In most cases, a honey is considered as coming predominantly from a given botanical origin (unifloral honey) if the relative frequency of the pollen of that
taxon exceeds 45%. Therefore, the following terms are used in order to characterize the relative frequency of the main pollen types: predominant pollen >45%, secondary pollen 16–45%, important minor pollen 3–15%, and minor pollen <3% [
16].
During the microscopic examination of honeys, variations in characteristic pollen of clover and citrus honeys were monitored (
Table 1). Based on the frequency of pollen grains encountered, the botanical origin of clover honey samples was in accordance with package labeling (average value of predominant pollen
Trifolium alexandrinum was ca. 69%). The average number of citrus pollen grains was ca. 32%, covering the group of secondary pollen. However, there were cases where citrus pollen grains were under-represented in numerous European citrus honeys (range of pollen grain percentages between 2–42%) [
16]. In that sense, melissopalynological data may be combined with those of physicochemical or sensory data for the accurate characterization of a monofloral honey [
16]. Therefore, Egyptian honeys analyzed in the present study were categorized as clover and citrus based on the melissopalynological and physicochemical parameter analyses carried out.
3.1.1. Physicochemical Parameter Values of Egyptian Honeys
Data regarding physicochemical and color parameters of Egyptian honeys are summarized in
Table 2. Clover and citrus honeys recorded significant variations in TDS, salinity, EC, moisture, ash, moisture, FA, TA, TDS/TA, and ash values. These honey types belong to blossom honeys, hence, physicochemical parameter analysis showed significant differences on the aforementioned parameters (
p < 0.05). However, pH, total sugar content, browning index (BI), LA, and L/FA did not vary significantly according to floral origin of honeys (
Table 3).
Free acidity, moisture content, along with electrical conductivity values conform to the European directive relating to blossom honey [
20]. What is remarkable is the fact that clover and citrus honeys showed very low acidity values, indicating possible regional characteristics
Moisture content of Egyptian clover honeys is in agreement with that of Argentinean [
21] and Pakistanean [
22] clover honeys and within the range reported for Algerian blossom honeys [
9]. However, clover honey from India possessed higher moisture content [
23].
FA and TA values of Egyptian clover honeys are significantly lower than those reported in the aforementioned studies [
9,
21,
22,
23]. However, LA values of Egyptian clover honeys are in excellent conformity with those of India [
23].
At this point, it should be noted that there is no data in the literature involving L/FA ratio for Egyptian clover honeys. EC values of Egyptian clover honeys revealed that they are a typical blossom honey [
20].
pH values are in agreement with those of clover honey from Pakistan [
22]. Finally, ash content is much lower compared to Pakistanean and Indian clover honeys [
22,
23].
Egyptian citrus honeys recorded lower FA values than citrus honeys from Morocco [
4,
17], Pakistan [
22], and Greece [
11]. However, lactonic acidity was much higher compared to citrus honeys from Pakistan [
22] or Greece [
11] and within the range of that reported for Moroccan citrus honeys [
22]. L/FA was lower than that of Moroccan citrus honeys [
12] and higher of that reported for Greek citrus honeys [
11]. Furthermore, EC and ash values of Egyptian citrus honeys are lower compared to those of Greek citrus honeys [
11]. Finally, pH values are within the range reported for Moroccan [
4,
12] and Greek citrus honeys [
11].
3.1.3. Color and Browning Index Values of Egyptian Honeys
Honey color is the primary criterion of quality, acceptance, and preference among different types of consumers. It varies from light to almost black amber tones, with the most common being bright yellow, orange, or reddish. Parameters that may affect honey color are: (i) botanical origin; (ii) storage time; (iii) flavonoid content; (iv) ash content; (v) the temperature of honey at the hive, (vi) the use of new or already used (old) hives for honey collection, etc., [
24].
Egyptian citrus honeys analyzed were the brightest honeys (had the higher mean
L* values compared to the mean values of clover or thyme honeys) (
Table 2). The results regarding the
L* color parameter were in line with those of Petretto et al. [
15] involving Moroccan citrus honeys. Color parameter
a* had negative values (green components) for all the analyzed honeys (
Table 2). The
a* values were within the range reported previously for citrus honeys produced in Greece [
11], but significantly lower than those reported for native Mexican honeys [
10].
Additionally, color parameter
b* (yellow components) had positive and rather constant values for all honeys analyzed. Present
b* values for clover and citrus honeys from Egypt are higher than those reported for Moroccan blossom honeys [
5] and within the range reported for Greek citrus honeys [
11].
Browning index (BI) is a measure of the development of brown color in foodstuffs. Recently, Tornuk et al. [
25] reported that the brown pigment development in honey was associated with thermal processing due to the non-enzymatic browning such as the Maillard reaction. At the same time, the Maillard reaction depends, to a great extent, on the presence of a high concentration of sugar and amino acids under thermal conditions. Based on the aforementioned, a browning reaction could occur as a consequence of increased temperature during honey processing. However, it is well known that browning may also arise from prolonged honey storage at room temperature.
