74 °C. A double-channel VIS detector with the inner cell volume of 5 μL was set to two wavelengths: 440 and 570 nm. A solution of ninhydrin (Ingos, Czech Republic) was prepared in 75% v/v methyl cellosolve (Ingos, Czech Republic) and in 2% v/v 4 M acetic buffer (pH 5.5). Tin chloride (SnCl2) was used as a reducing agent. The prepared solution of ninhydrin was stored in an inert atmosphere (N2) in darkness at 4 °C. The flow rate was 0.25 (mL.min-1) and the reactor temperature was 120 °C.
Vitamin C concentration was determined according to the method described by
The method described by
Analysis Tocopherols and tocotrienols were analyzed by normal-phase HPLC using a Merck-Hitachi system (Merck, Darmstadt, Germany) with fluorescence detection (excitation: 292 nm, emission: 330 nm). Chromatographic separation was achieved within 45 min at 35 ±10 °C using a Eurospher-100 DIOL column (250 × 4.0 mm, 7 μm), preceded by a Eurospher-100 DIOL guard column (5×4.0 mm, 7 μm) (both from Knauer, Berlin, Germany). An isocratic mobile phase of n-hexane/MTBE (98:2, v/m) was used at a flow rate of 1.5 mL.min-1. Individual tocochromanols were identified by comparing their retention times with those of external standards and quantified by 5-point calibration curve of externalstandards.
All reagents and standards were of analytical grade. Vitamins standards were from Sigma-Aldrich with a purity ≥99%. Potassium hydroxide (KOH), concentrated sulfuric acid (H2SO4), and acetic anhydride was purchased from Mikrochem (Pezinok, Slovakia). Ethyl acetate, n-hexane, and sodium sulphate anhydrous were purchased from Centralchem s.r.o. (Bratislava, Slovakia). Methanol, HPLC grade was purchased from Fisher Chemical (Loughborough, UK).
Preparation 1 mg.mL-1 of working standards i. e. thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, and folic acid were dissolved in methanol. Solutions were further serially diluted to the concentration of 5 – 10 μg.mL-1 for thiamine chloride hydrochloride, 1.5 – 2 μg.mL-1 for riboflavin, 5 – 10 μg.mL-1 for pyridoxine hydrochloride, and 1 – 2 μg.mL-1 for folic acid. 50 μl of each solution was used for analysis.
Preparation. Each sample was weighed and triturated into fine powder. The powdered sample was suspended in 50 mL of methanol, sonicated for 15 min, and dilute to 100 mL with methanol. 50 μL of each was for HPLC analysis.
Chromatographic Conditions Shimadzu LC system (LC 20A pump) equipped with UV/Visible detector was used for analysis. A single wavelength of 254 nm was used for simultaneous detection. A Promosil HPLC column C18 was used at 35 ºC. Methanol – 0.05 M sodium acid phosphate (80 + 20, v/v) at pH 7.0 was used as mobile phase. The flow rate was adjusted to 0.8 mL.min-1. 50 μL of sample and standard solutions were injected in a single injection.
Freshly flowers (male catkins) were collected randomly from the three genotypes at the ballon stage (June 2020). Pollen samples released from male flowers (catkins) were further dried under laboratory conditions at 20 – 25 °C and prepared for analysis.
A total of 5 honey samples were collected from Ukrainian beekeepers. The samples were stored at room temperature in the dark and analyzed within the month after extraction. To confirm the beekeeper’s denomination, the samples were subjected to preliminary analyses. Uniflorality of samples was confirmed according to electrical conductivity.
