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Bee wax samples were collected from local Apis mellifera apiaries in Daegu (Am1), and Andong National University bee lab (Am2), and A. cerana apiaries in Cheonan (Ac1) and Yean, Andong (Ac21, Ac22, Ac23) in 2021 (Fig. 1).

Fig. 1. 
Beeswax samples (Photo take by Aman Dekebo on September 21, 2021) of Apis mellifera; Am1 from Daegu, Am2 from ANU bee lab, and Apis cerana; Ac1 from Cheonan, Ac21-23 from yean, Andong, Korea in 2021.

Extraction, fractionation and derivatization of beeswax were done based on the method reported by Svečnjak et al. (2019) with some modification.

Isolation and analysis of different beeswax samples is a time and resources consuming process. Tulloch (1980) reported a quicker method which could be used to compare large numbers of beeswax samples by thin-layer chromatography (TLC) and gas liquid chromatography (GLC). The advantage of TLC is that it is the equipment is simple and inexpensive, and that all classes of components can be screened however its disadvantage is that the composition is analyzed only qualitatively. Additionally, TLC has the advantage in that a small amount of sample (less than 100 μg) is applied on a TLC plate, and solvent (chloroform or benzene) is used as a developing solvent to separate the components and 50% sulfuric acid is used as spraying reagent.

GC-MS analysis was done by using a GC (7890B, Agilent Technologies, USA) coupled with an MS (5977A Network, Agilent Technologies). The GC had an HP 5MS column (non-polar column, Agilent Technologies), 30 m×250 μm internal diameter (i.d.) and 0.25 μm film thickness. The injection volume was 1 μL. The carrier gas was helium flowing at a rate of 1 mL/min. The injector temperature was 280℃ and the injection mode was split mode with split ratio 20 : 1. The initial oven temperature was 120℃ and it was raised to 320℃ at 10℃/min held at this temperature for 5 min. Mass spectra were recorded in EI mode at 70 eV, scanning the 50-550 m/z range. The identification of compounds was performed by comparing the mass spectra of the compounds with those in the database of NIST11 (National Institute of Standards and Technology, Gaithersburg, USA). Relative amounts of detected compounds were calculated based on the peak areas of the total ion chromatograms (TIC).

Acid value, ester value, melting point and saponification values were done according to procedure reported by Svečnjak et al. (2019) and Iodine number was performed based on thee method of Bernal et al. (2005). The acid value determination is one of the most effective and simple methods for detecting beeswax adulteration especially with paraffin and stearic acid. Acid value decreases with the addition of paraffin (Bernal et al., 2005; Maia and Nunes, 2013; Svečnjak et al., 2015), and increases with the addition of stearic acid (Bernal et al., 2005).

Physicochemical properties of beeswax samples of two A. mellifera and four A. cerana were shown in Table 1. Melting point of the beeswax analyzed were comparable to each other. The acid value was higher for A. mellifera beeswax samples (18.1±0.23-19.5±0.43). The saponification and ester values were higher for A. cerana than A. mellifera beeswax samples. Iodine absorption values of A. mellifera is relatively higher than those of A. cerana. Our physicochemical parameters analysis results for all samples are comparable with those of international standard and the samples do not have adulterants. As the beeswax is the honeybee product from wax gland of worker bees, beeswax profiles would be more influenced by the honeybee species than floral, environmental or surrounding vegetation structures which are their foraging resources. Physicochemical properties were shown more influential between species. Biochemical or functional properties could be differed among honeybee species and relative to the local conditions such as floral composition and season.

Fig. 2 shows TLC profile of beeswax samples from A. mellifera and A. cerana. Our TLC analysis profile shows presence of typical spots of beeswax components comparable with those of Tulloch (1980).

