The sugar content profile of honey produced by the Indonesian Stingless bee, Tetragonula laeviceps, from different regions

Agussalim, Ali Agus, Nurliyani and Nafiatul Umami

Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta - 55281, Indonesia
aliagus@ugm.ac.id

Abstract

The objective of this study was to determine the sugar content profile of honey produced by the Indonesian stingless bee, Tetragonula laeviceps, from different regions. Honey was obtained from three regions, the Faculty of Animal Science Universitas Gadjah Mada (UGM), Nglipar Gunungkidul, Yogyakarta and North Lombok, West Nusa Tenggara. The sugar content profile of honey was analyzed for glucose, fructose, and sucrose contents; reducing sugars; the sum of fructose and glucose; and the ratio of fructose to glucose. All data were statistically analyzed using one-way analysis of variance (ANOVA), and significant differences between the means were identified with Duncan’s multiple range test (DMRT).

Region had a significant effect on glucose and sucrose contents, and reducing sugars, but not on the sum of fructose and glucose (p>0.05). In addition, region had a highly significant effect on fructose content and the ratio of fructose to glucose. Honey from the Faculty of Animal Science UGM had the highest sugar content profile compared with the honey from Lombok and Nglipar.

Keywords: flavor, geographical regions, nectar, processing

Introduction

Indonesia is an archipelagic country with different geographical regions. The different geographical regions affect meliponiculture, species of stingless bees and the types of plants that serve as food sources influence honey quality (chemical composition). In Indonesia, stingless bee species can be found nesting in bamboo, sugar palm stalks, tree trunk or wood and in the ground (Agussalim 2015; Agussalim et al 2015). Tetragonula laeviceps also known as Trigona bees, are a stingless bee species found in Indonesia that nests in bamboo (Agussalim et al 2017a). The honey from Indonesian stingless bees has been commercialized by beekeepers but has not been studied with regard to its chemical composition; therefore, information about it is lacking.

The main products of the stingless bee Tetragonula laeviceps are honey, bee pollen, bee bread, and propolis (Agussalim 2015; Agussalim et al 2015, 2017a). Honey is defined as the naturally sweet substance produced by honey bees or stingless bees from the nectar of plant flowers (floral nectar), the extrafloral nectar of plants and honey dew (Codex Alimentarius 2001). Honey is a natural food that is mainly composed of sugars and other constituents, such as enzymes, amino acids, organic acids, carotenoids, vitamins, minerals, and aromatic substances (Alqarni et al 2012; da Silva et al 2016). Honey from Tetragonula laeviceps has varying flavors consisting of sweet, sour, bitter, sour to sweet (acidic) and a mixture (Agussalim 2015; Suntiparapop et al 2012; Chanchao 2013). The properties and chemical composition, color, aroma, and flavor of honey depends mainly on the flowers sources of nectar, geographical regions, climate, and honey bee species involved in its production. Honey properties are also affected by postharvest treatment, such as weather exposure, processing, manipulation, packaging, and storage time (da Silva et al 2016; Chanchao 2013; da Costa Leite et al 2000; Juan-Borrás et al 2014; Escuredo et al 2014; Tornuk et al 2013).

Sugars are the main constituents of honey, comprising approximately 95% of honey dry weight (Bogdanov et al 2004). The sugars present in honey are responsible for properties such as energy value, viscosity, hygroscopicity, and granulation (da Silva et al 2016; Kamal and Klein 2011). The following sugar profiles of honey have been studied by scientists: fructose, glucose, sucrose, rhamnose, trehalose, nigerobiose, isomaltose, maltose, maltotetraose, maltotriose, maltulose, melezitose, melibiose, nigerose, palatinose, raffinose, erlose and others (da Silva et al 2016; Fuente et al 2011). The relative amount of the two monosaccharides fructose and glucose is useful for the classification of unifloral honey, as well as the fructose to glucose and glucose to water ratios (Bogdanov et al 2004). Furthermore, the sum of fructose, glucose, the fructose to glucose ratio, and the glucose to water ratio are important factors related to honey quality. The Fructose to glucose ratio indicates the ability of honey to crystallize (Manikis and Thrasivoulou 2001; Buba et al 2013; Kaškoniené et al 2010). The objective of this study was to determine the sugar content profile of honey produced by the Indonesian stingless bee, Tetragonula laeviceps, from different regions.

