Honey contains various microorganisms, including microscopic fungi, which may, under certain circumstances, affect its quality and safety.

A total of 27 different honey samples were mycologically monitored (20 samples from the year 2018 and 7 samples from the year 2019). A list of honey samples with their characteristics is given in Table 1. All samples were obtained directly from beekeepers.

Each honey sample in the present study was tested for the occurrence of yeasts and filamentous microscopic fungi using a plate dilution method and appropriate culture media.

In the second part of the study, were (under laboratory conditions) prepared samples of honey with the addition of various fruits in various forms and concentrations. The fruits used in this study were blueberries (frozen, dried, and lyophilized), cranberries (fresh, dried, and lyophilized), and red currants (frozen, dried, and lyophilized). Table 2 provides a more detailed description of the raw materials used to prepare the products. Weighed fruits were mixed with rapeseed pasted honey, particularly in the concentrations shown in Table 3, and have been thoroughly homogenized. Concentrations were designed according to the expected water content of the used fruit. With decreasing water concentration in fruits, the content of their addition was increased. The products were stored in glass containers in a cool and dark place. Mycological analyses were performed both on individual raw materials and final products, all variants in 3 repetitions.

Mycological analyses of honey samples, fruit samples, and honey samples with additives were aimed at determining the total counts of yeasts and filamentous microscopic fungi. A plate dilution method was used for the above analyses. The procedures were followed according to Slovak technical standards - STN ISO 21527-1 (2010) and STN ISO 21527- 2 (2010). Two nutrient media were used according to the water activity of the particular sample analyzed. For samples with water activity greater than 0.95, DRBC nutrient medium (Dichloran Rose Bengal Chloramphenicol Agar; Himedia, M1881) was used.

In the case of samples with a water activity less than or equal to 0.95, the DG18 medium (Dichloran 18% Glycerol Agar; Himedia, M1129) was used. Cultivation was performed under aerobic conditions at 25 °C for 5 – 7 days. Grown colonies were converted to colonies forming unit (CFU), using the formula (1)

(1) N=ΣC/ [ ( 1   x   n1 ) + ( 0.1   x   n2 ) ] d

where N = number of colonies per milliliter or gram, ΣC = sum of all colonies on all plates counted, n1 = number of plates in lower dilution counted, n2 = number of plates in next highest dilution counted, d = dilution from which the first counts were obtained.

Filamentous microscopic fungi were subsequently identified into the genera. The identification was based on the observation of macro- and micromorphological features, using respected mycological keys (Samson et al., 2002; Pitt and Hocking, 2009; De Hoog, Guarro, and Gene, 2000).

The reference device, LabMaster-aw from Novasina, was used to determine the water activity of the raw materials and honey products. The measurements were performed at a constant temperature of 25 °C, in 2 replicates and the average values were taken into account.

Counts of microorganisms were reported as mean values calculated from the results of 3 replicates of mycological analyses of each sample. These were then converted to decimal logarithms, referred to as log CFU.g^-1.

The obtained mycological results were evaluated and expressed in isolation frequency (Fr), which is defined as the percentage of samples within which the species or genus occurred at least once. These values were calculated according to (González et al. 1996) using the formula (2):

(2) Fr (%) = (ns / N) x 100

where ns = number of samples within a genus; N = total number of samples.

