**3.14. Antimicrobial activity**

produced mainly by aphids and some scale insects as they feed on plant sap. According to Mifsud et al. [35], aphids are present on carob and citrus during autumn, particularly *Aphis* (Aphis) *gossypii* on carob, *Aphis* (Aphis) *craccivora* and *Toxopteraaurantii* on citrus. This may explain the presence of honeydew honey within the floral honey during autumn. Honeydew honey may be produced during summer and autumn [36]. However, due to limitations in tree numbers, this cannot be produced on a large scale in Malta. Therefore, a possible indicator of pure autumn honey may be

The mean total polyphenolic content in mg TAE/100 g honey for spring (56.943 ± 7.027) was significantly lower (*p*< 0.05) than for the other two seasons (AU: 79.692 ± 8.000 and SU: 69.598 ± 3.208 mg TAE/100 g honey) (**Table 7**). It was observed that the darker the honey colour, the higher was the total polyphenolic content. This was the case with autumn honey samples [12].

In spite of the higher flavonoid content for some autumn honey samples (212.86 and 197.57 mg RE/100 g), there was no statistical difference in content for honey samples from the three seasons (**Table 7**). The mean flavonoid content for the three seasons was 31.154 ± 17.729, 37.651 ± 8.460, and 31.420 ± 11.373 mg RE/100 g honey, respectively. As compared to similar studies, the Maltese honey samples contained superior flavonoid content to that observed in other similar studies with quantities ranging between 1.35 and 9.78 mg RE/100 g honey [37, 38].

The mean DPPH inhibition in mg AEAC/100 g honey for autumn, spring and summer was 9.300 ± 1.292, 5.805 ± 0.610 and 5.238 ± 0.657, respectively (**Table 7**). The autumn honey samples had a superior DPPH inhibitory activity with respect to the other two seasons (*p*< 0.01). This may be due to the presence of carob nectar in autumn honey which contains high amounts of

Spring 56.943 ± 7.027\* 37.651 ± 8.460 5.805 ± 0.610 16.600 ± 1.979 Summer 69.598 ± 3.208 31.420 ± 11.373 5.238 ± 0.657 14.250 ± 0.035 Autumn 79.692 ± 8.000 31.154 ± 17.729 9.300 ± 1.292\*\* 12.67 ± 1.093

**TPC TF DPPH Red Pow**

The Maltese honey samples had antiradical activity values between 3.33 and 15.62 mg AEAC/100 g honey (**Table 7**). The mean reducing power in mg AEAC/100 g honey for the

**Table 7.** Total polyphenolic (mg TAE/100 g honey), total flavonoid (mg RE/100 g honey), DPPH (AAE-DPPH mg AEAC/100 g honey) and reducing power (mg AEAC/100 g honey) values for honey samples from the three seasons.

melezitose, although this needs to be further investigated.

**3.12. Radical scavenging activity: the DPPH assay**

polyphenols and tannins, as noted in Ref. [39].

**3.13. Reducing power**

\* p < 0.05. \*\*p < 0.01.

**3.10. Polyphenolic content**

184 Honey Analysis

**3.11. Total flavonoid content**

Maltese honey exhibited MIC values ranging between 0.067 and 0.205 g/ml (**Table 8**). In spite of the statistical insignificance, the spring honey samples showed the best MIC values compared to the other two seasons. The honey samples were compared against artificial honey as highlighted earlier. Only spring samples against *S. aureus* and *P. aeruginosa* showed a significantly lower MIC than the artificial honey (*p*< 0.001 and *p*< 0.05, respectively) [41]. *S. aureus* strains are known to be involved in acquired and nosocomial infections, while *P. aeruginosa* may cause diabetic ulcers, wound infections and urinary tract infections [42]. Therefore, Maltese honey may be potentially useful for the topical treatment of microbial infections particularly associated with wounds and ulcers.


**Table 8.** Total minimum inhibitory concentrations (g/ml) for honey samples from the three seasons.

#### **3.15. PCA analysis of physicochemical parameters and sugar content**

It was observed from the scree plot that the first three components accounted for 50.31% of the total variance. However, the parameters studied fall within different components. The scores plot (**Figure 6**) shows the physicochemical parameters of honey samples in the space of the two new variables, F1 and F2. The parameters plot shows that brix and moisture are inversely related, while acidity, diastase and proline are particularly inversely related to HMF and pH.

Moving along F1, it was observed that the honey samples were distributed by those with low moisture and brix contents on the left and those with the highest moisture and brix contents on the right. From left to right, the samples moved from summer to spring to autumn.

To determine any possible clustering for the seasonal honey, the sugar content was used subjected to principal component analysis. It was observed from the scree plot that the first two components accounted for 71.843% of the total variance. The parameters studied fell within the first two components. The scores plot (**Figure 7**) shows the sugar content of honey samples in the space of the two new variables, F1 and F2. The parameters were grouped as factor 1 for the most common sugars in honey (fructose, glucose and sucrose). This analysis shows that fructose and glucose are inversely related to sucrose.

Moving along F1, it was observed that the honey samples were distributed by those with a high fructose and glucose and low sucrose on the left and those with the lower fructose and

**Figure 6.** Score plot of seasonal honey analysed by PCA (physicochemical).

**Figure 7.** Score plot of seasonal honey analysed by PCA (sugars).

glucose and higher sucrose on the right. From left to right, the samples moved from autumn to summer to spring. Autumn honey samples were particularly distinctive from the other two seasonal honey types. This may be due to the fact that autumn honey samples contained melezitose as opposed to the other two types.

#### **3.16. Concluding remarks on Maltese honey**

To determine any possible clustering for the seasonal honey, the sugar content was used subjected to principal component analysis. It was observed from the scree plot that the first two components accounted for 71.843% of the total variance. The parameters studied fell within the first two components. The scores plot (**Figure 7**) shows the sugar content of honey samples in the space of the two new variables, F1 and F2. The parameters were grouped as factor 1 for the most common sugars in honey (fructose, glucose and sucrose). This analysis shows

Moving along F1, it was observed that the honey samples were distributed by those with a high fructose and glucose and low sucrose on the left and those with the lower fructose and

that fructose and glucose are inversely related to sucrose.

186 Honey Analysis

**Figure 6.** Score plot of seasonal honey analysed by PCA (physicochemical).

**Figure 7.** Score plot of seasonal honey analysed by PCA (sugars).

The physicochemical characterisation of Maltese honey was conducted over a period of 4 years, in order to determine any typical similarities and differences that may be attributed to the different seasonal characteristics. The main characteristics are meteorological conditions that typify the season and the seasonal floral diversity. Data is not shown for the latter parameter, as the floral distribution is beyond the scope of this present study. The typical characteristics of seasonal Maltese honey are as follows.

Spring honey is typically reddish yellow in colour with a liquid consistency. It may be classified as a multifloral honey, featuring nectar and pollens from a vast number of plant that flower during spring. Summer honey is usually yellowish coloured with a viscous consistency. This typically features thyme due to the translocation of hives to areas (North of Malta) rich in thyme during early summer. Autumn honey is dark (reddish-brown) in colour and may contain honeydew due to the tree-related nectars. Therefore, it may contain Melezitose as a minor sugar. It usually has a higher conductivity in relation to the other seasonal honeys, but HMF tends to be high too. This typically contains carob and eucalyptus nectar and pollens. **Table 9** illustrates the typical physicochemical parameters for the three seasonal honey types. In conclusion, **Figure 8** shows a radial plot for the three seasonal honey-types.


**Table 9.** Typical ranges for physicochemical parameter for spring honey.

**Figure 8.** A radial plot for seasonal honey.
