Mycotoxins and Mycotoxin Producing Fungi in Pollen: Review

Due to its divergent chemical composition and good nutritional properties, pollen is not only important as a potential food supplement but also as a good substrate for the development of different microorganisms. Among such microorganisms, toxigenic fungi are extremely dangerous as they can synthesize mycotoxins as a part of their metabolic pathways. Furthermore, favorable conditions that enable the synthesis of mycotoxins (adequate temperature, relative humidity, pH, and aw values) are found frequently during pollen collection and/or production process. Internationally, several different mycotoxins have been identified in pollen samples, with a noted predominance of aflatoxins, ochratoxins, fumonisins, zearalenone, deoxynivalenol, and T-2 toxin. Mycotoxins are, generally speaking, extremely harmful for humans and other mammals. Current EU legislation contains guidelines on the permissible content of this group of compounds, but without information pertaining to the content of mycotoxins in pollen. Currently only aflatoxins have been researched and discussed in the literature in regard to proposed limits. Therefore, the aim of this review is to give information about the presence of different mycotoxins in pollen samples collected all around the world, to propose possible aflatoxin contamination pathways, and to emphasize the importance of a regular mycotoxicological analysis of pollen. Furthermore, a suggestion is made regarding the legal regulation of pollen as a food supplement and the proposed tolerable limits for other mycotoxins.


Introduction
Pollen grain, as a male gametophyte of flowering plants, is produced and released from anthers during pollination [1]. Two of the most important pollinators are insects (in the case of entomophilous plants it is, above all, the honey bee (Apis mellifera L.)) and, in the case of anemophilous

Contamination of Pollen with Aflatoxins-Possible Ways
Pollen often presents a suitable substrate for the proliferation of various microorganisms due to its favorable moisture content, water activity (aw), and pH-value. External conditions such as relative humidity and temperature, different stages of pollen production, and storage conditions have been shown to lead to microbiological contamination of pollen [35]. According to data found in the literature, pH-value ranging between 4.0 and 6.5 have been shown to be suitable for the development of bacteria, mold, and yeast while the minimal aw-values sufficient for the growth of Aspergillus and Penicillium spp. have been shown to be 0.71 to 0.96 [53] i.e., 0.55 in the case of pollen [54]. Microbiological contamination is strongly pH and temperature dependent and is also conditioned by the type of microorganism [53]. If proper conditions have been achieved in any phase of pollen production, the growth of microbes will occur which can cause aflatoxin production and the contamination of pollen. In addition to production process and human hygiene practices, which are the most important sources of aflatoxin contamination, sometimes microbe growth can be triggered by infected flowering plants [25,48]. Namely, during the flowering and the pollination process, Aspergillus spp. spores can germinate on female flower parts. Following this, the toxigenic fungal spores placed in the pollen tubes will grow and further infect the egg-cells [25]. If bees visit these flowers, the contaminated pollen grains will be transferred into the hives. Since there is intensive contact between bees when in the hive (due to highly organized bee societies) their "home" is the third possible source of aflatoxin pollen contamination [48]. As aflatoxins show detrimental effects on bee health, the incidence of these compounds in hives is undesirable. It is for this reason that the occurrence and production of propolis in hives is an effective way for bees to deal with AFs toxicity [55,56] which could indicate that this source of pollen contamination with aflatoxins is at

Contamination of Pollen with Aflatoxins-Possible Ways
Pollen often presents a suitable substrate for the proliferation of various microorganisms due to its favorable moisture content, water activity (a w ), and pH-value. External conditions such as relative humidity and temperature, different stages of pollen production, and storage conditions have been shown to lead to microbiological contamination of pollen [35]. According to data found in the literature, pH-value ranging between 4.0 and 6.5 have been shown to be suitable for the development of bacteria, mold, and yeast while the minimal a w -values sufficient for the growth of Aspergillus and Penicillium spp. have been shown to be 0.71 to 0.96 [53] i.e., 0.55 in the case of pollen [54]. Microbiological contamination is strongly pH and temperature dependent and is also conditioned by the type of microorganism [53]. If proper conditions have been achieved in any phase of pollen production, the growth of microbes will occur which can cause aflatoxin production and the contamination of pollen. In addition to production process and human hygiene practices, which are the most important sources of aflatoxin contamination, sometimes microbe growth can be triggered by infected flowering plants [25,48]. Namely, during the flowering and the pollination process, Aspergillus spp. spores can germinate on female flower parts. Following this, the toxigenic fungal spores placed in the pollen tubes will grow and further infect the egg-cells [25]. If bees visit these flowers, the contaminated pollen grains will be transferred into the hives. Since there is intensive contact between bees when in the hive (due to highly organized bee societies) their "home" is the third possible source of aflatoxin pollen contamination [48]. As aflatoxins show detrimental effects on bee health, the incidence of these compounds in hives is undesirable. It is for this reason that the occurrence and production of propolis in hives is an effective way for bees to deal with AFs toxicity [55,56] which could indicate that this source of pollen contamination with aflatoxins is at least probable. In the past, aflatoxin occurrence in feed and food was a characteristic of tropic or sub-tropic regions due to favorable climatic conditions. Recently, with climatic changes, which extensively influences weather conditions in temperate areas (such as the majority of Europe), the presence of aflatoxins in these areas is becoming more frequent. The detection of aflatoxins in samples of pollen from the most diverse parts of the world (Table 1) is in accordance with this fact and is becoming a growing problem. Interestingly, in our previous investigation [48] the majority of examined pollen samples were sterile but all were contaminated with AFB 1 . This situation confirms three hypotheses: - There are different ways of pollen contamination with aflatoxin(s). -These toxins remain in samples with or without presence of appropriate fungi. -It is extremely important to always perform mycotoxicological analysis together with microbiological characterization of pollen.