Materials and methods

Honey from the stingless bee, Tetragonula laeviceps, was obtained by meliponiculture for four months in three regions. This study used a completely randomized design (CRD) with three treatments (different regions). The first location was the Faculty of Animal Science UGM, District of Depok, Sleman Regency, Yogyakarta. The second location was in Katongan Village, District of Nglipar, Gunungkidul Regency, Yogyakarta. The third location was in Sukadana Village, District of Bayan, North Lombok, West Nusa Tenggara. The honey was harvested, separated from the propolis and placed in a plastic bottle before analysis of the sugar content profile. The sugar content profile consistsed of glucose, fructose, and sucrose contents; reducing sugars; the sum of fructose and glucose; and the fructose to glucose ratio. For all analyses, the sugar content profile of honey was performed in duplicate for the three replicates.

Analyses of glucose and fructose contents

The glucose and fructose contents of honey were analyzed by high-pressure liquid chromatography (HPLC). The samples were prepared by adding 0.11 g of honey to 5 mL of aquadest, which was then extracted with an ultrasonic sonicator for 15 minutes. The samples were vortexed for 2 minutes and then centrifuged for 5 minutes. The supernatant was removed, and the pellet or residue was extracted again 3 times with aquadest. The supernatant was transferred to a 25 mL Erlenmeyer flask until it reached the limit mark, and then the supernatant was centrifuged for 5 minutes. It was filtered by a Millex 0.45 µM filter and then 20 µL was injected into the HPLC column. The sugar standard for fructose and glucose was determined with concentrations 12.5, 25, 100, 500, and 1,000 ppm and then injected into the HPLC column in 20 µL volumes. The standard curve for glucose is shown in Figure 1, and the standard curve for fructose is shown in Figure 2. The components of the HPCL system were a MetaCarb column at 87^oC, an H₂O eluent, a flow rate 0.5 mL/min, a temperature of 85 ^oC, and a refractive index detector (RID).

Figure 1. Standard curve to determine the glucose content of honey	Figure 2. Standard curve to determine the fructose content of honey

Analysis of sucrose content

The sucrose content was determined by the Luff Schoorl method as described by AOAC (1990) and divided into two steps. The first step before inversion, included adding approximately 2 g of honey to aquadest, then homogenizing in an Erlenmeyer flask (50 mL). The sample was added to 5 mL and 25 mL of Luff Schoorl solution plus 2 boiling stones, and the samples were cooled and heated for 10 minutes. The solution was cooled quickly, and 15 mL of KI 20%, followed by 25 mL of H₂SO₄ 26.5% was carefully added. Furthermore, titration using Na₂S₂O ₃ 0.2 N, which was standardized, was performed, and 2 to 3 mL of starch was added near the end point of titration. The blank consisted of a treatment without the sample, and the total sugar amount before inversion was calculated by equations 1 and 2 as follows:

Equation 1

Sugar with N Na₂S₂O₃ 0.2 N (mg) = ((A+C) x B) - A

Where

A = mg sugar in the table (small)

B = mg sugar in the table (big)

C = decimal of titration difference

Equation 2

In the second step, after inversion, 10 mL of filtrate was transferred to a 50 mL Erlenmeyer flask with the addition of 5 mL HCl 6.76%. The solution was then heated in a water bath at 60^oC for 10 minutes (rocked for 3 minutes). The solution was quickly cooled to 20^oC, and then a few drops of phenolphthalein (pp) indicator was added and neutralized by NaOH 20% until a red color appeared. A HCl 0.5 N solution was added dropwise until the red color vanished, and then the solution was diluted using aquadest up to the mark. Samples (50 mL) were transferred to an Erlenmeyer flask, and 25 mL of Luff Schoorl solution plus 2 boiling stones were added. The samples were then cooled and heated for 10 minutes. The solution was cooled quickly, 15 mL of KI 20%, followed by 25 mL of H ₂SO₄ 26.5%, was carefully added. Titration was performed using Na₂S₂O₃ 0.2 N and 2 to 3 mL of starch was added near the end of titration. The blank consisted of a treatment without the sample. The total sugar after inversion was calculated by equations 1 and 2, similar to the calculation of sugar total before inversion. The total sugar (%) was calculated by equations 3 and 4 as follows:

