**1. Introduction**

Honey is a sweet, supersaturated solution of carbohydrates, composed of glucose, fructose, oligo- and polysaccharides, water, and other substances, such as proteins, enzymes, vitamins, minerals, phenolic compounds and amino acids that are of nutritional and health significance [1]. The chemical composition of honey and physicochemical parameters are variable and related to the botanical origin, geographic area and environmental conditions [2,3].

Pine honey is a honeydew honey produced by honeybees from the secretions of the plant sucking insect *Marchalina hellenica* (Gennadius) (Coccoidea: Marchalinidae) feeding on living parts of *Pinus* species. It is a typical honeydew honey, with high ash content, pH value and electrical conductivity. Additionally, the fructose and glucose content are low; therefore, its tendency to crystallize is low. This type of honey is produced in the Mediterranean region and specifically in Greece and Turkey. It constitutes 60–65% and 50% of the total annual honey production in Greece and Turkey, respectively [4]. It is of high nutritional value due to, among others, its high content of minerals such as potassium, calcium, iron, phosphorus, magnesium, sodium and zinc [5].

Greek legislation has adopted the Council Directive 2001/110/EC [6] regarding the physicochemical properties of honey. In addition, the Government Gazette B-239/23–2-2005 [7] describes the required properties for monofloral Greek honey varieties, according to which the electrical conductivity of Greek pine honey must be higher than 0.9, while also the presence of honeydew elements must be significant.

In recent decades, scientific interest has been focused on the antibacterial activity of diverse types of honey against clinical and foodborne pathogens [8–16] as well as on antioxidant activity [12,17–20]. Recently, honey antibacterial activity has been proposed as a valuable parameter determining its quality which takes into account the biological properties of honey [21].

Hydrogen peroxide (H2O2) is often considered as the major antibacterial compound of honey and it is produced by the enzyme glucose oxidase, which converts glucose into gluconic acid [22]. Several studies have clearly demonstrated the strong correlation between the antibacterial activity and the presence of H2O2 in certain types of blossom honeys [23–27]. However, a recent study by Farkasovska et al. [25] reported a weak or no correlation between antibacterial activity against particular bacteria and H2O2 concentration in linden honey samples. Similarly, despite the high level of H2O2 measured in Slovak honeydew honeys, no significant correlation was found between their overall antibacterial activity and the level of H2O2 [28]. In addition to H2O2, peptides and proteins such as bee defensin-1 and MRJP glycoproteins have been isolated from various honeys exhibiting antibacterial activity through cell lysis [29,30]. Additionally, phytochemicals such as phenolic compounds may significantly contribute to the antibacterial and antioxidant activity of honey, in particular honeydew honey [31].

A small fraction of honey's composition (2–5% of honey dry weight) contains compounds responsible for a plethora of biological properties, such as anti-inflammatory, antimicrobial antimutagenic, antioxidant, antiproliferative and antithrombotic [32,33]. The antioxidant activity involves the deactivation of free radicals, and it is classified into two mechanisms: hydrogen atom transfer (HAT) and electron transfer (ET). In the former, a hydrogen atom is donated to the radical, while in the latter, a single electron is transferred [34]. Phenolics are a class of phytochemicals that are primarily responsible for

the antioxidant activity exerted by honey. Other non-phenolic compounds with the same activity are enzymes (catalase and peroxidase), ascorbic acid and carotenoids. Phenolic compounds that have been reported in honey include phenolic acids (coumaric, caffeic, ellagic, ferulic and chlorogenic acids) and flavonoids (chrysin, kaempferol, pinosembrin, quercetin, galangin, hesperetin, and myricetin) [35]. Very often, the presence of these compounds is expressed as total phenolic content, and it is positively correlated with the antioxidant capacity as well as antibacterial activity of the tested honey [27,34].

The aim of this study was to characterize unifloral pine honeys produced in Greece regarding their physicochemical parameters, antioxidant activity as well as their antibacterial activity against nosocomial and foodborne pathogens. To the best of our knowledge, this is the first in-depth attempt to investigate the antibacterial activity of pine honey from Greece and correlate it with various honey properties.

### **2. Materials and Methods**
