**3. Results and Discussion**

The first stage of the research included the melissopalynological analysis of the examined honey samples. For each honey, at least 300 consecutive pollen grains of nectarproducing plants, including *L. scoparium* pollen, were identified and counted, and then the percentage of *L. scoparium* pollen content was calculated on this basis. The microscopic image of *L. scoparium* pollen found in the analyzed honeys is presented in Figure 1. As can be seen, the pollen grain is small (~15–20 μm), triangular in polar view, isopolar, and tricolporate, which is consistent with the literature data [42,43]. It is also worth noting that the sides of the pollen sometimes appear concave, while the angles appear extended [43]. Most of the studies to date report a significant resemblance between manuka and kanuka pollen; consequently, melissopalynological studies tend to combine them [28,43,44]. Despite its imperfections, this method is still useful as it allows for the simple verification of the presence and quantity of manuka-like pollen in a honey sample.

**Figure 1.** Microscopic image of *L. scoparium* pollen under 400× magnification.

The results obtained from the pollen analysis of thirty honey samples labeled as manuka honeys are listed in Table 1. As shown, the percentage of *L. scoparium* pollen grains in the analyzed honeys varied from 45 to 90%. Importantly, as much as 47% of the tested honeys indicate a low content of manuka tree pollen, lower than the Moar's limit (70%) for honey to be called manuka honey [28]. The obtained results point to the wide diversity in the manuka pollen composition of commercially available manuka honeys, and simultaneously prove the importance of the melissopalynological analysis of purchased honeys, especially in order to verify the authenticity of honey without the use of advanced methods.


**Table 1.** The percentage of *L. scoparium* (*LS*) pollen grains in the analyzed honeys.

In the next step, the activity level of α-amylase was determined and plotted in Figure 2. While the DN was estimated for all analyzed honey samples, we focus our discussions on the results obtained for the melissopalynologically classified manuka honey samples; that is, honeys that contain at least 70% of manuka pollen (shaded area of the graph). As illustrated, the diastase level ranged from 1.8 to 15.2 Schade units, but was mostly lower than 8 Schade units. According to the European Directive that governs the standards for honey sold in the European market, a minimum diastase level of 8 Schade units is necessary for honey to be deemed of good quality and acceptable; thus, the vast majority of the analyzed manuka honeys do not meet this requirement [15]. In fact, merely five manuka honeys (31%) had a diastase activity higher than 8 Schade units. Nevertheless, honeys with naturally low enzymatic activity are known, such as *Acacia*, *Becium grandiflorum*, *Croton macrostachyus*, *Eucalyptus globulus*, *Hypoestes*, *Leucas abyssinica*, *Schefflera abyssinica*, *Syzygium guineense*, and *Vernonia amygdalina* monofloral honeys [15,45]. Such honeys are characterized by a DN value between 3 and 8 Schade units and an HMF content lower than 15 mg·kg−<sup>1</sup> [15,46].

**Figure 2.** The diastase number (DN) in the analyzed honeys.

Figure 3 provides the HMF content in the manuka honey samples evaluated in this study. Only two melissopalynologically classified manuka honeys showed the HMF content below 15 mg·kg<sup>−</sup>1, more precisely 13.5 ± 1.8 mg·kg−<sup>1</sup> (when the percentage of *L. scoparium* pollen grains was 76%) and 5.1 ± 1.5 mg·kg−<sup>1</sup> (when the percentage of *L. scoparium* pollen grains was 85%). However, the DN for these samples was 14.3 ± 2.9 Schade units and 15.2 ± 3.0 Schade units, respectively. This denotes that the low level of HMF present in this honey does not coincide with a low level of diastase activity, which would typically be expected in honeys with naturally low enzymatic activity. Thus, even though manuka honey is most often characterized by a low level of α-amylase activity, it should not be considered a honey with intrinsically low enzyme activity as it does not fulfill the second condition for such honeys [15]. The low DN may result from thermal treatments or longterm storage, as well as from storage under inappropriate conditions [47].

**Figure 3.** The hydroxymethylfurfural (HMF) content in the analyzed honeys.

Nearly all of the manuka honey samples with a high content of manuka pollen (shaded area in Figure 3) satisfy the maximum limit of HMF content allowed by the European Directive, which is 40 mg·kg−<sup>1</sup> [15]. Specifically, the HMF content ranged from 5.1 to 55.5 mg·kg−<sup>1</sup> and two samples (13%) exceeded the permissible limit. These two samples had HMF values of 45.2 ± 4.5 mg·kg−<sup>1</sup> and 55.5 ± 5.6 mg·kg−1, and were characterized by a DN equal to 2.1 ± 0.5 Schade units and 1.8 ± 0.5 Schade units, respectively. Such a combination of HMF content and DN clearly indicates that the honeys were of poor quality, presumably due to overheating or improper/prolonged storage [48]. According to European regulations, these honeys should be withdrawn from sale, especially since the HMF impact on human health is ambiguous [15,41,49,50]. Other melissopalynologically classified manuka honeys, based on the HMF content, may have been released for consumption.

To gain further insight into the characteristic of commercially available manuka honeys, the correlation between the DN value and HMF content was analyzed. As can be seen in Figure 4, showing a comparison of the regression graph between the diastatic activity and HMF content obtained for thirty honey samples labeled and sold as manuka honeys (Figure 4a) and for the manuka honeys with a high content of manuka pollen, that is, honeys that contain at least 70% of manuka pollen (Figure 4b); in both cases, the DN presents a substantial negative relationship with HMF. Regression equations for diastase activity to HMF content for all analyzed honey samples and for melissopalynologically classified manuka honeys were y = −0.2086x + 13.759 and y =−0.2762x + 14.361, respectively. This shows that the negative regression is even more significant when the manuka honeys with a high content of manuka pollen are considered. Notably, the obtained dependencies are in good agreement with the knowledge that a high HMF content is a marker of excessive heating or improper storage of honey, which in turn causes a decrease in the enzymatic activity. Similar behavior was reported in the study of the Ethiopian monofloral honeys [45]. As a result of the regression analysis of these honeys, the following regression equation for DN to HMF was obtained: y = −0.1389x + 6.3701 [45]. In addition, the study of honeys from Bosnia and Herzegovina and Algeria also revealed a negative correlation between the diastase activity and HMF content [51,52].

A concise summary of the results obtained for the melissopalynologically classified manuka honeys is presented in Table 2. As depicted, the average percentage of *L. scoparium* pollen grains in the analyzed honeys was 77.7 ± 5.7%, while the mean DN and the mean HMF content were 6.4 ± 4.0 Schade units and 29.0 ± 12.7 mg·kg−1, respectively. These results indicate there is a wide variety of manuka honeys available on the Polish market in terms of enzymatic activity and HMF content. In addition, a significant part of these honeys do not meet the requirements of the European Directive authorizing honey for use, mainly due to the low DN. The obtained results suggest that the analyzed manuka honeys were stored under inappropriate conditions for long periods of time or were intentionally heated, for example in order to achieve a higher content of methylglyoxal, a compound believed to be the predominant antibacterial constituent of manuka honey [53]. Thus, the physicochemical quality of these imported honeys is questionable.


**Table 2.** Summary of the characteristics of the melissopalynologically classified manuka honeys.

SD—Standard Deviation.

**Figure 4.** The correlation between the diastase number (DN) and hydroxymethylfurfural (HMF) content obtained for: (**a**) thirty honey samples labeled and sold as manuka honeys; (**b**) the part of the examined manuka honeys that contains at least 70% of manuka pollen.