Equation 3

Sugar total (% w/v) = the different sugar content after inversion and before inversion

Equation 4

Sucrose content (% w/v) = sugar total (% w/v) x 0.95

Analysis of reducing sugars

The reducing sugar analysis was performed using the Layne-Enyon method as described by AOAC (1990). Approximately 2.6 g of honey was weighed and transferred to a 500 mL volumetric flask. Five milliliters (5 mL) of standardized Fehling’s solutions A and B were transferred to a 250 mL Erlenmeyer flask containing 7.0 mL of water and 15.0 mL of honey solution. The Erlenmeyer flask was heated and 1.0 mL of methylene blue (0.2%) was added. Titration was carried out by adding the diluted honey solution until the indicator decolorized. The reducing sugar content was expressed in g 100 g^-1.

Statistical analysis

The data of the sugar content profile was analyzed with one-way analysis of variance (ANOVA) using SPSS (Windows version of SPSS, release 22) (SPSS 2013). Significant differences between the means were identified with Duncan’s multiple range test (DMRT) (Steel et al 1997). Values of p<0.05 and p<0.01 were considered statistically significant.

Results and discussion

Glucose content

The results showed that the different regions for the meliponiculture of the stingless bee Tetragonula laeviceps had a significant effect (p<0.05) on the glucose content of honey (Table 1). At 22.78% w/w, the highest glucose content was in honey from Nglipar Gunungkidul, followed by 20.25% w/w in honey from the Faculty of Animal Science UGM and at 11.49% w/w, the lowest glucose content was in honey from Lombok. The glucose content of honey from Nglipar and the Faculty of Animal Science UGM did not differ, but the glucose content of honey from Lombok did differ. The glucose content of honey is affected by botanical origin (the types of flowers as the source of nectar for the raw material to produce honey), geographical origin (for beekeeping or meliponiculture), climate, processing and storage (da Silva et al 2016; Escuredo et al 2014; Tornuk et al 2013). All of honey in the study included fresh honey so the floral source in each region is influence the glucose content. The predominant floral in each region is different which shown by Table 2 and plant types in the Faculty of Animal Science UGM and Nglipar were potentially the source of nectar for honeybees or stingless bees in Yogyakarta (Agussalim et al 2018, 2017b). Different flowers have an impact on the chemical composition of nectar, therefore, the chemical composition of honey will influence the glucose content. However, in our study, we did not an analyze the chemical composition of each nectar that was produced by each plant.

The glucose content from honey in the study ranged from 11.49 to 22.78% w/w (Table 1) is similar to those previously reported (Suntiparapop et al 2012; Oddo et al 2008) and was lower to those previously reported (Biluca et al 2016; Guerrini et al 2009; Souza et al 2006). The glucose content was lower than the international standard with minimum glucose content for floral honey is 60 g/100 g (Codex Alimentarius 2001) and Indonesian standard is 65% w/w (SNI 2018). The lower glucose content was related to the ability of stingless bee Tetragonula laeviceps to collect much more nectar from various plants just around the meliponiculture location with near distance and can not reached the long distance because they have the smallest body size (Michener 2007) and consequent the number of flowers can be visited also a little.

Fructose content

The results showed that the different regions for the meliponiculture of the stingless bee Tetragonula laeviceps had a highly significant effect (p<0.01) on the fructose content of honey (Table 1). The highest fructose content was in honey from the Faculty of Animal Science UGM, at 22.92% w/w, followed by honey from Lombok (20.76% w/w), and the lowest fructose content was in honey from Nglipar (7.79% w/w). The fructose content of honey from the Faculty of Animal Science UGM and Lombok did not differ, but the fructose content of honey from Nglipar did differ. The fructose content of honey is affected by botanical origin (the types of flowers as the source of nectar for the raw material to produce honey), geographical origin (for beekeeping or meliponiculture), climate, processing and storage (da Silva et al 2016; Escuredo et al 2014; Tornuk et al 2013). All of honey in the study included fresh honey so the floral source in each region is influence the fructose content. The predominant floral in each region is different which shown by Table 2. Different flowers have an impact on the chemical composition of nectar, therefore, the chemical composition of honey will influence the fructose content. However, in our study, we did not an analyze the chemical composition of each nectar that was produced by each plant.

