Abstract

Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS –PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230–180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110–85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, “protein-type” complexes were formed by protein cross-linking, while in the smaller, “polyphenol-type” complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the reaction of quinones with proteins and polyphenols could possibly be under assumed guidance of dirigent proteins.

Highlights

  • Honey processing at high temperatures or its prolonged storage triggers the Maillard reaction that is responsible for a formation of high molecular weight polymerized brown pigments called melanoidins [1,2]

  • The cascade of these redox reactions lead to the formation of high molecular weight sugar-derived protein adducts and crosslinks known as melanoidins or advanced glycation endproducts (AGEs)

  • Months showed that storage led to a significant decrease in protein content with an average decrease of 46.7% in all tested honeys (t-test, n = 24, p,0.002) (Table 2)

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Summary

Introduction

Honey processing at high temperatures or its prolonged storage triggers the Maillard reaction that is responsible for a formation of high molecular weight polymerized brown pigments called melanoidins [1,2]. According to current views and in vitro models, melanoidin structure and composition result from a condensation and polymerization of nitrogenous and heterocyclic compounds derived from sugar degradation intermediates; deoxyglucosones, Strecker aldehydes, and dicarbonyl compounds [3,4]. The cascade of these redox reactions lead to the formation of high molecular weight sugar-derived protein adducts and crosslinks known as melanoidins or advanced glycation endproducts (AGEs). The mechanism underlying loss of these functions in honey is not clear, the protein inactivation by complexation with polyphenols could be one of the logical cause worth to investigate

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