Isolation and Molecular Weight Characterization of Tetragonula laeviceps Honey Protein

Abstract

The concentration of protein in honey is lower than polysaccharide. However, recently the honey's protein is intensively studied and it showed that protein also have several biological activities such as antibacterial activity. The purpose of this research is to isolate and characterize Tetragonula laeviceps honey protein by determining the molecular weight. Honey protein of Trigona laeviceps was isolated using ultra-filtration with the membrane's size of 30 kDa, then concentrated using tube membrane size 10 kDa. Molecular weight was analyzed by SDS PAGE. From the analysis, there was major protein band in honey produced by Tetragona laeviceps identified. The produced molecular weight of major protein bands were about 87 and 65 kDa. Determining of the molecular weight of this protein could be used to detect the originality of Tetragonula laeviceps honey from Indonesia. AbstrakIsolasi dan Karakterisasi Berat Molekul Protein Madu dari Tetragonula laeviceps. Konsentrasi protein dalam madu sangat rendah dibandingkan dengan polisakarida. Namun, belakangan waktu ini protein madu secara intensif diteliti dan menunjukkan bahwa protein madu juga memiliki beberapa aktvitas biologi yaitu aktivitas antibakteri. Tujuan dari penelitian ini adalah untuk mengisolasi dan mengkarakterisasi protein yang berasal dari madu Tetragonula laeviceps dengan menentukan berat molekul. Protein dari madu Tetragonula laeviceps diisolasi menggunakan ulltrafitrasi dengan penambahan gas N 2 (ukuran membrane 30 kDa). Berat molekul protein dianalisis dengan menggunakan SDS PAGE. Dari hasil analisis, didapatkan band major protein dalam madu Tetragona laeviceps. Berat molekul dari major protein adalah 87 dan 65 kDa. Penentuan berat molekul dapat berguna untuk mendeteksi originalitas madu Tetragonu laevices dari Indonesia.

Purification and characterization of proteins in multifloral honey from kelulut bee (stingless bee)

“…Dialysis was performed within 24 h with 3 times of buffer changing. After reaching 80–100 mL, the dialyzed samples were then centrifuged at 5000 rpm and – 4 °C for 30 min (Sahlan et al., 2018a, Sahlan et al., 2018b). The supernatants were collected and the pellets were disposed.…”

“…The sample resulted from purification was concentrated using amicon® ultra centrifugal filters (Merck, Ireland) with capacity of 4mL and MWCO of 10 kDa in order to get the higher bands intensity (Sahlan et al., 2018a, Sahlan et al., 2018b). The concentrated proteins were collected from the bottom part of the filters.…”

“…The bands corresponding to honey protein subunits were then extracted, destained, and digested in-gel using trypsin. Next, tryptic peptides were extracted and then further processed in preparation for LC-MS/MS analysis (Chua et al., 2015; Sahlan et al., 2018a, Sahlan et al., 2018b). The peptide sequences resulted from LC-MS/MS were then analyzed proteome discoverer 2.1 .…”

“…The publication of kelulut bees has increased over the past 10 years from 288 to 1870 publications in 2018 (Avila et al., 2018). The growth of research in this field is based on the presence of beneficial bioactive compounds such as proteins that make honey have high antibacterial or antimicrobial and antioxidant activity (Alvarez-Suarez et al., 2018; Boorn et al., 2010; Jalil et al., 2017; Sahlan et al., 2018a, Sahlan et al., 2018b). It is known that the protein concentration in the Tetragonula sp.…”

“…It is known that the protein concentration in the Tetragonula sp. is very high, reaching 97 μg/mL (Sahlan et al., 2018a, Sahlan et al., 2018b). The high economic value, the many benefits, and the absence of quality standard of kelulut bee honey makes this honey very likely to be faked.…”