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The pH of samples was measured with pH-meter Thermo Scientific Orion Star A211 (Thermo Scientific Inc) with glass electrode. 2 g of each sample was dissolved in 15 mL of distilled water for 24 h at room temperature before analysis (Adaškevičiūtė et al., 2019). Calibration of pH-meter was performed with three different buffer solutions having pH values of 4, 7 and 10.

A Dionex ICS-3000 Reagent-Free Ion Chromatograph (Dionex Corporation, Sunnyvale, CA, USA) and column and Dionex IonPac (250 mm×4 mm) was used to identify the mineral levels using Ecem Bayram (Ecem Bayram, 2021). 0.6 g of sample was weighed and 7 mL of suprapur nitric acid (Sigma Aldrich, Germany) (65%) and 1 mL of hydrogen peroxide (Sigma Aldrich, Germany) (30%) were added. After that, the digestion procedures were carried out in a microwave digestion system (Milestone, Ethos Easy, Italy) according to instrumental parameters. The final volume of the samples removed from the microwave was completed to 50 mL with ultra-pure water. The column oven temperature was set at 30℃. The flow rate of 1 mL/min and injection volume 25 μL was used. The eluent used was 24 mM KOH for negative ion and 20 mM methanesulfonic acid (MSA) for positive ion (Huang et al., 2021). Detection system was set on suppressed conductivity, ASRS-URTRA II (4 mm) with recycle mode.

Total phenols content was determined by modified Folin-Ciocalteu colorimetry method (Kujala et al., 2000). 5 g samples were dissolved in 10 mL of distilled water, and 125 μL folin-Ciocalteu was added to the 125 μL of all samples. The mixture was incubated for 2 min at room temperature, and 1.25 mL of 7% Na₂CO₃ was added. Then, distilled water was added up to 3 mL of the final volume. This mixture was incubated for 90 min at room temperatuere then, centrifuged at 150 g for 10 min. The specific absorbance at 765 nm was immediately measured with UV/VIS Spectrophotometer (Perkin-Elmer Lambda 10). The standard curve was established with gallic acid.

Vitamin analysis is carried out for vitamin B₆ and vitamin C. For vitamin B₆ analysis, samples were prepared as follows (Aslam et al., 2008). For buffer preparation, 1.08 g of hexane sulphonic acid sodium salt and 1.36 g of potassium dihydrogen phosphate were dissolved in 940 mL of HPLC water and 5 mL of triethylamine was added to it and the pH was adjusted to 3.0 with orthophosphoric acid. Extraction solution was made by mixing 50 mL of acetonitrile with 10 mL of glacial acetic acid and the volum was finally made up to 1000 mL with double distilled water. 10 g of each sample was homogenized, and transferred into conical flasks and 25 mL of extraction solution was added, kept on shaking water bath at 70℃ for 40 min. Then, the sample was cooled down, filtered and finally the volum was made up to 50 mL with extraction solution. For vitamin C analysis, samples were prepared as follows. Homogenized by adding 10% methane phosphate solution to 10 g of sample, and extracted at 5℃ in the dark. This mixture was centrifuged at 12,500×g for 10 min and the supernatant was filtered with 0.45 μm filter (Hong et al., 2017). The samples were then analyzed using an Agilent 1200 series HPLC instrument (Agilent Technologies) under a 20℃-controlled column chamber. For HPLC analysis, 60 μL of each sample was injected into a waters symmetry C18 column, 4.6 mm×150 mm, 5 μm (Innopia Technologies, Korea). For vitamin B₆ analysis, the mobile phase was used with a linear gradient of Buffer/methanol (96 : 4) and filtered through 0.45 μm membrane filter. The flow of injection into the syetem of HPLC was 1 mL/min, using two channels simultaneously at a wavelength of 210 nm and 280 nm. For vitamin C analysis, the mobile phase was 0.05 M KH₂PO₄/ Acetonitrile (95/5) at pH 6.8. The flow rate of injection into the system of HPLC was 0.5 mL/min, and detection UV 254 nm.