Quantification of Aflatoxins in Pollen Samples
Results of different studies about the determination of aflatoxin content in pollen samples with diverse palynological (botanical) and geographical origins are given in Table 1.

Ochratoxins
Ochratoxins (OTs) are a group of chemical compounds ( Figure 2) derived from shikimic acid metabolic pathway with ochratoxin A (OTA) as a major food contaminant [57]. The main OTs-producers are different Aspergillus species with a special emphasis on Aspergillus niger strains since they are industrially important due to their applications for enzyme and citric acid production. Furthermore, one species (P. verrucosum) belonging to Penicillium genus can be the source of ochratoxins [24]. OTA belongs to the IARC 2B group which means that it is a possible carcinogen for humans [51]. The kidneys are the most vulnerable organs effected by OTA. OTA has been noted as having a strong influence on the endemic disease 'Balkan nephropathy', as well as porcine nephropathy, which has been documented in several Scandinavian countries [24].

Ochratoxins
Ochratoxins (OTs) are a group of chemical compounds ( Figure 2) derived from shikimic acid metabolic pathway with ochratoxin A (OTA) as a major food contaminant [57]. The main OTs-producers are different Aspergillus species with a special emphasis on Aspergillus niger strains since they are industrially important due to their applications for enzyme and citric acid production. Furthermore, one species (P. verrucosum) belonging to Penicillium genus can be the source of ochratoxins [24]. OTA belongs to the IARC 2B group which means that it is a possible carcinogen for humans [51]. The kidneys are the most vulnerable organs effected by OTA. OTA has been noted as having a strong influence on the endemic disease 'Balkan nephropathy', as well as porcine nephropathy, which has been documented in several Scandinavian countries [24].

Ochratoxins in Pollen
Besides many types of food (nuts, meat products, barley, oats, rye, wheat, wine, dried fruits, coffee, and coffee products) where the presence of OTA has been recorded [24,57], in some herbs, bottled water [57], and pollen samples, this mycotoxin has also been observed. Xue et al. [45] conducted an examination of 20 bee pollen samples from North China for the presence of OTA p by LC-MS/MS analysis. The obtained results showed that none of the studied pollen samples were contaminated with OTA. These results can be associated with the dry weather conditions during the collection period. The same situation was observed in the case of 20 bee pollen samples that originated from Spain [37]. However, HPLC analysis of 90 Spanish and Argentinian bee pollen samples in [38] confirmed the presence of several Aspergillus (A. carbonarius, A. ochraceus and A. niger), and Penicillium (P. verrucosum) species with the ability to produce OTA. Significant contamination of bee pollen was determined in a case of Slovakian samples [41]. In total, 45 samples were divided in three groups of 15 samples originating from poppy, rape, and sunflower plants. Determined OTA concentration ranges in poppy, rape, and sunflower pollen samples were 6.12 to 10.98 µg/kg, 3.24 to 9.87 µg/kg, and 0.23 to 6.93 µg/kg, respectively. In Spain, by analyzing the toxigenic potential of A. ochraceus in various substrates (bee pollen, maize, wheat, and rice) Medina et al. [36] found that OTA production in bee pollen was statistically significantly higher than that found in the production of tested cereals, regardless of the incubation time (7, 14, 21, 28 days). Likewise, positive correlations have been found between the proportion of bee pollen added to the yeast extract sucrose broth inoculated with spores of A. ochraceus and OTA level [36]. Based on all of the above, it can be assumed that bee pollen may represent a significant risk factor for the occurrence of OTA in the food chain.