Basic statistical analyses were performed using PAST 2.17. Data were presented as mean ±standard error of the mean (SEM) and differences between means were considered at
Bee pollen contains high amounts of carbohydrates, amino acids, fatty acids, minerals, and vitamins. The contents of these ingredients depend on the botanical origin of the pollen. The presence of these components shows that bee pollen can be used for human nutrition (
In honey, the content of proteins, including the enzymes, is relatively low and has a minor nutritive significance. On the other hand, the proteins, including the enzymes, are usually used as honey quality evaluation parameters. This is because protein content and enzyme activities vary regarding the botanical and geographical origin of the honey (
The phytochemical compounds in samples of pollen, staminate catkins, and honey of
Contents of some phytochemical compounds in pollen, staminate catkins and honey of
Components | Pollen ( |
Staminate catkins ( |
Honey ( |
---|---|---|---|
|
|||
954.6 ±29.32 | 190.6 ±3.10 | <1 | |
138.2 ±2.86 | 911.3 ±19.66 | – | |
– | – | 14.43 ±0.11 | |
169.0 ±1.60 | 69.8 ±1.67 | 6.5 ±0.16 | |
55.4 ±0.15 | 30.0 ±0.11 | 0.3 ±0.01 | |
3.0 ±0.04 | 2.4 ±0.009 | 2.0 ±0.01 | |
<0.1 | <0.1 | <0.1 | |
<0.1 | 24.0 ±0.13 | <0.1 | |
1.5 ±0.04 | 5.8 ±0.03 | 2.1 ±0.04 | |
<0.5 | 1.3 ±0.01 | 0.5 ±0.03 | |
<0.5 | 3.2 ±0.01 | 1.1 ±0.009 | |
95.0 ±2.10 | 71.0 ±2.17 | 55.0 ±0.96 | |
0.7 ±0.02 | 1.8 ±0.02 | 0.6 ±0.01 | |
<0.5 | 5.8±0.05 | 10.9 ±1.21 | |
3.0 ±0.01 | 4.1 ±0.05 | 38.0 ±1.32 | |
1.8 ±0.04 | 2.2 ±0.02 | 32.5 ±0.68 | |
4.6 ±0.05 | 2.1 ±0.04 | 6.1 ±0.06 |
Note:
Three free sugars, namely, fructose, glucose, and sucrose, were detected in all samples, while the honey samples have the highest content of carbohydrates (fructose 38 g.kg-1, glucose 32.5 g.kg-1, and sucrose 6.1 g.kg-1). Saccharides represent the main components of honey, and many papers have been published for using sugars as an indication of adulteration but cannot be used to identify the floral or geographical origins of honey (
Regarding the vitamin content, vitamin C was the most represented in all samples in the following amounts in pollen 95 mg.kg-1, staminate catkins 71 mg.kg-1, and honey 55 mg.kg-1.
The fat content predominates in pollen and staminate catkins against chestnut honey in amounts 55.4, 30.0, and 0.3 g.kg-1, respectively.
The fat content of the polyfloral bee pollen from different plant species varied in the intervals 10.0 to 130.0 g·kg-1 DW (
Data presented in Figure
Amino acid composition from pollen and staminate catkins of
According to many authors in bee pollen predominate aspartic acid, glutamic acid, glycine, and leucine (
The most abundant amino acid in staminate catkins was found to be aspartic acid (6.16 g.kg-1), slightly below glutamic acid (4.35 g.kg-1), which is 13.19 and 9.30% of the sum of total amino acid.
The main amino acids found for honey were proline (0.59 g.kg-1), which is 22.52% of the sum of total amino acids. Lower but also important amounts of glutamic acid (0.34 g.kg-1), aspartic acid (0.30 g.kg-1), tyrosine (0.24 g.kg-1) and valine (0.21 g.kg-1) were present (Figure
Amino acid composition from the honey of
Note: * – methionine, tryptophan and cysteine determined at the trace level (<0.01).
