**4. Discussion**

Pollen is a fundamental element in the analysis and quality evaluation of honey [24]. Melissopalynological analysis of honey provides the identification of the pollen types and potential plant source of honey [25]. This knowledge of pollen species is expedient in elucidating the sources of floral nectar that bees forage to produce honey of specific geographical and botanical sources [26]. In the present study, the pollen spectra from honey samples revealed that local honey had a relatively wide variety of botanical families than the imported honey. The possible explanation for the diversity in pollen content taxa between local and imported honey is because of different geographical regions, as well as the treatment of fine filtration. The local honey was without any fine filtration; unlike the imported honey, which might be commonly exposed to fine filtration to remove most of its pollen content before commercialization. According to USDA standards, commercial honey is filtered to remove suspended particles, including pollen grains [27]. Ponnuchamy et al. [28] reported the diversity of pollen spectra in the honey collected from one area at different times of the year.

We found that the color diversity among the honey samples (local and imported) ranged from white to dark amber, which is in accordance with the Pfund scale [21]. The diversity in honey color is common, and a previous study also reported the diversity ranged from colorless to amber and dark amber to black [14]. It is evident that the commercially available honey varied greatly in quality due to its color, flavor, and density over the globe [29]. Honey color is closely connected with botanical origin, and is an imperative to assess the honey quality [30]. Light-colored honey has a mild flavor, while dark honey has a more concentrated and rich flavor [31]. Furthermore, darker honey also has a high content of manganese, iron, phenolic compounds, and copper [14,32]. Many factors such as the environment, season, mineral, Maillard reaction, phenolic content, pollen, wax used, floral origin, and length of storage can affect the color of honey [25,32–34]. The Pfund color scale of local and imported honey was within the suggested range (0–150 mm) of the International Codex [9,10,23] and depended on the botanical origin.

The moisture content in the honey is important to determine honey quality, stability, resistance to spoilage, resistance to fermentation, and granulation during storage [25]. We found a relatively higher mean moisture content in imported honey than local honey, but both were within the acceptable limit (<20%) of international standards [9,10,23]. The prevalent subtropical climate conditions of high temperature and low humidity in Saudi Arabia could be the reason for the low moisture level in local honey. Moisture level is also vulnerable to geographical moisture conditions (temperature and humidity) during honey production, level of honey maturity in the hive, content of floral nectar, harvesting time, processing techniques, storage conditions, and apiary management [35–37]. The low moisture content would be an advantage for long storage with the prolonged shelf life of honey [25,38]. Other studies also found comparable findings of low moisture content in Saudi honey [24,39,40].

The level of EC is an important indicator of the quality of honey [41]. Our result showed that the EC value of local nectar honey exceeded the permitted limit (0.8 mS/cm) of international and Gulf standards [9,10]. The level of EC depends on the presence of mineral contents, storage time, floral origin, proteins, and organic acids in honey [14,40,42]. The higher level of these contents resulted in the higher EC, and vice versa [43]. EC is the most appropriate parameter for differentiating the geographical source and identification of flora of honey [41]. The level of EC showed great variation depending on the floral origin of honey [38]. The storage, floral sources, and color of honey also affect the EC values, as dark honey provides a higher EC than light-colored honey due to differences in the levels of minerals [43]. The local Saudi honey was exceptional nectar honey, which is characterized by naturally higher EC, and likewise, previous studies also presented higher EC in Saudi honey [25,40,43].

The pH value is linked with the number of organic acids present in the honey. It can also be influenced by various other factors such as the presence of inorganic ions, as well as extraction and storage conditions, which affect the structure, stability, and shelf life of honey, as well as the fermentation process [14,37]. In the present study, the mean pH values of local and imported honey (5.0 ± 0.2 and 4.5 ± 0.2, respectively) were within the permitted limit (3.4–6.1) of standards [9,10,23]. Generally, our results regarding pH values are in agreement with those described in the literature from different countries [38,39]. We also found that few samples of local Sidr honey (*Ziziphus sp*.) exhibited higher pH (>6.1) than standard limits (3.40 to 6.10), which is in line with previous studies where Sidr honey revealed high pH [40].

FA is a characteristic that originally depends on the floral source, geographical origin, and climatic conditions. We demonstrated that the mean FA (18 ± 2.1 meq/kg) of imported honey samples was within the permitted range (≤50 meq/kg) of standards) and was in agreement with previous studies [44]. Conversely, the mean FA of local honey samples (61 ± 8.3 meq/kg) was higher than that of imported honey and exceeded the permitted limits (≤50 meq/kg). The high mean FA value in local honey was due to the honey samples that originated from *Acacia* spp. plants, which had high FA because of the nature of floral source [45]. The high FA value exceeding the permitted standards in honey originated from *Acacia* plants were in agreement with previous studies conducted in different Gulf countries, such as Oman, Saudi Arabia, and Yemen [33,40,46]. Irrespective of geographical origin, *Acacia* honey has distinctive acidic properties. The nature of the *Acacia* nectar and the effect of the honey harvest season of hot summer, as well as high-salinity soils, could be the possible reason for the acidity of *Acacia* honey [47]. In honey, essential acid gluconic acid is produced by oxidation of glucose with an enzyme glucose oxidase, which makes honey slightly acidic [20]. Thus, the increase in FA may be due the presence of a high level

of gluconic acid in *Acacia* flowers as a rich source of nectar [48]. The variations in FA among local and imported honey samples might be due to differences in geographical conditions; the presence of organic acids, particularly gluconic acid; inorganic ions (phosphate and chloride); floral sources; the fermentation process; and the bee species [33]. A high FA value in *Acacia* honey samples could therefore be a feature of honey related to the floral origin of honey.