Fig. 2. 
Thin layer chromatography (TLC) profiles of Beeswaxes of Apis mellifera; Am1 from Daegu, Am2 from ANU bee lab, and Apis cerana; Ac1 from Cheonan, Ac21-23 from yean, Andong, Korea in 2021 (A), compared with Tulloch (1980, B), where M; European A. mellifera; A; African A. m. adansonii; D, C, F; Asiatic (A. dorsata, A. cerana, A. florea, respectively). S is a mixture of 1, C₃₀ hydrocarbon (12 mg); 2, C₄₆ monoester (14 mg); 3, C₅₈ diester (5 mg); 4, C₇₆ triester (3 mg); 5, C₂₆ alcohol (3 mg); 6, C₄₀ hydroxy monoester (2 mg); 7, C₅₈ hydroxy polyester (2 mg); 8, C₂₄ acid (6 mg). Adsorbent is silica gel diluted twice with benzene; components were detected by spraying with 50% sulphuric acid.

GC-MS analysis of the hydrocarbon fraction showed the alkane C27 (heptacosane) was the most abundant and common (14.9-27.2%). The highest composition of the compound was recorded in Am1 and the lowest in Ac21. Another predominant compound in Am1 were nonacosane (C29), 1-docosene and octadecane (Table 2, Fig. 3). Similar trend of the percentage composition of the hydrocarbons were observed for those samples of ACs, but the detailed composition was differed even among those from the same apiary but different hives (Fig. 4).

Fig. 3. 
GC of the hydrocarbon fraction of Apis mellifera beeswax sample, Am1 collected from Daegu, South Korea; 1: Heptacosane (27.18%); 2: Nonacosane (18.35%); 3: Octadecane (15.92%); 4: 1-Docosene (16.11%); Am2 from Andong National University bee lab); 1: Heptacosane (15.41%); 2: Heneicosane (13.90%); 3: Octadecane (11.93%).

Fig. 4. 
GC of the hydrocarbon fraction of Apis cerana beeswax samples Ac1 from Cheonan, Korea; 1: Pentadecane, 2,6,10,14-tetramethyl- (12.39%); 2: Heptacosane (15.68%); 3: Nonacosane (9.54%); 4: Octadecane (9.00%) 5: Heptadecane, 9-octyl (6.45%). Ac21 collected from hive 1, Yean, Andong, South Korea; 1: Heptacosane (14.87%); 2: Nonacosane (9.13%); 3: Octadecane (8.04%); 4: Heptadecane, 9-octyl (8.33%); 5: Docosane, 11-butyl- (8.80%); 6: Docosane (6.4%). Ac22 collected from hive 2, Yean, Andong, South Korea; 1: Heptacosane (15.18%); 2: Nonacosane (9.48%); 3: Hexadecane (7.28%); 4: 2-methyloctacosane (8.77%); 5: Heptadecane, 9-octyl (7.98%). Ac23 collected from hive 3, Yean, Andong, South Korea; 1: Heptacosane (23. 07%); 2: Nonacosane (6.19%).

A. cerana beeswax constitute 88.6% hydrocarbon and the remaining 11.4% include nitrogen and silicone derivatized hydrocarbons. Hydrocarbon compounds such as heptadecane (C17), tetracosane (C24), pentacosane (C25), eicosane (C20), nonadecane (C19), heptacosane (C27), octadecane, 2-methyl (C19), heneicosane (C21), hexadecane (C16), 2-methyloctacosane (C29), octadecane (C18), docosane, 11-butyl-(C26), 6,6-diethylhoctadecane (C22), tetratriacontane, 17-hexadecyl-(C50), Anthracene and 9-ethyl-9,10-dihydro-9,10-dimethyl-(C18) were identified in the beeswax (Table 2). The principal compounds in the hydrocarbon fraction were heptacosane (15.7%) and 2-methyloctacosane (9.0%) (Table 2 and Fig. 4). The previous report indicated heptacosane (C27) is a wasp queen pheromone that decreased ovary activation in queenless workers (Oi, 2022). Since the structural basis of the cell consists only of wax, the wax must have suitable physical properties. Presumably some degree of plasticity and knead ability are also desirable. The unsaturated hydrocarbons of beeswax may act as plasticizers (Tulloch, 1970). Live bees are warm and produce a wide range of hydrocarbons. Dead bees cool rapidly and some of the volatile hydrocarbon levels decrease in the immediate vicinity of the corpse. The undertaker bees specifically monitor the levels of (at least) heptacosane and nonacosane as a means of discriminating between live and dead bees. Within 30 min of death local heptacosane and nonacosane levels have dropped below a level associated with life and the undertaker bee removes the corpse. This study was conducted on Apis cerana. A. mellifera, may use the same necrophoresis signals (The Apiarist, https://theapiarist.org/the-scent-of-death/).

