What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

Rocco Rossano,Paolo Riccio,Marilena Larocca,Anna Maria Perna,Teresa Polito,Giuseppe Martelli,Maria Carmela Padula,Rizwan Hasan Khan

doi:10.1371/journal.pone.0049164

Abstract

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

Highlights

Honey is the natural product processed by honeybees (Apis mellifera L.) from flower nectars or plant secretions, by aid of their own secretions
2-D zymography (2-DZ) represents a useful technique to detect the whole pattern of proteolytic enzymes present in the sample under study [18,19,20].This study describes for the first time the proteolytic enzymes present in honey and their effects on honey proteins
The highest protein content was found in eucalyptus honey, whereas the highest values of total proteolytic activity were detected in the extracts obtained from chestnut honey

Summary

Introduction

Honey is the natural product processed by honeybees (Apis mellifera L.) from flower nectars or plant secretions, by aid of their own secretions. The chemical composition of honey - either from single or multi-floral species – depends mainly on beekeeper ability, on the botanical and geographical origin of the nectars, and on the different climatic and environmental conditions. Several natural components of honey have been used as a marker to determine floral, geographical and botanical origin of the product: phenolic compounds [1,2], proteins [3], aminoacids [4], oligoelements [5] and carbohydrates [6]. Honey proteins can originate from nectar and pollen of flowers, or occasionally from the sap of plants, but more often they derive from secretions of cephalic glands of honeybees. MRJP1 is likely to promote liver regeneration and to have a cytoprotective action on hepatocytes [7], MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo [8], and both MRJP4 and MRJP5 are important sources of essential aminoacids [9]

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