The origin of rare reducing sugar, trehalulose, in Australian stingless bee honey

Abstract

Trehalulose, an atypical glucose-fructose disaccharide with 𝛼𝛼-(1→1) glycosidic bond, has known low glycemic index, and acariogenic properties (Tian et al. 2019), as well as antioxidant activity (Kowalczyk et al. 2015). It has been discovered as the dominant sugar (13%-44%) in Australian, Malaysian and Brazilian stingless bee honey samples (Fletcher et al. 2020). Floral nectar is an aqueous mixture of glucose, fructose and sucrose, and the natural presence of trehalulose in stingless bee honey is highly curious. This study aimed to investigate the effect of feeding sugars to stingless bees on the sugar composition of their honey. By feeding sucrose and glucose/fructose solutions to confined stingless bees, this study established the origin of trehalulose and gained insight into optimization of its level in stingless bee honey. Microcolonies of the Australian stingless bee species Tetragonula carbonaria were created by transferring brood, adult bees and cerumen from established colonies into specially designed boxes with large viewing windows, which allowed detailed observation of all parts of the nest throughout the feeding experiments. Transferred stingless bees were permitted free forage for 3 days with small amount of supplementary food. During the feeding experiment, the microcolonies were confined to the box, and external liquid feeders were attached to the entrance. Sucrose solution (50:50 sucrose/water) or 1:1 glucose/fructose solution (25:25:50 glucose/fructose/water) were placed into the external feeder. During each period of confinement, bees were observed concentrating the sugar solutions into honey and filling honey pots. Honey samples were transferred by pipette from the honey pots, which were filled and capped during the confinement. The sugar composition of these honey samples was analysed through liquid chromatography-mass spectrometry (LC-MS/MS) and ion chromatography (IC-PAD). Table 1. Sugar composition (% of total sugar) of honey from confined stingless bee sugar feeding experiments Sugar fed Sample code Glucose (%) Sucrose (%) Fructose (%) Trehalulose (%) Erlose (%) Sucrose S1 0.4 ND 10.3 71.6 17.8 Sucrose S2 ND ND 8.8 68.7 22.5 Sucrose S3 1.0 ND 10.3 67.7 21.0 1:1 glucose/fructose GF1 45.0 ND 55.0 ND* ND* 1:1 glucose/fructose GF2 47.6 ND 52.4 ND* ND* 1:1 glucose/fructose GF3 48.6 ND 51.4 ND* ND* ND, not detected. Trehalulose was the most abundant sugar (67.7–71.6%) formed in the sucrose feeding experiments S1, S2 and S3 (Table 1). A novel trisaccharide erlose was detected for the first time, at 17.8–21.0%. Monosaccharides, glucose and fructose, were detected in low amounts. In the 1:1 glucose/fructose feeding experiments GF1-GF3 (Table 1), trehalulose, sucrose and erlose were not detected. The resultant ‘honey’ contained glucose and fructose in the same 1:1 ratio as per the feeding solution. From these feeding experiments, it was demonstrated that sucrose can be directly converted into trehalulose by live stingless bees. Feeding sucrose solutions resulted in honey that contained mostly trehalulose, erlose and fructose. In contrast, feeding 1:1 glucose/fructose solutions to stingless bees resulted in no conversion to trehalulose. In conclusion, stingless bees are able to produce trehalulose from the isomeric disaccharide sucrose, not directly from the monosaccharide components, glucose and fructose. Improving the trehalulose levels in stingless bee honey would involve locating nectar high in sucrose and optimizing bee access to that nectar. Further work using isotopically labelled sugars is required to elucidate whether the conversion of sucrose to trehalulose is an intermolecular or intramolecular process.