*3.2. Physicochemical Parameters*

The average physiochemical parameters of pine samples are presented in Table 1 (full data are available as Supplementary Materials Table S1). Moisture content, pH value and free acidity averaged 16.1 ± 1.0%, 4.7 ± 0.2 and 25.5 ± 4.6 meq/kg, respectively, values in accordance with previous publications regarding pine honey produced in Greece and Turkey [54–58]. Electrical conductivity values ranged between 0.91 and 1.31 mS/cm, averaging 1.11 ± 0.13 mS/cm. Moisture, free acidity, and electrical conductivity values are within legal requirements [6,7] for unifloral Greek pine honey (less than 20%, less than 50 meq/Kg honey and more than 0.9 mS/cm, respectively). Color values averaged 91.9 mm Pfund but presented high variability among samples (SD = 15.9). HMF was as low as 1 ± 1.25 mg/Kg of honey, with seven samples having no HMF at all, with these values being well below the legal limit of 40 mg/Kg. It is known that Greek pine honey has a low tendency to form HMF [59], which may be attributed to the less acidic nature of pine honey compared to other honey varieties. Fallico et al. [60] reported that, among others, pH values were correlated with the formation of HMF in honey and high pH honeys had a lower formation of HMF. Diastase activity averaged 20.9 ± 8.71 DN which is in accordance with previous works with Greek pine honey [56,57], but visibly higher than values reported for Turkish pine honey [55,58].

It should be stated that a certain number of samples showed relatively low diastase activity, two of them being slightly lower than the legislation limit of 8 DN (see Table S1). Honey enzyme is affected by storage and exposure to high temperatures [61]; however, this is not the case here as the samples are fresh. Fluctuation within a certain honey variety of fresh honey samples is known and can be explained by poor processing of nectar by the bees during an abundant nectar flow or seasonal activity of the pharyngeal glands [62], age of the bees, environmental conditions and beekeeping practices [61].

Fructose concentration was higher than glucose, ranging from 25.87 to 39.22%, while glucose from 14.38 to 33.20%. The average value for the sum of fructose and glucose was found to be 57.75 ± 6.15%, characteristic for a honeydew honey, like pine honey is. In all samples this value (min = 45.65%, max 69.76%) covers the legislation level for honeydew honeys "more than 45 g/100 g honey" [6]. Fructose/glucose ratio averaged 1.27 proving that pine honey has a moderate tendency to crystallize [63].

#### *3.3. H2O2 Concentration and Total Protein Content of Pine Honeys*

Antibacterial activity of honey is mainly attributed to accumulated H2O2 in diluted honey. The average value of accumulated H2O2 in 40% of honey solutions after 24 h was 3341 ± 921 μM and ranged from 1911 to 5620 μM (Table 1). The observed average ability of pine honey samples to generate high levels of H2O2 is substantially higher than in other types of honeydew honey, whereas total phenolic content was similar in both groups of honeydew honey samples [28]. In our previous study, the average value of H2O2 of Slovak honeydew honeys and blossom honeys was 1800 and 743 μM, respectively [27,28]. We assume that polyphenolic compounds, including specific flavonoids, contribute to higher levels of H2O2 by their pro-oxidant activities and/or the ability to act as electron acceptors in enzymatic reactions [28].

Apart from H2O2, defensin-1, an antibacterial bee-derived peptide found in honey, can contribute to the overall antibacterial activity of honey [30,64]. However, the effective concentration of defensin-1 at MIC of honeydew honeys is rather low and its contribution seems to be significant only in blossom honey [28].

Although the overall protein content can vary from honey to honey, the profile of the honey's most abundant proteins is similar among natural honey of different botanical and geographical origin. In the present study, the average of total honey protein content was 552 ± 128 μg/g, ranging from 301 to 827 μg/g (Table 1). The SDS-PAGE analysis showed an identical protein pattern among pine honeydew honeys where MRJP1 was the most dominant protein (Figure S2). These observations are in accordance with other studies where MRJP1 protein was found to be the most prominent band in all tested honeys, including medical-grade ones [65–67]. In fact, all major proteins identified in honey are of bee origin [66,68] and are secreted by hypopharyngeal glands into the nectar during collection and processing [69]. It has been proposed that some of these bee proteins and peptides including MRJP1 and defensin-1 could be considered as markers of honey quality and authenticity [30,70].

### *3.4. Total Phenolic Content (TPC) and Antioxidant Activity*

Antioxidant activity is the outcome of different pathways which are not fully elucidated. Numerous assays are commonly employed to determine the antioxidant effect of a substrate. The lack of validated and standardized antioxidant protocols poses a challenge when authors compare their data [42,71]. Therefore, data regarding the antioxidant activity of pine honey are limited and discrepancies are observed.