The fructose content from honey in the study ranged from 7.79 to 22.92% w/w (Table 1) is lower to those previously reported (Biluca et al 2016; Suntiparapop et al 2012; Guerrini et al 2009; Oddo et al 2008; Souza et al 2006). The fructose content was lower than the international standard with minimum 60 g/100 g for floral honey (Codex Alimentarius 2001). The lower fructose content was related to the ability of stingless bee Tetragonula laeviceps to collect much more nectar from various plants just around the meliponiculture location with near distance and can not reached the long distance because they have the smallest body size (Michener 2007) and consequent the number of flowers can be visited also a little.

Sum of fructose and glucose

The results showed that the different regions for the meliponiculture of stingless bee Tetragonula laeviceps had no significant effect (p>0.05) on the sum of fructose and glucose in honey (Table 1). At 43.16% w/w, the highest sum of fructose and glucose was detected in honey from the Faculty of Animal Science UGM, followed by 32.25% w/w in honey from Lombok, and the lowest sum, at 30.57% w/w, was in honey from Nglipar. The sum of fructose and glucose obtained in the study ranged from 30.57 to 43.16% w/w (Table 1). This value is lower than the international standard by Codex Alimentarius (2001), in which the minimum sum of fructose and glucose content for floral honey is 60 g/100 g and also was lower than previously reported (Suntiparapop et al 2012; Guerrini et al 2009; Oddo et al 2008; Souza et al 2006). The sum of fructose and glucose depends on fructose and glucose content.

Fructose to glucose ratio

The results showed that the different regions for the meliponiculture of stingless bee Tetragonula laeviceps had a highly significant effect (p<0.01) on the fructose to glucose (F/G) ratio of honey (Table 1). At 1.99, the highest fructose to glucose ratio of honey was from Lombok, followed by 1.13 in honey from the Faculty of Animal Science UGM, and the lowest fructose to glucose ratio, at 0.34, was from Nglipar. The average of fructose to glucose ratio obtained in the study ranged from 0.34 to 1.99, which is similar to those previously reported (Suntiparapop et al 2012; Souza et al 2006) and lower was reported by Biluca et al (2016). The fructose to glucose ratio may reflect the ability of honey to crystallize (Suntiparapop et al 2012) and honey crystallization is slower when fructose to glucose ratio exceeds 1.3 (Amir et al 2010). Bases on the value honey from Lombok and Faculty of Animal Science might be slower crystallization than honey from Nglipar but until now all of honey not yet crystallize.

In addition, the average of fructose to glucose ratios of Tetragonula laeviceps honey were obtained from Lombok, the Faculty of Animal Science UGM, and Nglipar were 1.81:1, 1.13:1, and 1:2.93, respectively. The average of fructose and glucose ratio is 1.2:1, but this ratio depends largely on the source of the nectar collected by worker bees as the raw material to produce honey. This ratio is used to evaluate the crystallization of honey due to the lower solubility of glucose than fructose in water (da Silva et al 2016; Escuredo et al 2014, Tornuk et al 2013; Fuente et al 2011). Thus honey from the Faculty of Animal Science UGM was balanced, while the honey from Lombok was higher in fructose, and the honey from Nglipar was higher in glucose.

Sucrose content

The results showed that the different regions for the meliponiculture of the stingless bee Tetragonula laeviceps had a significant effect (p<0.05) on the sucrose content of honey (Table 3). At 4.49% w/v, the highest sucrose content of honey was from the Faculty of Animal Science UGM, followed by Lombok (3.00% w/v) and Nglipar (2.56% w/v). The sucrose content of honey from the Faculty of Animal Science UGM was different from that of Lombok and Nglipar. Sucrose content of honey is affected by botanical origin (the types of flowers as the source of nectar for the raw material to produce honey), geographical origin (for beekeeping or meliponiculture), climate, processing and storage (da Silva et al 2016; Escuredo et al 2014; Tornuk et al 2013). All of honey in the study included fresh honey so the floral source in the each regions is influence the sucrose content. The predominant floral in each region is different which shown by Table 2. Different flowers have an impact on the chemical composition of nectar, therefore, the chemical composition of honey will influence the sucrose content. However, in our study, we did not an analyze the chemical composition of each nectar that was produced by each plant.