In the first part of the study, we analyzed 27 honey samples of Slovak origin. Of these, a total of 21 samples (78%) were positive for the presence of yeasts and 14 samples (52%) were positive for the filamentous microscopic fungi occurrence. In 6 samples (22%) no presence of microscopic fungi was found at all. The counts of detected micromycetes are shown in Figure 1. The highest number of yeasts (3.07 log CFU.g^-1) was recorded in flower honey sample no. 19. According to (Snowdon and Cliver 1996), most samples of honey contain detectable levels of yeasts. Although yeast counts in many honey samples are below 100 CFU.g^-1, yeasts can grow in honey to very high numbers. (Bogdanov 2011) states that water content is an important factor for yeast development in the substrate. Honey generally contains osmophilic (sugar– tolerant) yeasts in greater or lesser amounts. If the moisture content is high enough and storage temperature is favorable, they can ferment substrate (Subramanian, Umesh Hebbar and Rastogi, 2007). Fermentation is an irreversible phenomenon that can run in honey mainly during storage, causing significant economic losses. In such a case, honey presents a characteristic odor, increasing acid flavor and gas bubbles (Perez-Perez, Rodriguez-Malaver and Vit, 2007). (Bogdanov 2011) expresses the relationship between fermentation risk and water content as follows. If the water content is 17% or less - honey is safe regardless of the number of yeasts. If the water content is 17.1 – 18% - honey is safe for yeasts up to 1000 CFU.g^-1 (3 log CFU.g^-1). If the water content is 18.1 – 19% - honey is safe for yeasts up to 10 CFU.g^-1 (1 log CFU.g^-1). If the water content is 19.1 – 20% - honey is safe for yeasts up to 1 CFU.g^-1 (<1 log CFU.g^-1). And finally, if the water content is over 20% - it is a constant risk of fermentation in honey. Similarly, Subramanian, Umesh Hebbar and Rastogi (2007) mention, that raw honey sample containing more than 20% moisture readily undergoes fermentation irrespective of the initial yeast count and reduction of moisture content below 17% is considered to be a safe level for retarding yeast activity. The water content in honey is one of the most important criteria of honey quality. The lower the water content, the more viscous, thick and unchanged honey is since such conditions in honey are unsuitable for the growth of osmophilic yeasts and thus prevent fermentation of honey (Korošec et al., 2016; Čermáková, Chlebo and Husáriková, 2017). A previous study (Kňazovická et al., 2020) on the physico-chemical parameters of honey from 2018 (our first 20 samples) concluded that the water content of the honey did not exceed 20% and its average value was 17.3%.

From this perspective, except for the flower honey sample no. 19, we can consider our honey as safe.

In this study, we did not perform yeast genera identification, but the most common yeasts isolated from honey include for example Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces (Silva et al., 2017; Tančinová et al., 2017). Among the isolated Portuguese honey samples, Rhodotorula mucilaginosa, Candida magnoliae, and Zygosaccharomyces mellis were the predominant species. Candida spp. represented more than 45% of the honey isolates (Carvalho et al., 2010). Authors (Sinacori et al. 2014) gave information about the isolation of Debaryomyces hansenii, Zygosaccharomyces rouxii, Zygosaccharomyces mellis, Aureobasidium pullulans, and Cryptococcus uzbekistanensis species from honey. Among them, Cryptococcus species were associated with human pathogenicity. Cryptococcus neoformans is characterized as opportunistic human pathogen, able to infect the central nervous system (Ashbee and Bignell, 2010). Čadež et al., (2014) isolated from bee bread and honey in Hungary five yeast strains representing a hitherto undescribed yeast species. The name Zygosaccharomyces favi sp. nov. is proposed for this new yeast species, which based on phenotype can be distinguished from related Zygosaccharomyces species by its obligate osmophilic nature.

In terms of filamentous micromycetes, the highest numbers we recorded in honeydew honey samples no. 24 and 25 (2.44 a 2.39 log CFU.g^-1). Honeydew kinds of honey are generally reported to contain fungal hyphae and algae when microscopically examined because bees collect honeydew, which is the product of aphids inhabiting green parts of plants. Bees along with honeydew collect additional structures, such as hyphae or spores of fungi, plant pathogens, and algae (Escuredo, Fernández-González and Seijo, 2012). However, we have found that honeydew honey does not necessarily show higher numbers of viable filamentous fungi, because in 4 of the 9 honeydew honey samples we did not detect any occurrence of filamentous fungi. Moreover, no fungi (and hence yeasts) were recorded in one of them.