Additionally, we detected presence of some Si-derivatized compounds as minor constituents of the beeswax of A. cerana sample. Zimnicka and Hacura (2006) analyzed chemical composition of beeswax of Apis mellifera using vibrational spectroscopy (FT-IR and Raman) and reported presence of esters and hydrocarbons as principal compounds together with alcohol and amides, inorganic compounds of Si, P, and S. The source of inorganic compounds of Si, P, and S were detected from samples collected from industrial area which was attributed to pollution of bees’ environment (Zimnicka and Hacura, 2006). Therefore, the presence of Si in the beeswax of A. cerana might be attributed by some environmental contaminants.

GC of the derivatized fraction of beeswax samples solution using the BF₃ solution in methanol enable identification of several class of compounds with methyl palmitate was the most abundant and common compound (8.46-28.66%). The percentage of this compound was higher for Apis mellifera (Am1). Heptacosane was also common and among major compounds in all beeswax analyzed.

The n-hexane fraction of sample of beeswax obtained from a local market in Sudan was reported to have heptacosane (37%) (Ebrahim, 2015) as a major compound along with other constituents even though its honey bee source was unknown. Aichholz and Lorbeer (1999) investigated the chemical composition of beeswax honeybee species Apis mellifera, Apis cerana, Apis dorsata, Apis laboriosa, Apis florea and Apis andreniformis using high-temperature GC/MS and reported the alkanes C₂₇ greater than 5% in beeswaxes of A. mellifera, A. cerana and A. florea (Aichholz and Lorbeer, 1999). Heptatriacontene (C₃₇) (5.4%) was reported as a major compound in A. cerana. However, this compound was absent in the beeswax sample we investigated. Previous reports showed that variation in chemical composition of beeswax depends on type the genetics of bees, the age of the wax and the climatic circumstances of its product (Negri et al., 2000; Maia and Nunes, 2013) which might be the reason for the absence of heptatriacontane in the A. cerana beeswax we analyzed.

Zhang et al. (2019) reported the number of hydrocarbons extracted from larvae and larval food at different larval instars of worker larvae of Apis mellifera. Pentadecane and heptadecane were major hydrocarbons which accounts (69.7-88.1%) in A. mellifera larvae and larval food. The source of pentacosane which was found in all bees waxes we analyzed in this study might be their larvae and larval food.

The gas chromatogram data of the mandibular gland pheromone of A. florea foragers revealed that the principal component of the gland pheromones of A. florea foragers was 1-eicosanol at (29.98 μg/bee) (Zhang et al., 2019). The other main components of its mandibular gland pheromones were nonadecane (5.775 μg/bee), 2-hexyl-1-decanol (4.181 μg/bee), 2-butyl-1-octanol (3.771), eicosane (1.978 μg/bee), heneicosane (1.804 μg/bee), limonene (1.663 μg/bee), 1-butyl-3-methyl acetate (1.646 μg/bee) and 2-heptanol (0.241 μg/bee). A. andreniformis foragers use 0.1% (w/v) 1-eicosanol, 10.0% (v/v) eicosane, 1.0%, 5.0% and 10.0% (v/v) heneicosane and 0.1% (v/v) 2-heptanol as attractant substances to mark rich food sources (Zhang et al., 2019).

