*3.6. HMF and Diastase Activity*

Regularly used indicators to assess the freshness of honey and to provide details on processing and storage conditions include HMF content and diastase activity [11]. In general, all tested samples before storage complied with the provisions of the regulations concerning the level of HMF and diastase activity. The minimum permitted value of diastase activity in all kinds of honey (except from baker's honey) is 8 DN, as is stipulated by regulations [36,42]. According to the data presented in Table 1, it is possible to verify the initial freshness of honey, since HMF content and diastase activity were in compliance with European and national legislations. The initial mean HMF level in examined sunflower honeys was 2.08 mg/kg, while it was in the range from 0.82 to 4.41 mg/kg (Table 1). The mean diastase activity was 17.35 DN, with the minimum value being 10.14 DN (Table 1).

Over the course of the 18 months of sunflower honey storage, there was a significant increase in HMF concentration and a decline in diastase activity (Table 3, Figures 4 and 5).

**Figure 4.** Effect of storage time on HMF content in sunflower honey.

**Figure 5.** Effect of storage time on diastase activity in sunflower honey.

The most significant changes after storage for 18 months can be observed in the content of HMF (*<sup>p</sup>* = 1.9 × <sup>10</sup>−31) and diastase activity (*<sup>p</sup>* = 2.8 × <sup>10</sup>−13). Four samples (16.7%) of the 24 honey samples examined did not meet the national regulation for honey in terms of HMF level (maximum permitted is 40 mg/kg, mean HMF content 34.67 ± 5.36 mg/kg) (Table 3). HMF was present in concentrations ranging from 26.98 to 44.65 mg/kg. Diastase activity was lower than the minimum allowed (8 DN) in 13 out of 24 samples (54.2%). However, 18 months after being kept at room temperature, the HMF content increased by about 17 times (from a mean of 2.08 ± 0.91 to a mean of 34.67 ± 5.36 mg/kg). At the same time, diastase activity decreased by 2 times (from a mean of 17.35 ± 3.97 to a mean

of 8.18 ± 1.99 DN). Nevertheless, 18 months after being kept at room temperature, nine samples (37.5%) of sunflower honey still presented acceptable values for HMF and diastase activity. Similar results of honey stability testing at room temperature during 18 months of storage were published by Seraglio et al. [41], Czipa et al. [5], Soares et al. [11], Korkmaz and Küplülü [49], Hasan [50], and Fallico et al. [51]. Da Silva et al. [4] and Chou et al. [19] reported that HMF and diastase activity did not change significantly 18 months after being kept at room temperature (20 ± 4 ◦C and 25 ◦C, respectively).

The potential for HMF formation is greater in honey that is more acidic than in those that have a higher pH value, such as darker honey [49,52]. Generally, the pH of honey is typically between 3.2 and 4.5 and depends on the content of organic acids. The mean pH value in the present paper was 3.66 ± 0.14 (ranging from 3.46 to 3.99). Based on this, it can be concluded that sunflower honey belongs to the group of honeys that are more acidic. The increase in HMF after storage is a possible consequence of the lower pH value of fresh honey. However, the small range of pH values was not suitable for confirming the linear dependence of pH and HMF. Our assumption that the increase in HMF concentration after storage for 18 months could be a consequence of the low initial pH value that was not confirmed by statistical analysis (R2 = 0.0289).