Considering the genera representation of filamentous fungi, we confirmed the occurrence of the following genera: Alternaria, Arthrinium, Aspergillus (including previously named as Eurotium), Aureobasidium, Cladosporium, Mucor, Penicillium, and Stemphilium. Isolates of the genus Penicillium were recorded with the highest isolation frequency (41%). For comparison, research by authors (Sinacori et al. 2014), performed with honey samples of different blossoming, pointed out the occurrence of different fungal species - Alternaria alternata, Aspergillus niger, Aspergillus proliferans, Aspergillus spelunceus, Chaetomium globosum, Cladosporium cladosporioides, Daldinia concentrica, Emericella discophora, Emericella qinqixianii, Penicillium corylophilum, Penicillium decumbens, Penicillium polonicum, and Penicillium echinulatum, of which P. corylophilum and A. niger were the most frequent, but in the low count, indicating that the honey is capable of containing multiplication of these fungi. (Rodríguez-Andrade et al. 2019) have surveyed and evaluated the presence of xerotolerant and xerophilic fungi in a set of honey bee samples collected from across Spain. From 84 samples, a total of 104 fungal strains were isolated. They identified 32 species distributed across 16 genera, most of them belonging to the ascomycetous genera Aspergillus, Bettsia, Candida, Eremascus, Monascus, Oidiodendron, Penicillium, Skoua, Talaromyces, and Zygosaccharomyces. As a result of this survey, eight new taxa were proposed. Other fungi rarely found in their study were Alternaria multiformis, and the mucoralean Cunninghamella bertholletiae, Mucor plumbeus, and Rhizopus oryzae. These probably represent environmental contaminants. Martins, Martins and Bernardo (2003) tested 80 multi-species flower kinds of honey, commercially available in Portugal. They focused on the spores of bacteria and fungi. Yeasts and microscopic filamentous fungi were detected in 88.8% of the samples, identifying 3 filamentous fungus genera - Aspergillus, Penicillium, and Mucor and 2 yeast genera - Saccharomyces and Candida. Aspergillus spp. fungi are ubiquitous and associated with disease in many insects, plants, animals, and man. They are regarded as opportunistic pathogens that require immunocompromised hosts to establish infection. Microbiological studies have shown a high prevalence of Aspergillus spp. in apiaries which occur saprophytically on hive substrates. However, the specific conditions required for pathogenicity to develop remain unknown (Foley et al., 2014). The results of (Foley et al. 2014) confirm the ubiquity of Aspergillus spp. in the apiary environment and highlight their potential to infect both larvae and adult bees unknown. (Bignell 2010) reported, that food with acidic pH, low humidity, and high concentration of sugars, such as honey, are sources for growth of the fungi Aspergillus glaucus. In our study, Aspergillus species were isolated from 19% honey samples.

The analysis of filamentous microscopic fungi is now highly emphasized (Kačániová et al., 2006). This is because they can produce mycotoxins that adversely affect human health and reduce the hygienic quality of food. However, the presence of fungi does not imply the presence of mycotoxin; it has necessary ideal conditions such as high water activity, the presence of sugars, and the presence of organic acids capable of reducing pH. Necessary conditions for fungal growth are not always the necessary conditions for the production of mycotoxins (Barkai-Golan and Paster, 2008). Several of the fungi found in honey samples (Aspergillus spp. and Penicillium spp.) are potential producers of mycotoxins, but this does not mean that the honey may represent a risk to the health of the consumer, because (in general) the production of mycotoxins or the fungal growth is suppressed at water activities lower than 0.70, as is the case of honey (a_w of 0.60 or less) (Rodríguez- Andrade et al., 2019). Despite inappropriate condition found in honey for mycotoxin production, it is important to say that the presence of the fungus can also cause disease in different ways, as induction of allergic responses and infections (An, 2004).

It is generally known that the raw materials must be of excellent quality to produce a good quality product. If poor quality raw materials with certain defective elements are used, these defects will also pass to the final product. Highquality mature honey is considered to be a food with a minimum number of microorganisms and many beneficial effects in terms of human nutrition compared to other foods and no or negligible health risks. In this part of the study, we focused at first on mycological analysis of the raw materials used for the preparation of honey with the addition of fruit. Secondly, mycological analyses of the final products were carried out. Information concerning the counts of isolated fungi (Figure 2) is supplemented by information on the water activity of the individual samples (Figure 3), which has a significant impact on their occurrence.

Analysis of the basic raw material - rapeseed pasted honey did not show the presence of microscopic fungi. The water activity of this honey sample was 0.567. Finola, Lasagno and Marioli (2007) reported that the low number of filamentous micromycetes in honey is a good indicator of suitable conditions in the apiary environment.