The sucrose content of Tetragonula laeviceps honey obtained in this study ranged from 2.56 to 4.49% w/v, which is acceptable according the international standard (Codex Alimentarius 2001) and Indonesian standard (SNI 2018) with maximum 5 g/100 g for floral honey. The sucrose content of Tetragonula laeviceps honey in this study is similar was reported by Guerrini et al (2009), was lower to those previously reported (Suntiparapop et al 2012; Souza et al 2006) and was higher than reported by Chutong et al (2016) and Oddo et al (2008). The sucrose content is a very important parameter to evaluate the honey maturity and to identifying any improper manipulation of honey. The high sucrose content may indicate a variety of adulterations such as adding cheap sweetenres like cane sugar or refined beet sugar; early harvest, indicating that the sucrose was not completely transformed into glucose and fructose; or prolonged artificial feeding of honeybees or stingless bees with sucrose syrups (da Silva et al 2016; Escuredo et al 2013; Puscas et al 2013; Tornuk et al 2013). Thus, honey in the study included mature and pure honey.

Reducing sugar content

The results showed that the different regions for the meliponiculture of the stingless bee Tetragonula laeviceps had a significant effect (p<0.05) on the reducing sugar content of honey (Table 3). At 60.14 g/100 g, the highest reducing sugar content was in honey from the Faculty of Animal Science UGM, followed by 50.83 g/100 g in honey from Lombok, and the lowest reducing sugar content, at 44.07 g/100 g, was in honey from Nglipar. The reducing sugar content of honey from the Faculty of Animal Science UGM was different from that of Lombok, which was also different from that of Nglipar. The reducing sugar content of honey from the stingless bee Tetragonula laeviceps ranged from 44.07 to 60.14 g/100 g, which is lower or similar than the international standard with minimum 60 g/100 g for floral honey (Codex Alimentarius (2001). The reducing sugar content of honey from the Faculty of Animal Science UGM was acceptable by Codex Alimentarius, while honey from Lombok and Nglipar was not acceptable.

Reducing sugar content of honey is affected by botanical origin (the types of flowers as the source of nectar for the raw material to produce honey), geographical origin (for beekeeping or meliponiculture), climate, processing and storage (da Silva et al 2016; Escuredo et al 2014; Tornuk et al 2013). All of honey in the study included fresh honey so the floral source in each region is influence the reducing sugar content. The predominant floral in each region is different which shown by Table 2. Different flowers have an impact on the chemical composition of nectar, therefore, the chemical composition of honey will influence the reducing sugar content. However, in our study, we did not an analyze the chemical composition of each nectar that was produced by each plant. The reducing sugar content of honey obtained in this study is similar was reported by Guerrini et al (2009) and was lower to those previously reported (Biluca et al 2016; Souza et al 2006).

In general, the sugar composition of honey is affected by botanical origin (the types of flowers used by the bees to produce honey), geographical origin, climate, processing (weather exposure, heating processes, manipulation, packaging) and storage time (da Silva et al 2016; Escuredo et al 2014; Juan-Borrás et al 2014; Tornuk et al 2013; Chanchao 2013; da Costa Leite et al 2000)

Conclusions

• Different regions of meliponiculture influence the sugars content profile of honey from the Indonesian stingless bee, Tetragonula laeviceps.
  
  
• The honey from the Faculty of Animal Science UGM had the highest sugar content profile compared with the honey from Lombok West Nusa Tenggara and Nglipar Gunungkidul, Yogyakarta.

Significant statements

This study discovered that honey from the Indonesian stingless bee, Tetragonula laeviceps, has a good quality and is characterized by a low sugars content, it can be beneficial as a functional food in the therapy of diabetic patients. This study will help researchers to uncover the sugar profile of honey from the Indonesian stingless bee, Tetragonula laeviceps, which have not yet explored. Thus, a new theory on these properties and other possible benefits of honey may be formulated.

Acknowledgments

We would like to thank the Directorate of Research and Community Service, Ministry of Research, Technology, and Higher Education of the Republic of Indonesia for financial support of the research through Penelitian Terapan Unggulan Perguruan Tinggi (PTUPT), Penelitian Disertasi Doktor (PDD), and Directorate of Research Universitas Gadjah Mada (UGM) through Rekognisi Tugas Akhir (RTA) 2019.

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Received 14 February 2019; Accepted 20 May 2019; Published 4 June 2019

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