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The free sugars from samples were analyzed using Dionex ultimate 3000 (Dionex). Free sugars were identified using a refractive index detector (Ri-101, Shodex) and a Sugar-pak column (Waters). The mobile phase was deionized water at a flow rate of 0.5 mL/min and the oven temperature was set to 80℃.

The organic acids were analyzed using Dionex DX500 ion chromatograph (Dionex). Organic acids were identified using an electro conductivity detector and an amount to 20 μL of each sample was injected into the ICE-AS6 column. The mobile phase was 0.4 mM heptafluorobutyric acid at a flow rate of 1 mL/min with Anion-ICE micromembrane suppressor as a suppressor and 5 mM tetrabutylammonium hydroxide as a regenerant.

The amino acids from each sample were analyzed using o-phthalaldehyde (OPA)/mercaptopropionic acid (MPA) and fluorenylmethyl chloroformate (FMOC) derivatization. The samples were mixed in borate buffer with OPA and FMOC. Samples were then analyzed using an Agilent 1200 series HPLC instrument (Agilent Technologies). After derivatization, an amount to 1 μL of each sample was injected into the INNO C18 column, 4.6 mm×150 mm, 5 μm (Youngjin Biochrom) at 40℃. Ultraviolet rays were detected at λ=338 nm by connected UV detectors. The emission and excitation wavelengths measured using fluorescence were 450 and 340 nm for the OPA derivative and 305 and 266 nm for the FMOC derivative. The mobile phase was comprised of solutions A (10 mM Na₂HPO₄ and 10 mM Na₂B4O₇·10H₂O, pH 8.2) and B (water : acetonitrile (ACN) : methanol (MeOH)=10 : 45 : 45, v/v%) with a flow rate of 1.5 mL/min. The gradient of A: B was initially set to 100 : 0 (v/v%), 55 : 45 at 26~28 min, 0 : 100 at 28~30.5 min, and 100 : 0 from 30.5 min.

The nutritional value of pollen often is evaluated by protein concentrations and amount of essential amino acids (Brodschneider and Crailsheim, 2010). To find protein sources for development of artficial bee feed, we searched twelve canditate protein sources which were industrial by-products. The sources were analysied for crude fiber, crude protein and essential amino acids. Depending on the results, brewer’s yeast and soybean oil meal were chosen for protein sources of substitute diet, which is named as Test A. The contents of Test A and the price of each ingredient was listed in Table 1. In test A, brewer̓s yeast and soybean oil meal was mainly used for protein suppliment. Soybean oil meal is suitable and most used feed for all livestock. The protein content is 50% after removing the skin of soybean, but 44% if the skin is not removed. Soybean oil meal is the standard for comparing the price or quality of different protein feeds. In soybean oil meal, the protein content or amino acid composition is uniform compared to other plant protein feeds. But soybean oil meal lacks of amino acids such as methionine, lysine and cystine, making it a limited source of amino acid for unit animals. Thus we mixed Brewer̓s yeast in Test A, it contain a lot of proteins, nucleic acids, carbohydrates, minerals, and vitamins.

Carbohydrates are primary energe source of honey bees (Brodschneider and Crailsheim, 2010). Nectar provides main source of carbohydrates for most bee species (Vaudo et al., 2015). Therefore sucrose is added in Teast A. Free sugar composition of honey accounts for an average of 38% fructose, 31% glucose which are the only monosaccharides in honey and other di and trisaccharids (Doner, 1977). Bee larvae require carbohydrates for normal development, often in the form of bee bread, however, a large quantity of carbohydrate-rich nectar is required by adult foragers (Brodschneider and Crailsheim, 2010). An adult honey bee worker require approximately 4 mg of utilizable sugars per day for survival (Barker and Lehner, 1974). Workers at foraging age have the enzymes necessary to use polysaccharides (starch) for flight metabolism (Hrassnigg et al., 2005). A lack of carbohydrates limits the number of larvae reared, when nectar sources are poor and winter storage has already depleted, or after harvesting the honey without adequate replacement of carbohydrates. Thus, carbohydrates are fed with sucrose solution to colonies in routine (Brodschneider and Crailsheim, 2010).

Canola pollen, mixed pollen, bee bread, Megabee and Test A were analysed for free sugar and organic acid concentration (Fig. 1 and Fig. 2). In Megabee and Test A, sucrose content showed the largest amounts with 46.15% and 59.60%, respectively. Sucrose was not found in canola pollen, mixed pollen and bee bread. Sucrose can not be used by honey bees and it’s molecule must be broken into two simple free sugars of glucose and fructose. This is done by an enzyme called sucrase, found in the digestive cavity stomach. Glusoce, as free sugar, plays a vital role in the functioning of brain, providing muscle energy for locomotion and body cells funtioning. Canola pollen and mixed pollen has a high content of glucose with 46.21% and 46.31%, respectively. Fructose, although an important free sugar for providing energy, can cause a problem like hydroxymethylfurfural (HMF) which is kown to be toxic for honey bees. Acids, like acetic acid (vinegar), citric acid (lemon juice) and tartaric acid (cream of tartar), produces HMF when mixed fructose. Bee bread showed the largest content of fructose with 68.06% among the 5 samples. In canola pollen and mixed pollen accounted for the largest content of fructose with 53.79% and 52.52%, respectively. However, fructose was not found in Megabee and Test A.

Fig. 1. 
Contents of free sugar on five samples (%).

Fig. 2. 
Contents of organic acid on five samples (%).

In the result of organic acid analysis, ctric acid accounted for the largest content in all five samples. Among them, Test A showed the highest amount of citric acid with 92.33%. Citric acid is a white powder used to give a sour taste to beverages and food products. It is also used as a preservative to prevent spoilage because it increases the acidity of products and restricts the bacterial growth responsible for food spoilage. It is also used in dietary supplements, as it enhaces the bioavailablity of the minerals.

De Groot reported in 1953 that arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine are the essential amino acids for honeybees (De Groot, 1953). Table 2 enlists the functions and requirements of each essential amino acid for honey bee. Among them, isoleucine accounted the highest content with 4.5%. Dietary sources of essential amino acids are used for growth, somatic maintenance and reproduction. Nicolson and Human in 2013, reported the essential amino acid concentrations in bee-pollens, expressed as the percentage of the total amino acids ranges from 34.59% to 48.49%. The most abundant amino acids in pollen proteins are proline, leucine, lysine, glutamic acid and aspartic acid (Nicolson and Human, 2013). The results of amino acid content in five samples are listed in Table 3. Canola pollen, mixed pollen and bee bread accounted for the highest content of proline with 1159.21 mg/L, 998.90 mg/L and 783.73 mg/L, respectively. Proline, a non-essential amino acid, is an important amino acid for the laying of queen bees and also necessary for wing muscles development in insects. It is swiftly metabolized and produces several nicotinamide adenine dinucleotide phosphate. Thus, prolin is considered as a fovourable ingredient of honeybees (Kim et al., 2020). Among the five samples, Test A showed the highest amount of lysine with 471.30 mg/L. Lysine is directly involved in nitric oxide synthesis, a known neurotransmitter to affect memory in bees and moths (Gage et al., 2020).