Present results showed that all honeys were not thermally treated, since browning index values were much lower as compared to thermally or ultrasound treated commercial honeys [
26]. This comprises an additionally quality criterion for Egyptian clover and citrus honeys for the domestic or international markets. It should be stressed that this is the first report in the literature regarding BI values of clover and citrus honeys produced in Egypt. Finally, browning index determination could serve as: (i) a fast qualitative criterion of honey thermal treatment investigation, since it is free and simpler to carry out, compared to HMF or diastase number determination and (ii) the determination of honey storage time at room temperature.
3.1.4. Discrimination of Egyptian Honey According to Floral Type Based on Selected Physicochemical Parameter Values
Eight selected significant physicochemical parameters values, namely
L*,
a*, TDS, salinity, moisture, FA, TA, and TDS/TA (
Table 3) were subjected to linear discriminant analysis. Results showed that one discriminant function was formed: Wilks’ Lambda = 0.204, X
2 = 25.418, df = 8,
p < 0.01. The discriminant function 1 was used for the classification of Egyptian honeys according to floral origin, since it explained 100% of total variance providing an eigenvalue of 3.897 and a good canonical correlation equal to 0.892. In addition, the standardized canonical discriminant function coefficients for each of the significant physicochemical parameters that contributed to the floral discrimination of Egyptian honeys are given in
Table 4. The overall correct classification rate was 95.5% for the original and 90.9% for the cross validation method, considered a satisfactory discrimination rate for this method. The higher discrimination rate was provided for clover (93.3%) followed by citrus (85.7%) honeys.
Discrimination ability of conventional physicochemical parameters, ease of application, and reproducibility, have been previously reported in the literature in studies involving Spanish [
3,
6,
13], Moroccan [
12], and Greek [
11] unifloral honeys, in agreement with the present results. What is remarkable, is that the discrimination rate obtained for citrus honeys is in great agreement with the results (classification rate of 82%) reported by Terrab et al. [
4] involving native Moroccan honeys (
Citrus sp.,
Lythrum sp. and Apiaceae). Serrano et al. [
6] classified Spanish eucalyptus and citrus honeys using electrical conductivity and water activity values in combination with linear discriminant analysis. The overall correct classification rate, based on the cross validation method, was higher than that of the present study (96.6%). However, the number of the investigated parameters (i.e., botanical origin, physicochemical parameters, etc.) may affect the overall correct classification rate.
3.1.6. External Validation of the Developed Statistical Model for the Differentiation of Egyptian Honeys According to Floral Type
In order to investigate the robustness of the statistical model developed for the classification of clover and citrus honeys from Egypt, unpublished data involving specific physicochemical parameters of honeys from Greece were introduced into the set of data and a new statistical analysis was carried out. Honeys from Greece served as the ‘’unknown’’ honey samples. The common physicochemical parameter values taken into account from our database were moisture, free acidity, total sugars (°Bx), browning index, pH, electrical conductivity, CIE color (
L*,
a*,
b*), TDS, and salinity. Thus, these physicochemical parameters served as the independent variables while botanical origin (clover, citrus, and unknown honeys) was taken as the dependent variable. The total number of honey samples was increased to 28 prior to discriminant analysis. Based on CIE color parameter analysis, visual color estimation, electrical conductivity, and ash content values, the unknown honey samples from Greece could be classified as honeydew honeys (
Table 5).
Discriminant analysis showed that two discriminant functions were formed: Wilks’ Lambda = 0.003, X
2 = 121.611, df = 20,
p < 0.001 for the first and Wilks’ Lambda = 0.246, X
2 = 28.774, df = 9,
p = 0.001 for the second. However, discriminant function 1 was the basic function for the classification of Egyptian and unknown honeys according to floral origin, since it explained 96.8% of the total variance providing a high eigenvalue (91.630) and a high canonical correlation (0.995) in comparison with those of the discriminant function 2 (eigenvalue of 3.070 and canonical correlation of 0.869). Respective group centroid values, representing the discriminant functions created, were (−4.220, −1.334), (−5.766, 2.666), and (17.277, 0.224), for clover, citrus, and unknown honeys, respectively (
Figure 1).
In
Figure 1 it is also shown that clover, citrus, and unknown honeys are well differentiated. The overall correct classification rate was 100% for the original and 92.9% for the cross validation method, which is considered a very satisfactory discrimination rate for this method. The higher discrimination rate was provided for citrus (100%) followed by clover (93.3%), and unknown (83.3%) honeys.
Table 6 lists the discriminatory power of the developed statistical model.