The Application of SDS-PAGE Protein and HPTLC Amino Acid Profiling for Verification of Declared Variety and Geographical Origin of Honey

Abstract

Proteins and amino acids are minor components of honey that are rarely used for its quality evaluation, although these components create its biological activity as well as can serve for overheating detection. The aim of the study was to use these indicators to confirm a declared on the label variety. Fifty-eight honey samples of 8 different varieties meeting the commercial quality requirements were used, including 28 local Polish and 30 commercial (mainly imported from EU and non-EU) honeys. For honey protein profiling previously used, polyacrylamide electrophoresis in denaturing conditions (SDS-PAGE) was applied whereas the free amino acid profile was analyzed by high-performance thin-layer chromatography (HPTLC) for the first time. As auxiliary indicators, the colorimetric determination of the protein content by the Bradford method, the activity of 5 glycolytic enzymes, including diastase, β-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-glucosidase, and the content of proline were used. It has been shown that based on the determined model protein SDS-PAGE profiles for selected monofloral honeys, it is possible to detect honeys of questionable variety based on the lack of specific protein bands or their diverging intensity. The HPTLC amino acid analysis can serve as a supporting control tool, capturing differences in the amino acid profile. Due to a great variation of multifloral honey, such assays are effective for monofloral honey only. The colorimetric assays, especially for total protein and β-galactosidase, can be also useful. The applied tools can be proposed for the initial verification of honey variety for cost reduction of officially recognized melissopalynological analysis.

Coniferous Honeydew Honey: Antibacterial Activity and Anti-Migration Properties against Breast Cancer Cell Line (MCF-7)

“…It is commonly accepted that dark honeys are richer in protein than pale ones [33]; however, previously we found great variation in this parameter regarding honey variety which ranged from 34.3 (acacia honey) to 452.7 (heather) mg/100 g. In contrast, for honeydew honey, a lower protein value was found in our previous paper (94-102 mg/100 g) [33].…”

“…Soluble protein fraction was determined in honey extracts (20% w/v) using the Bradford method according to D żugan et al [33]. The calibration curve in the range of 6.25-200 µg/per 20 µL was prepared using bovine albumin as standard and was applied to calculate the protein content in mg of protein per kg.…”

“…The activities of three glycosidases were evaluated using the p-nitrophenyl glycoside as substrate: N-acetyl-βglucosaminidase (NAG), β-galactosidase (β-GAL), and α-glucosidase (α-GLU). The analytic protocol has been described earlier [33]. Raw absorbance values were recalculated into enzymatic units mU (nmol/min/g).…”

“…The tested honeydew samples were compared in terms of protein content and enzymatic activity (Table 4). These parameters were formerly proposed as auxiliary indicators of honey quality [33], especially in terms of its overheating. Among the tested enzymes, diastase activity, included in the obligatory set of parameters used for quality control, as well as three further acid glycosidases applied to the honey activity evaluation were determined.…”

Novel Biocement/Honey Composites for Bone Regenerative Medicine

“…Our analysis revealed the presence of proteins with M w equal to 6, 10, 25, 55, 78, 110, and 160 kDa in both honeys, but the manuka-based honey contained a high number of proteins with M w of about 26 kDa. The identified proteins represent the major royal jelly proteins with M w ~48-70 kDa and proteins originated from nectars or pollen between M w 20-30 kDa, and proteins with M w equal 5 and 10 kDa can be assigned to apisimin and defensin-1 [31,32]. The total polyphenol contents of the honeys, which were determined colorimetrically, were 1.45 and 0.99 mg/g for the manuka and chestnut honeys, respectively.…”