Protein colloids in Manuka honey

A good update on the colloids in honey is given in the following paper.

Figure 1: Manuka honey spread under a light microscope

The presence of colloids in honey was identified in 1930's by Paine et al (1934). The colloid material was present in various honeys at a concentration ranging from 0.1% to 1%.  The honeys with higher colloid content were darker in colour and thixotropic in nature which indicates that the viscosity of the honey is influenced by the percentage colloid present.  The colloid also influenced colour, taste, and some biological properties of honey.  It was shown by Lothrop and Paine (1931) that heating the colloid resulted in its decomposition.  Dark Buckwheat honey had lower economic value due to its strong flavour and dark colour due to high colloid content.  Several methods were used to remove the colloid from the honey to lighten its colour and produce a more palatable product.  However, we and others have found that the colloid material which is responsible for the high viscosity of the BuckWheat and Manuka honey and Bush honey in Australia is also responsible for a considerable amount of the biological properties of the honey. 

The colloid composition is complex in nature and consists of plant phenolics, bee proteins, lipids, sugars and minerals.  The composition being enriched for components found in honey.  The formation of this complex colloid structure is of biological origin, whereby the bee through working the honey by sucking the nectar through the proboscis generates the colloid.  The bee proteins present within the colloid include apisimin, and MRJPs, defensin which are mixed with the nectar and pollen phenolic components which form the colloid structure. 

The colloid has unique biological characteristics related to its composition.  It is hypothesized that its biological properties assist in the splitting of water via photo-Fenton mediated hydrolysis reaction, and this exothermic process provides the heat needed to warm the hive.  The bees are able to utilise UVA mediated photo-Fenton reactions to warm their bodies and their hive and this property is linked directly with the colloid present within the honey and produced by the bee.

The colloid has a hydrophobic character which enables it to be stable in honey.  Under bright field microscopy the colloid looks like a bubble within the honey.  The photo-fenton chemistry responsible for the generation of the hydroxyl radical, which is responsible for the anti-microbial properties of honey², is elevated in Manuka honey due to the higher phenolic content as well as high mineral content of Manuka honey.  It is postulated that the phenolic and minerals are co-located predominately within the colloid structure providing an environment for photo-Fenton chemistry contained within the structural colloid.  Here we report on the further characterisation of the colloidal material from honey and its complex behavioural properties as a unique biomaterial from bees.

The colloid was shown to contain a complex between the protein apisimin, MRJPs, and perform photo-redox reactions with the reduction of iron to generate Fe(ii) which reacts to produce a hydroxyl radical.  The generation of soft X-rays from hole formation after K+ electron capture of coordinated iron is also proposed. 

Figure 2: MRJP apisimin X-ray structure

The production of high-energy short-lived species such as the hydroxyl radical via the colloid component of Manuka honey presents a significant challenge in the analysis of this biomaterial, any exposure to light of high energy leads to its deconstruction via rapid radical reactions and loss of biological activity.  Extraction into water after four days at room temperature led to the formation of a precipitate at the bottom of a glass vial and changes to Strong Cation Exchange SCX-HPLC analysis profile.

It is proposed that the hydroxyl radical is a regulatory compound that modulates the balance between oxidation and reduction in a temporal and spatial fashion.   The radical has regenerative properties when contained within the colloid whereby materials are initially deconstructed to form precursor colloidal systems which respond to light of different energies by changing shape to form minimized energy structures that accommodate the energy present in a given environment and allow tissue regrowth and regeneration. 

Upon dissolving Manuka in water whilst observing such an event under the Evos FL microscope it was evident that the colloid within Manuka honey remains intact.  This was confirmed by zeta sizing analysis.  However, the difficulty in observing such colloid in the aqueous mixture was due to its refractive properties as well as its size in the nm range. 

Zeta size analysis of honey colloids.  Many of the samples had several peaks present in the colloidal analysis indicating multiple colloid materials were present. 

Figure 3: Colloid analysis in different honeys

Figure 4: Colloid 100x magnification

Figure 5: Colloid 100x magnification

Strange optical properties were noted. 

Characterisation and analysis of the Manuka honey colloids (MRJPs)

The colloid isolated from Manuka honey was able to focus light and acted as a lens.  These optical properties of the Manuka honey colloid suggested that it may have had biological relevance which had not been recognised previously.  A biological lens such as this has the ability to concentrate light at a focal point.  At the focal point two photons of light of a particular wavelength come together increase the eV intensity.  If UVA light was used to image the colloid, we observed the formation of bubbles on the microscope slide when the LED was set to high power after the Manuka honey was dissolved in water.  This did not occur when the Texas red LED was used, or the bright field white LED was used.  However, the formation of bubbles in the dissolved Manuka honey was observed at 10 x magnification when the blue light LED was used which corresponded to the Qdot Long pass LED filter cube for the Evos FL epi-fluorescent microscope.

This observation prompted further investigation of what was responsible for the bubbling.  The increase in reaction rate as wavelength of the light used changed from red through to blue and into UVA at 357 nm suggested that light was responsible for this observation.  It is known that UVA light can split water? at pH 1.0 to produce hydrogen and oxygen gas.  The pH of the honey was determined to be 4.3.  So, it was not expected under these conditions that water could be split to form oxygen and hydrogen. I found the answer to be Fenton chemistry.

Fenton chemistry is fun to watch as it generates strange moving bubbles that appear out of nowhere. Methyl glyoxal, iron sulphate and hydrogen peroxide reaction driven by light to create hydroxyl radicals and superoxide. The components of Manuka honey have everything that is needed for photo-Fenton chemistry to occur. It is optimal at around pH 4.0 which is similar to the pH of Manuka honey. I am suggesting that photo-Fenton chemistry and iron coordination is key for the antimicrobial activity of Manuka honey.

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