Honey contains approximately 0 – 1.5% amino acids. The proline is the main amino acid in pollen (
The present study, bioaccumulation, and biosorption of minerals and heavy metal concentration (K, P, Ca, Mg, Na, Fe, Mn, Zn, Cu, Ni, Sr, Sn, Hg, Se, Sb, Li) were observed in pollen, staminate catkins and chestnut honey (Table
The concentration of the determined macro- and microelements and heavy metals in the pollen, staminate catkins and honey samples of
Components (mg.kg-1) | Pollen ( |
Staminate catkins ( |
Honey ( |
---|---|---|---|
|
|||
7400 ±117 | 7760 ±219 | 981 ±77 | |
3845 ±87 | 1819 ±109 | 96 ±1.6 | |
0.27 ±0.010 | 5940 ±227 | 170 ±1.7 | |
1730 ±66 | 2040 ±169 | 69 ±1.4 | |
98 ±1.8 | 31.7 ±0.07 | 25.1 ±0.5 | |
461 ±56 | 109 ±13 | 13.6 ±0.6 | |
478 ±43 | 247 ±34 | 3.7 ±0.3 | |
126 ±19 | 30.1 ±0.8 | 2.1 ±0.05 | |
16.7 ±0.9 | 10.4 ±0.3 | <1 | |
41.5 ±1.1 | –* | –* | |
12.8 ±0.5 | –* | –* | |
1.9 ±0.03 | –* | –* | |
0.027 ±0.001 | –* | –* | |
<0.05 | –* | –* | |
<0.25 | –* | –* | |
<0.5 | –* | –* |
Note:
Macro- and microelement percentage composition in the pollen, staminate catkins and honey samples of
Macro-elements (Na, K, Ca, Mg, P) are the most represented group, dominated by potassium contained in pollen (7400 mg.kg-1), staminate catkins (7760 mg.kg-1) and honey (981 mg.kg-1). Potassium is the main mineral element with an average of one-third of the total. Some research has suggested that the trace element content in honey is mainly based on the botanical origin of honey, for instance, light blossom honey having a lower content than dark honey such as honeydew, chestnut, and heather (
Calcium is the 2nd most abundant element in the staminate catkins (5940 mg.kg-1) and honey (170 mg.kg-1), while in pollen it is only in a small amount (0.27 mg.kg-1). Calcium was the prominent element in the honey sample in the amount of 170 mg.kg-1. According to the data, the Ca contents of the honey sample in the present study was higher than the ones established in Serbia (20.1 mg.kg-1) (
We can compare the percentages of individual components, which show that potassium was the most represented element in the honey (72%), pollen (53%), and staminate catkins (43%). Phosphorus was the second most abundant element in pollen (28%) of the total minerals, while calcium represented in staminate catkins 33% and the honey samples 13% of the total minerals.
Microelements (Mn, Zn, Cu, Fe, Se, and Ni) are the second represented group of biogenic elements, where the content of manganese and iron prevailed in all samples. The content of manganese and iron predominate in pollen (478 mg.kg-1 Mn and 461 mg.kg-1 Fe) and staminate catkins (247 mg.kg-1 Mn and 109 mg.kg-1 Fe) than in the honey samples (13.6 mg.kg-1 Fe and 3.7 mg.kg-1 Mn).
Heavy metals (Hg, Sr, Sn, Sb, Li) are present only in the pollen samples with the most abundant of Sr (12.8 mg.kg-1), Sn (1.9 mg.kg-1), Hg (0.0027 mg.kg-1) content and other in the trace level (<0.05 mg.kg-1).
Heavy metals such as Cd, Pb, Cr, and Ni in honey can be used as indicators suggesting the environmental pollution status in the region (
The chemical composition, amino acid content, and minerals determined in pollen, staminate catkins, and honey obtained from the
The publication was prepared with the active participation of researchers in the international network AgroBio
These studies were supported by from Bilateral Scholarship of the Ministry of Education, Science, Research and Sport (Slovak Republic), SAIA and Visegrad Scholarship Fund (#52010544, #52010762) for the scholarship grants for the research stay during which the presented knowledge was obtained.
The authors declare no conflict of interest.
This article does not contain any studies that would require an ethical statement.