HMF is one of the most important criteria to monitor the freshness of honey, beekeeping practices, honey exposure to high temperature, and storage conditions [49]. In fresh honey, the level of HMF is naturally in small quantities, but its concentration increases with storage duration and prolonged heating [50]. HMF is an indicator for poor storage conditions at high temperature [18]. Our result revealed that mean HMF content of the local honey samples (3.8 ± 1.5 mg/kg) was lower than that of the imported honey samples (23.0 ± 5.0 mg/kg), but both were within the permitted limits (≤40 or 80 mg/kg) of honey standards [9,10,23]. Only in a few imported honey samples did HMF exceed these standards. These high HMF values in the present results might have been due to storage time and honey exposure to heating [20]. The accepted level of HMF in honey differs among countries, i.e., being greater in hot tropical countries, and should not exceed 80 mg/kg, whereas in other countries, 40 mg/kg is the maximum accepted level [9,10,23]. Our results are in agreement with the findings of previous studies [24,51]. The production of HMF can be increased with the presence of simple sugars (glucose and fructose), many acids and minerals in honey, in addition to honey processing practices or long storage [52].

The diastase enzyme is a significant enzyme secreted by bees during the conversion of nectar into honey, in addition to its floral source. It is greatly affected by the floral origin, climate, poor storage, and exposure of honey to heating; the activity of the diastase enzyme indicates the freshness of honey [53]. The storage duration and honey exposed to heating can modify the diastase activity of honey [54,55]. Our results exhibited that the diastase activity (11.9 ± 1.4 DN) of local honey met the requirements of international and local standards (≥8), with the exception of only two samples with lower diastase activity than the standard limits. Comparable values for diastase activity have been reported for Ethiopian, Argentinian, and Omani honey [56]. The mean diastase activity of imported honey was 7.6 ± 1.6 DN, and the majority of samples were out of the standard limits of international and local standards [9,10,23]. These results indicated that imported samples were either older, stored in poor conditions, or exposed to heating [55] that degraded the enzyme and resulted in decreased diastase activity. The diastase activity values are in agreement with the findings of Mesallam and El-Shaarawy [51].

The level of sugar content in honey is an important for its quality assessment [24,25]. Our results revealed that fructose, glucose, and sucrose were the most important sugars found in the analyzed honey samples, and the levels of these sugars were significantly different among local and imported honey samples. These sugars were sourced from the floral nectar that bees forage and consume during honey production, and the floral source can be identified from sugar analysis [57]. The mean levels of reducing sugar in local (71.2 ± 1.3%) and imported honey (72 ± 2.8%) were within the permitted range (≥60) of honey standards [9,10,23], and these outcomes were in confirmation with previous findings [24,51]. In the present study, fructose was the main sugar in the honey samples compared to glucose and sucrose. The mean fructose level was 39.7 ± 0.6% in local honey and 39.2 ± 2.3% in imported honey, and they were within the permitted range (31–42%) of honey standards [9,10,23]. Only two samples of imported honey had less fructose but higher glucose, indicating that these samples were possibly adulterated [24,58]. The mean percentage of glucose in our data was 31.5 ± 0.7% in local honey and 31.8 ± 0.8% in imported honey, being within the permitted range (23–32%) of honey standards [9,10,23]. These values are in agreement with findings of previous studies [56].

In our data, the local honey had a higher sucrose percentage (2.8 ± 0.9%) than that of imported honey (0.7 ± 0.2%) and was within the permitted range (≤5%) of honey standards [9,10,23]. Tigistu et al. [33] also found high sucrose (2.54 ± 0.40%) in Ethiopian honey. The normal levels of sucrose in most samples indicate that these honey samples were highly matured [50]. Some Sidr honey samples in our data had a higher level of sucrose, which could be attributed to the fact that some beekeepers harvest their honey before the complete sealing of honeycombs. This early harvest is related to the two short peaks of honey flow during Sidr flowering season [45,59,60].

The adulteration of commercial honey is a continued concern worldwide. Generally, adulteration of honey involves the addition of different sugary syrups such as C3 and C4 sugars and certain oligosaccharides. The common source of C4 sugar is sugarcane and corn, with C3 sugar coming from rice and beetroot, while starch-based polysaccharides come from rice and corn [61,62]. Resin technology, a new kind of adulteration, is also being used to produce adulterated honey, hide its origin, and eliminate any trace of contamination and antibiotics. The FDA has notified that the honey going through resin technology should not be labelled as honey. Resin technology can eliminate/alter the chemical components of honey color, flavor, and aroma; pollen; antibiotics; and residues. It also helps the commercial companies to customize the color, aroma, and flavor of honey [63]. The modified sugar syrups are difficult to catch because they are designed not to be detected by the regular testing sugar methods. An advanced global standard specialized testing using nuclear magnetic resonance (NMR) is under debate, which might be needed in order to verity the quality of honey, as well as to analyze the presence of modified sugars in the honey. NMR is a powerful analytical tool that can detect the presence and structure of different substances in honey [64].

We proposed a recommendation for the implication of NMR testing at the country level for the export and import of honey in order to authenticate the honey. A thorough surveillance with solid custom regulations could help to alleviate the import of honey adulterated with common sugars. A regular inspection of honey processing units for sampling honey and testing with NMR is also recommended because there is a high probability that adulterated honey with C3, C4, polysaccharides, and fructose syrups could bypass the normal purity tests.