In the present work, the total phenolic content was estimated to be 451.38 ± 120.38 mg GAE/kg, which is in line with the majority of previously published data. Cavrar et al. [72] studied pine honeys from northern Turkey and reported TPC values of 496 ± 148 mg GAE/kg. Similarly, Nayik et al. [73] determined the TPC of pine honeys from the Kashmir valley and found values of 598.4 ± 3.3 mg GAE/kg. In another study of Turkish pine honeys, Can et al. [33] found similar TPC values of 614.2 ± 55.9 mg GAE/kg. Higher TPC values were found by Karabagias et al. [74] (1583 ± 338 mg GAE/kg) regarding pine honey from Greece. In the lower range were the values by Ozkok et al. [75], reported for pine honeys from Turkey (155.55 ± 2.04 mg GAE/kg).

The antiradical activity of pine honeys was expressed as the percentage of the absorbance reduction (RSA%) of the stable DPPH radical at 517 nm. The values we measured ranged from 42.43 to 79.33% which are in excellent agreemen<sup>t</sup> with previously published data from Ekici et al. [76] (57.49 ± 20.15%) and Nayik et al. [73] (55.37 ± 6.8%). The reducing power of pine honeys was determined with the FRAP reagen<sup>t</sup> and expressed as mmolFe+2/kg. The values ranged from 1.87 to 9.43 mmol Fe+2/kg, and in general were higher than those reported by Can et al. [33] (1.48 ± 0.83 mmol Fe+2/kg).

### *3.5. Correlations of Parameters*

Correlations were looked into among physicochemical parameters using Spearman's rho test and the results are presented in Table S2. Moisture content was negatively correlated with H2O2 value (r = −0.471, *p* = 0.013). Free acidity and pH were negatively correlated (r = −0.447, *p* = 0.019), which is logical since free acids contribute to a more acidic nature of honey. In addition, free acidity was positively correlated with several parameters, namely color (r = 0.577, *p* = 0.002), protein content (r = 0.556, *p* = 0.003), TPC (r = 0.592, *p* = 0.001), DPPH (r = 0.410, *p* = 0.034) and FRAP (r = 0.493, *p* = 0.009). Color has been found to correlate with TPC and DPPH [77]. Apart from free acidity, color was positively correlated with the electrical conductivity (r = 0.482, *p* = 0.011), which was expected since minerals considerably contribute to the color of honey [78] and has been demonstrated in the literature [79]. In addition, positive correlations of color were found with H2O2 (r = 0.381, *p* = 0.050), TPC (r = 0.399, *p* = 0.039) and FRAP (r = 0.547, *p* = 0.003). Dark honeys are known to have higher antibacterial potential, which is partly connected to the H2O2 value [80], ye<sup>t</sup> this was not shown in our work. In addition, phenolic compounds contribute to honey color [81] and FRAP has been related to color intensity [82–84]. Diastase activity was negatively related to fructose (r= −0.425, *p* = 0.027) for no apparent reason, and positively to protein content (r = 0.590, *p* = 0.001), which was expected due to the protein nature of enzymes. The HDE/P ratio was negatively correlated with fructose content (r = −0.422, *p* = 0.028) and DPPH (r = −0.417, *p* = 0.030). H2O2 value was strongly positively correlated with protein content (r = 0.501, *p* = 0.008). Higher amount of protein could relate to a higher amount of glucose oxidase which is involved in the production of hydrogen peroxide. As expected, TPC, DPPH and FRAP showed a very strong positive correlation with each other (r = 0.637 and 0.684, *p* < 0.001). In the literature, correlations among these three parameters depend on the honey type [17,81,85]. These data indicate that the antioxidant activity is mainly attributed to phenolic compounds. FRAP values were positively correlated with fructose/glucose ratio (r = 0.412, *p* = 0.033) and H2O2 value (r = −0.497, *p* = 0.008). Finally, HMF was not correlated with any other physicochemical parameter.

### *3.6. Antibacterial Activity of Pine Honey*

Nosocomial infections are a major cause of high morbidity and mortality both in developing and developed countries. Most common nosocomial pathogens include *A. baumannii, P. aeruginosa*, *S. aureus* and *K. pneumoniae*. Infections caused especially by hypervirulent strains of those pathogens are very difficult to treat due to multidrug resistance. Therefore, alternative therapeutic approaches to combat nosocomial infections are urgently needed [86]. On the other hand, *S.* Typhimurium-related serotypes are implicated in salmonellosis, the second most common gastrointestinal infection in Europe, thus leading to serious public health issues and economic losses in the food industry [87].

It is generally acknowledged that MIC measurement in broth is a more sensitive and quantitatively accurate method to study honey antimicrobial activity in comparison to an agar-well diffusion assay due to slower diffusion rates of active substances in agar [9,33]. Therefore, MICs of honeys were determined in broth using a spectrophotometric-based assay.

