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Pollen of four Quercus spp. (QA: Q. acutissima, QM: Q. mongolica, QS: Q. serrata, QV: Q. variabilis), collected from the National Institute of Forest Science in Suwon were used in this study. A voucher specimen was deposited at the National Institute of Forest Science, Suwon, Korea.

The content levels of sugars (sucrose and glucose) in the samples were analysed by a modified procedure of a reported LC method (Godin et al., 2011). A total of 2g of freeze-dried pollen was extracted with 100 mL of water in a cooled ultrasonic bath (40kHz) for 60 min. Then the extract was centrifuged (15,000 rpm, 4°C, 10 min) and the supernatant was separated with membrane filter (0.45μm) and transferred to vials. Samples were analysed on a Dionex Ultimate 300 HPLC system. A Sugar-pak (waters, 300×6.5mm) column with deionised water as the mobile phase was used for separation of the sugars. The flow rate was optimized as 0.5mL/min and a Shodex RI-101 Detector was used for identification. Quantification was performed on the basis of linear calibration plots of the logarithm of peak areas versus the logarithm of concentrations. The concentrations were expressed in grams per 100g of dry weight (DW). The summary of HPLC conditions for free sugar analysis in this study was listed in Table 1.

Approximately, 3g of freeze-dried pollen were transferred to Eppendorf tube with 5mL of distilled water. Then the extract was centrifuged (15,000 rpm, 4°C, 10 min) and the supernatant was separated with membrane filter (0.45μm) and transferred to vials. Organic acids in pollen were analyzed by a Dionex Ultimate 300 HPLC system. The chromatographic separation used for organic acid detection employed 0.01 N H₂SO₄ as the mobile phase, with a flow rate of 0.5mL/min, and the samples were injected into an Aminex 87H (300×10mm) column (Bio-Rad, USA). Organic acids were detected with a refractive index (RI) detector (ERC, RefractoMax, Japan).

Sweetness index in this study was calculated based on the amount and sweetness properties of individual carbohydrate in fruit (Keutgen and Pawelzik, 2007). The contribution of each carbohydrate was calculated, based on the fact that fructose is 2.30 and sucrose 1.35 times sweeter than glucose. Accordingly,

sweetness index = [1.0 (glucose) + 2.30 (fructose) + 1.35 (sucrose)]

All experiments were performed at least three times and the results were reported as mean±standard deviation (SD). Data were statistically evaluated by one-way ANOVA analysis and Duncan’s multiple range test. Results of tables and figure were presented as mean±SD.

The HPLC quantitative analytical data of the free sugars in pollen are listed in Table 2. Sugar composition is one of the most important parameters for food energy level. Glucose and sucrose were the major sugar components of pollen in Quercus spp. While fructose was detected in other pollens (Martins et al., 2011), there was no fructose detected in pollen of Quercus species in the present study. While other samples were pollen which obtained by bee collecting, we used pollen obtained directly from flower. This is the reason why we did not detected fructose from the pollen.

Glucose content are between 0.27 and 0.41g/100g, and sucrose contents are between 14.35 and 16.03g/100g. The highest sucrose content of pollen was 16.03g/100g in Q. variabilis.

It is well known that fruit sweetness depends not only on the content of each sugar, but also on the ratios of the main individual sugars (Zheng et al., 2016). It is also found that the sweetness index, which plays important role of taste, increased with fruit growth and development (Fig. 1). The highest sweetness of pollen was 21.98 in Q. variabilis while the lowest sweetness of pollen was 19.65 in Q. acutissima.

Fig. 1. 
Sweetness index of pollen of four Quercus spp. (QA: Q. acutissima, QM: Q. mongolica, QS: Q. serrata, QV: Q. variabilis).

Organic acids represent further compounds contributing to taste and flavour of pollen. Three organic acids in pollen, citric acid, malic acid, and fumaric acid, were detected by HPLC (Fig. 2). The organic acids of pollen in four Quercus spp. are shown in Table 3.

Fig. 2. 
Chromatogram of a pollen sample (Q. mongolica) for organic acid analysis by HPLC.

In this study, malic acid showed the main organic acid in pollen followed by citric acid and fumaric acid. There were different organic acids in different kinds of foods. Although citric acid dominated in lemons, limes and orange, the malic acid was the main organic acid in pollen (Albertini et al., 2006). Like pollen, malic acid was the most common in ripe apple, sweet cherry and blackberry (Zhoa et al., 2015). Organic acid are intermediates in the tricarboxylic acid (TCA) cycle, one of the respiratory pathways.

Citric acid content in pollen of Q. mongolica was the highest as 3.11g/kg. Fumaric acid were the third-most common organic acids, and occupied 0.03~0.17g/kg according to species. Citric acid can be used for proper mineral supplementation of food and as a flavoring ingredient. It is also reported that citric acid ensures proper functioning of the kidneys and prevents the formation of kidney stones.

Recently, it is found that organic acids have been used in food preservation and as a new generation of growth promotors instead of antibiotics (Kim and Rhee, 2015).

In this study, we analyzed the free sugars and organic acid using HPLC. To our knowledge, this is the first study investigating the content of free sugars and organic acids in pollen of four different Quercus species. From the results of this study we found that the glucose and sucrose were the major sugar components of pollen. The sweetness index was also calculated from the content of free sugars. The highest sweetness of pollen was 21.98 in Q. variabilis. Organic acid in pollen of Quercus spp. were mainly composed of citric acid, malic acid and fumaric acid. The highest citric acid and malic acid content in pollen were 3.11g/kg (Q. mongolica) and 5.62g/kg (Q. mongolica), respectively. The results would be helpful for using food and functional food products for pollen, due to the beneficial effects of free sugar and organic acid for human health such as antioxidants and anticarcinogenic properties.