Extraction and identification of proteins and their antioxidant activities from Acacia, Tualang and Gelam honey samples

Abstract

Honey protein is mostly studied from royal jelly samples. A relatively limited study was carried out on protein from honey samples. This is because usually protein in honey presents in minute amount. However, the presence of protein in honey might also contribute to the pharmacological activities of honey, especially its anti-oxidant property. Therefore, this study investigated the extraction methods of honey protein such as dialysis, ammonium sulphate and sodium tungstate precipitation methods. The results showed that the dialysis method produced the highest amount of protein content for all types of honey samples such as Acacia (6.755 µg/µL), Tualang (5.675 µg/µL) and Gelam (3.515 µg/µL) honey using Bradford assay. The crude protein samples were then separated by 12% polyacrylamide gel and exhibited six (20.1-76.7 kDa), seven (30.3-79.5 kDa) and three (30.7-56.3 kDa) protein bands on the gel for Acacia, Tualang and Gelam honey samples, respectively. The protein bands were excised and digested by trypsin before analysed by a liquid chromatography integrated with tandem mass spectrometer (LC-MS/MS). The mass spectrometric data was matched with the universal protein database of Uniprot using the algorithm of BLAST (Basic Local Alignment Search Tool). Interestingly, the Acacia honey contained a number of major royal jelly proteins (MRJPs) such as MRJP 1 (76.7 kDa), MRJP 2 (44.7 kDa) and MRJP 5 (64.9 kDa) with the probability higher than 95% and score value higher than 3. The Tualang honey contained MRJP 7 precursor. Besides the MRJP, Tualang and Gelam honey contained a few uncharacterized proteins from honey bees (Apis mellifera). The low score of honey protein from both Tualang and Gelam honey could be due to the detection of single peptide. In line with the results of Bradford assay and LC-MS/MS, Acacia honey protein showed the highest antioxidant properties, followed by Tualang and Gelam honey protein samples based on the results of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assays