Author: Eleonora Chirico Francesca Santangelo
Date: 04/07/2012


Honey has been renowned for its wound-healing properties since ancient times .
At least part of its positive influence is attributed to antibacterial properties. With the advent of antibiotics, clinical application of honey was abandoned in modern West- ern medicine, although in many cultures, it is still used.
These days, however, abundant use of antibiotics has resulted in widespread resistance.
With the devel- opment of novel antibiotics lagging behind, alternative antimicrobial strategies are urgently needed.
Has been demonstrated the potent in vitro activity of honey against antibiotic-resistant bacteria and its successful application in treatment of chronic wound infections (The Evidence Supporting the Use of Honey as a WoundDressing, 2002).
The broad spectrum antibacterial activity of honey is multifactorial in nature. Hydrogen peroxide and high osmolarity (honey consists of about 80% (w/v) of sugars) are the only well-characterized antibacterial f.actors in honey.
High concentrations of the antibacterial compound methylglyoxal (MGO) were found specifically in Manuka honey, derived from the Manuka tree Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand, 2008.

How honey kills bacteria

Recently, a group of researchers determined that Revamil medical-grade honey, produced under standardized conditions in greenhouses, has potent, reproducible bactericidal activity (Medical-grade honey kills antibiotic-resistant bacteria in vitro and eradicates skin colonization, 2008).
In 2010, the same group identified all bactericidal factors in the honey used as source for this product and assessed their contribution to honey bactericidal activity (How honey kills bacteria, 2010).
To accomplish this, they used a novel approach of successive neutralization of individual honey bactericidal factors combined with activity-guided identification of unknown factors.
All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10–20% (v/v) honey, whereas >40% (v/v) of a honey-equivalent sugar solution was required for similar activity.
Honey accumulated up to 5.62 mM H2O2 and contained 0.25 mM methyl- glyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity:

-The addition of catalase reduced H2O2 to negligible levels and markedly reduced the bactericidal activity against all bacteria tested, except B. subtilis. However, H2O2-neutralized honey exerted stronger bactericidal activity than equivalent sugar solutions. This indicates that H2O2 is important for the bactericidal activity of honey, but that additional factors must also be present. As B. subtilis was the most susceptible bacterium for nonperoxide bactericidal activity, they used it for identification of additional bactericidal factors.

-The honey bactericidal compound MGO can be converted into S-lactoylglutathione, a nonbactericidal product, by glyoxalase I.
Neutralization of MGO or H2O2 alone did not alter bactericidal activity of honey, but simultaneous neutralization of MGO and H2O2 in 10% honey reduced the killing of B. subtilis by 4-logs . At higher concentrations of honey, the bactericidal activity was not affected by neutralization of H2O2 and MGO, indicating that still more factors were involved.

Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, they discovered the killer segret “ingredient” of honey: bee defensin-1

Next, they assessed the contribution of bee defensin-1 to the bactericidal activity of honey against B. subtilis.
As previously observed, 20% honey retained bactericidal activity when H2O2 and MGO were neutralized.
Additional neutralization of bee defensin-1 strongly reduced the bactericidal activity of 20% honey but did not affect the activity of 30 and 40% honey .
So, bee defensin-1 contributed to the bactericidal activity of honey, but still other bactericidal factors were involved.
Honey has a low pH, mainly because of the conversion of glucose into hydrogen peroxide and gluconic acid by glucose oxidase. This low pH might also contribute to the bactericidal activity of honey. Titration of the pH of –10% honey from 3.4–3.5 to 7.0, combined with neutralization of H2O2, MGO and bee defensin-1, reduced the bactericidal activity of honey to a level identical to that of a honey-equivalent sugar solution.
In summary, H2O2, MGO, and bee defensin-1 differentially contributed to the activity of honey against specific bacteria, and their combined presence was required for the broad-spec- trum activity.
This study demonstrated for the first time that honey contains an antimicrobial peptide, bee defensin-1, and that this peptide substantially contributes to the bacte- ricidal activity.
Bee defensin-1 was previously isolated from royal jelly, the major food source for bee queen larvae (and then referred to as “royalisin”), and was identified in honeybee hemolymph.
Royal jelly is produced by young worker bees and contains their hypopharyngeal and mandibular gland secretions. Bee defensin-1 mRNA has been identified in the hypopharyngeal gland of young worker bees, suggesting this gland is involved in production of bee defensin-1 found in royal jelly.
When worker bees age, they become the major producers of honey. Major differences develop in morphology and protein expres- sion of their hypopharyngeal glands, e.g., several important carbohydrate-metabolizing enzymes, including glucose oxidase are expressed.
The bees add the secretion from their hypopharyngeal glands to the collected nectar.
The carbohydrate-metabolizing enzymes then convert sucrose to glucose and fructose, and glucose oxidase converts the glucose to hydrogen peroxide and gluconic acid.
These latter compounds presumably are involved in prevention of microbial spoilage of unripe honey. Since we have found bee defensin-1 in honey, this suggests that after the transition in hypopharyngeal gland function of the worker bees with age, the gland still produces bee defensin-1.
This peptide, therefore, likely contributes to protection of both royal jelly and honey against microbial spoilage.
It remains to be established whether bee defensin-1 is also present in other honeys.

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