The APICULTURAL SOCIETY OF KOREA
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Current Issue

Journal of Apiculture - Vol. 33 , No. 3

[ Original Article ]
Journal of Apiculture - Vol. 32, No. 4, pp.375-379
Abbreviation: J. Apic.
ISSN: 1225-0252 (Print)
Print publication date 30 Nov 2017
Received 17 Nov 2017 Revised 24 Nov 2017 Accepted 24 Nov 2017
DOI: https://doi.org/10.17519/apiculture.2017.11.32.4.375

Free Sugar and Organic Acid Contents of Pollens from Quercus spp. in Korea
Youngki Park* ; Jae-Hee Kim ; Sea-Hyun Kim
Department of Forest Genetic Resources, National Institute of Forest Science, Suwon, 16631, Korea

Correspondence to : * E-mail: woodpark@korea.kr


Abstract

In this study, we analyzed free sugar and organic acid content in pollens of four different species of Quercus in Korea. Glucose and sucrose were the major sugar components of pollen. The highest sucrose content of pollen was 16.03g/100g in Q. variabilis. The sweetness index, which plays important role of taste, was also calculated from the content of sucrose and glucose. The sweetness of pollen were different with other species. The highest sweetness of pollen was 21.98 in Q. variabilis. Main organic acids detected in pollen were 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.


Keywords: Citric acid, Fumaric acid, Glucose, Malic acid, Sucrose, Sweetness index

INTRODUCTION

The consumption of natural products has increased substantially, because of the recent consumers’ trends. This current concept is mentioned in recent researches, which report several bioactivities of pollens from flower direct or collected by bee (Morais et al., 2011; Kim et al., 2005). Pollen is a fine, powder-like material produced by flowering plants and it is the male reproductive cells of flowers. Generally, flower pollens contain different phytochemicals and nutrients and are rich in carotenoids, flavonoids and phytosterols. Pollen and pollen products have been successfully used for the treatment off oral desensitisation of children who have allergy (Basim et al., 2006).

Since the pollen obtained from flower and collected from bee has therapeutic properties such as antioxidant, anti-inflammatory, antibacterial and anticariogenic activities, it is important to know chemical constituents of pollen (Kalaycioglu et al., 2017). In this study, we analyzed the free sugar and organic acid content in pollens of four Quercus species in Korea namely Q. acutissima, Q. mongolica, Q. serrata, and Q. variabilis.

Q. acutissima is closely related to the turkey oak, classified with it in Quercus sect. Quercus mongolica, commonly known as Mongolian oak, is a species of oak native to Japan, southern Kuriles, Sakhalin, Manchuria, central and northern China, Korea, eastern Mongolia, and eastern Siberia. The species can grow to be 30 m tall. Quercus serrata is a deciduous oak tree reaching a height of 25m occupying elevations from 100 to 2,000m. Leaves are up to 17cm long by 9cm wide, leathery, elliptical in shape, with serrated margins. Q. variabilis is a mediumsized to large deciduous tree growing to 25~30m tall with a rather open crown, and thick corky bark with deep fissures and marked by sinuous ridges. The leaves are green above and silvery below with dense short pubescence.

While there are some data on the chemical constituents such as total phenol, flavonoid and vitamin C and biological activities of pollen of Quercus spp. (Ghosh and Jung, 2017; Hong et al., 2015), there are no studies of the free sugar and organic acid content of four individual species. Thus, in this study, we report the overall free sugar and organic acid content of pollen in four Quercus species. We aim to increase the understanding knowledge of nutrition values of Quercus spp. pollen, which may be useful for using pollen as food and functional food products.


MATERIALS AND METHODS
Sample collection and preparation

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.

Free sugar content analysis

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.

Table 1. 
The operating conditions of HPLC for analysis of free sugars
Instrument Dionex ultimate 3000
Shodex RI-101 Detector
Column Sugar-pak (waters, 300×6.5mm)
Solvent Distilled water
Flow rate 0.5mL/min
Injection volume 10μL

Organic acid analysis

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).

Estimation of pollen taste parameters

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)]

Statistical analysis

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.


RESULTS AND DISCUSSION
Free sugars analysis

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.

Table 2. 
Free sugar content of pollen of four Quercus species (g/100g)
Species Contents of analyte (mean±SD, n=3) Total
Sucrose Glucose
Q. acutissima 14.35±0.14nsz 0.27±0.02cy 14.62±0.14ns
Q. mongolica 14.54±0.53ns 0.41±0.02a 14.96±0.54ns
Q. serrata 15.65±0.17ns 0.30±0.01bc 15.95±0.17ns
Q. variabilis 16.03±0.15ns 0.33±0.03b 16.37±0.14ns
zns means not significant.
yDifferent letters indicate Duncan's multiple range test (Significant at p<0.01).

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 acid

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.

Table 3. 
Organic acid content of pollen of four Quercus species. (g/kg)
Species Contents of analyte (mean±SD, n=3) Total
Citric acid Malic acid Fumaric acid
Q. acutissima 1.91±0.14cz 2.04±0.06c 0.03±0.002c 3.98±0.18c
Q. mongolica 3.11±0.13a 5.62±0.02a 0.17±0.004a 8.89±0.30a
Q. serrata 2.59±0.02b 2.17±0.01bc 0.04±0.003b 4.79±0.03b
Q. variabilis 0.67±0.03d 2.30±0.05b 0.04±0.001b 3.00±0.05d
zDifferent letters indicate Duncan's multiple range test (Significant at p<0.01).

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.


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