Honey samples exerted antibacterial activity against all tested bacterial strains. MIC and MBC values are presented in Table 2. MIC values of tested honeys against *S. aureus* varied from 3.125% (*v*/*v*) to 12.5% (*v*/*v*). Nine honeys exhibited comparable MIC values to manuka honey with an MIC of 3.125% (*v*/*v*). Regarding *K. pneumoniae*, the MIC values of tested honeys varied from 6.25% (*v*/*v*) to 12.5% (*v*/*v*) while manuka's MIC value has been determined at 6.25% (*v*/*v*). Thirteen honeys demonstrated an MIC value equal to manuka honey. Similarly, for honeys that tested against *A. baumannii* and *S.* Typhimurium, the MIC values varied from 6.25% (*v*/*v*) to 25% (*v*/*v*), whereas the MIC value of manuka honey has been determined at 6.25% (*v*/*v*). Five and twelve honey samples, respectively, demonstrated MIC values equal to manuka honey. Interestingly, MIC values of honeys tested against *P. aeruginosa* ranged from 6.25% (*v*/*v*) to 25% (*v*/*v*) whereas the MIC value of manuka has been determined at 12.5% (*v*/*v*), meaning that two honey samples exerted

superior antibacterial activity against this particular pathogen, while twenty-one honeys were comparable to that of manuka.


**Table 2.** Antibacterial activity of pine honeys (*n* = 27) compared to manuka honey expressed as MIC and MBC values.

Values expressed as % (*v*/*v*). ND: Not Determined.

The variation in MICs could possibly reflect differential bacterial susceptibility due to distinct antibacterial mechanisms. Furthermore, it has been shown that *S. aureus,* a Grampositive bacterium was, in general, more susceptible to honey and other bee products compared to Gram-negative bacteria [12,88], which is in accordance with the present study. However, in a recent study that tested blossom honeys from the Greek island of Lemnos, it was demonstrated that Gram-positive bacteria were more resistant compared to the Gram-negative bacteria [89].

In order to find out whether honey samples exert bacteriostatic or bactericidal activity, MBC was determined (Table 2). The MBC values of all tested honey, including manuka honey, against all tested bacterial strains were identical to the MIC values, demonstrating that pine honey kills bacteria, not just inhibits their growth.

Overall, the antibacterial activity exerted by pine honeys, especially of those honeys demonstrating superior or comparable activity to manuka, warrants further investigation.

#### *3.7. Antibacterial Activity of Pine Honey Could Be Attributed to Multiple Mechanisms*

The underlying mechanisms that could contribute to exerted antibacterial activity were further assessed in those honey samples demonstrating comparable or superior antibacterial activity to manuka. In that respect, catalase-treated honeys demonstrated higher MIC values up to 16-fold in some cases (some honeys against *S. aureus*, for instance) compared to untreated samples (Figure S3). Of note, two honey samples tested against *P. aeruginosa* did not demonstrate higher MICs after catalase treatment.

Similarly, after proteinase K treatment, 2 out of 14 honey samples tested against *P. aeruginosa*, 9 out of 12 against *S.* Typhimurium, 10 out of 14 against *S. aureus* and 6 out of 11 against *K. pneumoniae* demonstrated higher MICs up to 4-fold. Surprisingly, no increase in MIC value was observed for all tested honeys against *A. baumannii* after proteinase K treatment, indicating that proteins present in honey that might exert antibacterial activity have no effect on this certain pathogen (Figure S3).

Spearman's analysis was performed to assess the correlation between the physicochemical parameters and antibacterial activity. It is shown (Table 3) that no correlation between pH, HMF content, H2O2 concentration and the antibacterial activity was observed. Surprisingly, there is a statistically significant positive correlation of moisture and MIC and MBC values against *K. pneuomoniae*, indicating that moisture negatively affects the antibacterial activity against *K. pneuomoniae*. A statistically significant positive correlation of antibacterial and antioxidant activity was observed for *K. pneumoniae* and *S.* Typhimurium. Interestingly, a statistically significant negative correlation was observed between DN and *S. aureus's* MIC and MBC values, indicating that higher diastase activity correlates with higher antimicrobial activity against *S. aureus.*


**Table 3.** Correlation coefficient (r) and significance (parenthesis) values calculated by Spearman's correlation analysis.

\* Correlation is statistically significant at the 0.05 level; \*\* Correlation is statistically significant at the 0.01 level.

Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey. This is further supported by our recent study, whereas RNA −sequencing analysis revealed that pine honey affected the transcriptomic profile of *P. aeruginosa* by inducing the expression of 189 genes and by suppressing the expression of 274 genes [90]. Pine honey treatment exerted a broad range of action on several pathways and biological processes, including oxidative stress, transmembrane transport and regulation of DNA-templated transcription, two-component regulatory systems, ABC transporters and SOS response. Interestingly, pine honey downregulates key physiological responses in *P. aeruginosa* such as quorum sensing, bacterial chemotaxis and biofilm formation [90].