The values of pH of the tested samples were identified (Fig. 2). Among them, bee pollen samples which were Canola pollen and Mixed pollen distinguished by the highest pH values with 5.12 and 5.18, respectively, while the lowest pH values showed beebread with pH 4.03 and Megabee with pH 4.05. The Test A was similar with beebread and Megabee with pH 4.30. Other studies in literature showed that the pH values of the bee pollen varied 4.30 to 6.30 and beebread from 4.11 to 4.44 (Siksna et al., 2014; Adaškevičiūtė et al., 2019). We found the results to be consistent with the data published by other authors. The royal jelly is crucial for the development of a bee larva into a queen (Kurth et al., 2019). In the natural system, hypopharyngeal gland secretion has a pH of 5.1, and just after addition of the mandibular gland secretion (pH 3.9) the pH of the final product is lowered to around pH of 4.0 (Hoffmann, 1960; Buttstedt et al., 2018). Thus, royal jelly is the mixture of the hypopharyngeal and mandibular gland secretions that reduces pH levels around 4.0 (Buttstedt et al., 2018). In addition, other research showed that the pH value of royal jelly varied from 3.6 to 4.1 (Adaškevičiūtė et al., 2019). The pH value of honey ranges from 3.4 to 4.1 (Jantakee and Tragoolpua, 2015). Since the pH of beebread, royal jelly, and honey, the main food for honey bee, is around pH 4.0, it is important to adjust the pH to around 4.0 when developing pollen substitute diets (Mureşan and Buttstedt, 2019).

Fig.2. 
Measurement of pH on five diets.

The elements chlorine (Cl), hydrogen sulfate (SO₄), monohydrogen phosphate (PO₄), nitrogen dioxide (NO₂), bromine (Br), lithium (Li), sodium (Na), ammonium (NH₄), potassium (K), magnesium (Mg), and calcium (Ca) was detected in the present study (Table 1).

The minerals that make up the body are essential micronutrient like amino acid, and it has a variety of physiological functions in the body. However, it is a substance that must be ingested through food because it is not synthesized in the body (Kim et al., 2021). In this study, five samples were examined in terms of concentrations of 11 different elements in total. The detailed mineral profile of five samples is presented in Table 1. Of these minerals, K was detected at the highest concentration with 5960.17 mg/kg, 6023.18 mg/kg, 5686.04 mg/kg, 9511.15 mg/kg in Canola pollen, Mixed pollen, Beebread and Megabee, respectively. The K with the highest content is involved in cell growth and helps to excrete sodium to maintain blood pressure and prevent osteoporosis (Ophir et al., 1983). In other study, K detected at the highest concentraion with 7400 mg/kg among the detected minerals on the pollen of Castanea sativa Mill (Chestnut) (Horcinova Sedlackova et al., 2021). Na acts as a water regulator and nerve stimulant in the body (Forbes, 1984). It contains 118.49 mg/kg, 148.53 mg/kg, 281.63 mg/kg, 1087.81 mg/kg, and 290.70 mg/kg in Canola pollen, Mixed pollen, Beebread, Megabee, and Test A, respectively.

The phenolic compound content of foods has become the focus of many studies due to their positive efects on health, especially antioxidant activity. The high antioxidant capacity of bee pollen is associated with its high content of phenolic compounds (Leja et al., 2007). In this study, the total phenol content of five samples was ranked from high to low as following: Canola pollen (16230 mg/kg), Bee bread (15660 mg/kg), Mixed pollen (9588 mg/kg), Megabee (4093 mg/kg), and Test A (3392 mg/kg) (Fig. 3).

Fig. 3. 
Contents of total phenol on five diets.

Bee pollen contains fat-soluble with 0.1% (A, E, D), water-soluble with 0.6% (B₁, B₂, B₆, C) vitamins, along with significant amounts of pantothenic acid, folic acid, rutin, and inositol (Komosinska-Vassev et al., 2015; Ecem Bayram, 2021). Although the vitamin content of bee pollen varies between 0.02 and 0.7% (Kieliszek et al., 2018). In this study, vitamin B₆ and C were analyzed (Fig. 4). Among the five samples only Test A and Canola pollen were detected vitamin B₆ and C with 83 mg/kg and 479 mg/kg, respectively. Average vitamin C contents of twelve domestic and three imported pollens from Spain, Vietnam and China were 554.7 mg/kg and 62.2 mg/kg, respectively (Lee and Ahn, 2019).Vitamin C content showed 9 times higher in domestic pollens than imported pollens. Chestnut trees are major honey plants in many countries and the vitamin C contents of its pollen was 95.0±2.10 mg/kg (Kim et al., 2020; Horcinova Sedlackova et al., 2021).

Fig.4. 
Composition of vitamin B₆ (A) and vitamin C (B) on five diets.