Aichholz and Lorbeer (1999) reported percentage composition and class of compounds identified in beeswax of A. mellifera and A. cerana. The percentage composition of hydrocarbons in the derivatized solution of our beeswax (Table 3) sample of A. cerana is higher compared with those reported for A. cerana beeswax by Aichholz and Lorbeer (1999) analyzed by high-temperature gas chromatography. We found the derivatized beeswax has monoesters (21.5%) among which hexadecanoic acid, methyl ester (methyl palmitate) (14.1%) was the most abundant monoester in the derivatized solution of beeswax (Table 3, Figs. 5 and 6). The percentage of monoesters identified in this study was relatively lower compared with those reported by Aichholz and Lorbeer (1999).

Fig. 5. 
GC of the derivatized fraction of Apis mellifera beeswax sample, Am1 collected from Daegu, South Korea; 1: Methyl palmitate (28.66%); 2: Heptacosane (10.62%); 3: Tetracosanoic acid, methyl ester (9.76%); Am2 from Andong National University bee lab); 1: Methyl palmitate (9.96%); 2: Heptacosane (15.08%); 3: Heptadecane (12.53%); 4: Eicosane, 9-octyl- (10.04%).

Fig. 6. 
GC of the derivatized fraction of Apis cerana beeswax samples Ac1 from Cheonan, Korea; 1: Methyl palmitate (8.46%); 2: Heptacosane (15.16%); 3: Heptadecane (8.44%); 4: Eicosane, 9-octyl- (7.34%). Ac21 collected from hive 1, Yean, Andong, South Korea; 1: Methyl palmitate (14.87%); 2: Heptacosane (10.21%); 3: Heptadecane (7.72%); 4: Eicosane, 9-octyl- (9.00%); 5: Tetratriacontane, 17-hexadecyl- (8.55%); 6: 2-methylhexacosane (7.22%). Ac22 collected from hive 2, Yean, Andong, South Korea; 1: Methyl palmitate (12.43%); 2: Heptacosane (14.60%); 3: Heptadecane (8.69%). Ac23 collected from hive 3, Yean, Andong, South Korea; 1: Methyl palmitate (18.60%); 2: Heptacosane (17.35%); 3: Cyclobarbital (11.79%).

Jackson and Eller (2006) reported the methanolysis reaction of beeswax from A. mellifera which was catalyzed by an immobilized lipase from Candida antarctica in supercritical carbon dioxide and the alcohols. Then the researchers isolated the fatty acid methyl esters by precipitation of the mixture from heptane (Jackson and Eller, 2006). Tulloch (1971) analyzed chemical composition of A. mellifera beeswax. The authors identified esters by thin layer chromatography (TLC), gas liquid chromatography (GC) and nuclear magnetic resonance spectroscopy (NMR) and by comparison with synthetic mono-, di- and triesters, hydroxy mono- and diesters and acid esters. Acids, hydroxy acids, alcohols and diols composition from each ester fraction were determined by gas liquid chromatography. Negri et al. (2000) alsp reported hydrocarbons and monoesters compounds constituents of beeswax samples. These workers hydrolyzed hydroxide esters fractions first by using alcoholic potassium and then derivatized prior to GC-MS analysis. The beeswax constitutes 15.1-23.5% aliphatic saturated and mono unsaturated hydrocarbons.

Maia and Nunes (2013) analyzed beeswax of Apis mellifera using high temperature GC-MS and chemometric. Major components of the beeswax were hydrocarbons, palmitate, oleate and hydroxypalmitate monoesters. Additionally, palmitate and oleate monoesters present in beeswax were obtained from 1-octadecanol and 1-eicosanol (Maia and Nunes, 2013). Blum et al. (1988) extracted volatiles oxygenated volatile organic compounds by steam distillation, and analyzed it by using GC-MS. The major oxygenated compounds identified were decanal (46%), 1-decanol (10%), nonanal (18%), octanal (6%), heterocyclic aldehyde furfural (10%) and benzaldehyde (10%). Additionally, hydrocarbons such as nonane and C₁₅-C₂₇ alkanes were also reported from the steam distillate (Blum et al., 1988). The detailed compositional characteristics would be useful for future development into the new biomaterial for functional pharmacological and cosmetic uses.