**2. Mechanism of antimicrobial activity of honey**

The antimicrobial activity of honey is highly complex and still remains not fully recognized. To date, it has been established that several components of this product play a crucial role for its antimicrobial properties [6–11]:


*Staphylococcus aureus* and *Paenibacillus larvae* (etiological agent of important bee larval diseases American foulbrood). While high concentration of sugars and low pH are universal antibacterial factors of all honeys, strong differences have been noticed in the case of amount of this peptide in different honey and royal jelly samples. The bees also produce at least three other antibacterial peptides as important components of their innate immune system. However, to date, they have not been detected in honey;

• glucose oxidase—the enzyme, oxidoreductase that catalyzes the oxidation of glucose to gluconic acid. The side product of this reaction, hydrogen peroxide (H<sup>2</sup> O2 ), is a strong antimicrobial agent. The detailed mechanism of this reaction is presented below (**Figure 2**). The enzyme is produced in honeybees' salivary glands and introduced to the collected nectar. It protects the ripening honey against the development of pathogenic microorganisms. Interestingly, the enzyme is present but not active in the mature honey; this product is sufficiently protected with high osmotic pressure and low acidity. When the honey is diluted, the enzyme regains activity, which is extremely important for honeybees and especially their larvae health. Honey is the most important component of honeybees' diet; however, before consumption, it is diluted in water. The generated by the enzyme H<sup>2</sup> O2 is a major antimicrobial defense factor for this diluted honey. Its production is also crucial for antimicrobial potential of honey used for treatment of skin and soft tissue infections, infected wounds or eradication pathogenic bacteria located within upper respiratory tract or *Helicobacter pylori* located in human stomach.

**Figure 2.** The reaction calalyzed by glucose oxidase—generation of hydrogen peroxide.

The unique antimicrobial properties have been identified for honey produced from the Manuka bush (*Leptospermum scoparium*) indigenous to New Zealand and Australia. In contrast to majority of other nectar and honeydew honeys, the crucial factor responsible for the bactericidal activity of this product is high concentration of 1,2-dicarbonyl compound methylglyoxal (MGO) [10–12]. Some authors also suggest that other phytochemicals, especially phenolic compounds, are important antibacterial ingredients of honey. Evident differences in activity of honeys produced from different botanical sources seem to support this hypothesis [13–15]. However, the observed differences could be also caused by differences in activity or concentration of glucose oxidase or concentration of defensin-1, which has not been investigated to date. The presence of phenolic compounds in honey has been confirmed in several independent studies. The results of these studies also revealed that concentrations of individual constituents are too low to substantially contribute to the antimicrobial activity of the product. It is possible, however, that combination of different ingredients, for example, phenolic compounds, might significantly contribute the activity of honey [6, 11, 16]. Quite satisfactory activity of composition of phenolic compounds extracted from several Malysian and Polish honeys was observed, respectively, by Aljadi and Yusoff [17] and Mazol and coworkers [18]. Interesting results in this area have been also presented by Mundo and coworkers (2004) [15], who observed non-peroxide activity against *Bacillus stearothermopnilus* in most of 27 honey samples diluted in water containing catalase (the enzyme degrading hydrogen peroxide). In contrast to *Bacillus*, the neutralization of H2 O2 with catalase resulted in loss of activity against *S. aureus* in the case of all tested honey samples except of two samples of horsemint honeys. This result could suggest the presence of some nonproteinaceous components in these honeys, which were responsible for inhibition of growth of *Bacillus* in the suspensions of honey not containing hydrogen peroxide [15]. The presence of antimicrobial components (combination of cationic and noncationic but not identified substances) other than methylglyoxal, glucose oxidase and defensin-1 in Manuka honey was confirmed in the studies of Kwakman and coworkers [10, 11]. These authors also investigated that the other honeys, assigned as RS (Revamil—medical grade honey) and completely opposite results, were obtained. In the case of this product neutralization of H2 O2 ,MGO, defensin-1 and subsequent titration of honey to neutral pH resulted in complete loss of antimicrobial activity [10, 11]. On the basis of current state of knowledge, it rather should be assumed that phytochemicals, except of methylglyoxal, are not crucial for antimicrobial potential of most honeys. However, in the case of honeys produced from some botanical sources, they probably substantially support the primary factors: pH, high osmolarity and defensin-1 (in the case of undiluted honey) and hydrogen peroxide in the case of diluted product. Thus, the contribution of phytochemicals to the antimicrobial activity of honey remains unclear and needs to be investigated.

*Staphylococcus aureus* and *Paenibacillus larvae* (etiological agent of important bee larval diseases American foulbrood). While high concentration of sugars and low pH are universal antibacterial factors of all honeys, strong differences have been noticed in the case of amount of this peptide in different honey and royal jelly samples. The bees also produce at least three other antibacterial peptides as important components of their innate immune

• glucose oxidase—the enzyme, oxidoreductase that catalyzes the oxidation of glucose to glu-

bial agent. The detailed mechanism of this reaction is presented below (**Figure 2**). The enzyme is produced in honeybees' salivary glands and introduced to the collected nectar. It protects the ripening honey against the development of pathogenic microorganisms. Interestingly, the enzyme is present but not active in the mature honey; this product is sufficiently protected with high osmotic pressure and low acidity. When the honey is diluted, the enzyme regains activity, which is extremely important for honeybees and especially their larvae health. Honey is the most important component of honeybees' diet; however, before consumption, it is di-

O2

this diluted honey. Its production is also crucial for antimicrobial potential of honey used for treatment of skin and soft tissue infections, infected wounds or eradication pathogenic bacteria located within upper respiratory tract or *Helicobacter pylori* located in human stomach.

The unique antimicrobial properties have been identified for honey produced from the Manuka bush (*Leptospermum scoparium*) indigenous to New Zealand and Australia. In contrast to majority of other nectar and honeydew honeys, the crucial factor responsible for the bactericidal activity of this product is high concentration of 1,2-dicarbonyl compound methylglyoxal (MGO) [10–12]. Some authors also suggest that other phytochemicals, especially phenolic compounds, are important antibacterial ingredients of honey. Evident differences in activity of honeys produced from different botanical sources seem to support this hypothesis [13–15]. However, the observed differences could be also caused by differences in activity or

**Figure 2.** The reaction calalyzed by glucose oxidase—generation of hydrogen peroxide.

O2

is a major antimicrobial defense factor for

), is a strong antimicro-

system. However, to date, they have not been detected in honey;

conic acid. The side product of this reaction, hydrogen peroxide (H<sup>2</sup>

luted in water. The generated by the enzyme H<sup>2</sup>

218 Honey Analysis

The investigation carried out by Lee and coworkers [4] revealed that honey is a promising source of bacteriocinogenic bacteria strains. The mentioned authors analyzed two Manuka honey samples from New Zealand and six domestic honeys from the United States of America. The 2217 isolates out of 2398 strains (92.5%) exhibited activity at least against one of the tested microorganisms. Among them, 1655 exhibited activity against *Listeria monocytogenes* and 1605 inhibited the growth of another important human and veterinary pathogen *S. aureus* [4]. Beside of that, at the moment, it is rather difficult to classify bacteriocins as the next important antibacterial component of honey. To date, only the strains producing these peptides have been isolated from honey, the presence of bacteriocins within the product has not yet been confirmed.