Fumonisins
Fumonisins (FBs) are a group of mycotoxins predominantly connected with maize (grown as endophyte in both vegetative or reproductive tissues) and maize products but can be found in many cereals and products made from these plants [24,58]. Although maize is an anemophilic plant due to its high pollen production [7] it is not a rare that bees collect its pollen during the pollen collection season [4]. In that sense, it is possible to find pollen samples contaminated with FBs. The first report about FB food contamination dates back to 1988. The main representative of this mycotoxin group is fumonisin B 1 (FB 1 ) [24,58]. It is sorted in IARC 2B group of carcinogenic substances [51]. Moreover, fumonisins B 2 , B 3 , and B 4 also exist ( Figure 3) [57]. Fungi belonging to Fusarium genus are the most important FBs producers, especially two species: F. proliferatum and F. verticillioides as well as A. alternata from Alternaria spp. It is important to point out that the presence of these microbes does not mean that FBs contamination is guaranteed [24]. In an investigation by Kačaniová et al. [41] the presence of both, F. proliferatum and F. verticillioides was confirmed in thirty i.e., forty-five bee pollen samples, respectively but FBs were quantified only in the samples originating from sunflower (fifteen samples). This observation confirms the previously mentioned hypothesis, that despite the presence of Fusarium spp. in some material, appropriate weather conditions or insect damage are necessary for FBs production [24]. The range of FBs concentrations in these samples is given in Table 2.    in crops and crop products [24]. According to IARC this macrocyclic lactone is classified in group 3 which means that it is not classifiable as to its carcinogenicity to humans [51]. In the case of pollen, the significant contamination with ZEN was recorded in Slovakian bee samples [41] (Table 2).
Toxins 2019, 11, FOR PEER REVIEW 9 which means that it is not classifiable as to its carcinogenicity to humans [51]. In the case of pollen, the significant contamination with ZEN was recorded in Slovakian bee samples [41] (Table 2).

Trichothecenes group of mycotoxins
In a study from Slovakia [41], the authors also reported the contamination of all examined bee pollen samples with T-2 toxin and deoxynivalenol ( Figure 4). Both toxins belong to trichothecene compounds, the sesquiterpenoid metabolites obtained after microbiological activity of several fungi from the following genera: Fusarium (primary source), Trichoderma, Myrothecium, Phomopsis, etc., [24]. Together with ZEN, they were the most dominant quantified mycotoxins in the pollen samples. Additionally, the presence of DON and T-2 toxin was checked in fifteen pollen samples from Spain, but the content of these mycotoxins was below limit detection of applied GC/MS method [43]. In the same study, the authors examined the presence of several other Fusarium spp. producing mycotoxins: 3-acetyl-deoxynivalenol, fusarenon-X, diacetoxiscirpenol, nivalenol, neosolaniol, and HT-2 toxin. All the above-mentioned compounds belong to trichothecene terpenoid's derivatives. It was determined that some of the samples were contaminated with neosolaniol and nivalenol (Table  2), while all other examined toxins were below limit detection. A report made by Cirigiliano et al. [46] should also be mentioned as their study was the first to detect seven specific mycotoxins (roridin A, verrucarrin A, α-β-dehydrocurvularin,phomalactone,6-(1-propenyl)-3-,4,5,6-tetrahydro-5-hydroxy-4H-pirane-2-on e, 5-

Trichothecenes Group of Mycotoxins
In a study from Slovakia [41], the authors also reported the contamination of all examined bee pollen samples with T-2 toxin and deoxynivalenol ( Figure 4). Both toxins belong to trichothecene compounds, the sesquiterpenoid metabolites obtained after microbiological activity of several fungi from the following genera: Fusarium (primary source), Trichoderma, Myrothecium, Phomopsis, etc., [24]. Together with ZEN, they were the most dominant quantified mycotoxins in the pollen samples. Additionally, the presence of DON and T-2 toxin was checked in fifteen pollen samples from Spain, but the content of these mycotoxins was below limit detection of applied GC/MS method [43]. In the same study, the authors examined the presence of several other Fusarium spp. producing mycotoxins: 3-acetyl-deoxynivalenol, fusarenon-X, diacetoxiscirpenol, nivalenol, neosolaniol, and HT-2 toxin. All the above-mentioned compounds belong to trichothecene terpenoid's derivatives. It was determined that some of the samples were contaminated with neosolaniol and nivalenol (Table 2), while all other examined toxins were below limit detection. A report made by Cirigiliano et al. [46] should also be mentioned as their study was the first to detect seven specific mycotoxins (roridin A, verrucarrin A, α-β-dehydrocurvularin, phomalactone,6-(1-propenyl)-3-,4,5,6-tetrahydro-5-hydroxy-4H-pirane-2-one, mycotoxins were obtained as result of Nigrospora sphaerica strains activity. Their structures were confirmed by 1D and 2D-NMR spectroscopy.

Mycotoxin Producing Fungi in Pollen
The microbiological quality of pollen is equally important as its chemical composition due to its safety use. Although the examination of mycotoxins in pollen began mostly in the last decade, the determination of different microbes (bacteria, mold, and yeast) present in pollen samples started much earlier-at the end of 1970s with studies by Gilliam [59,60]. Considering that a long period of time usually passes between collection of pollen samples and its application as food supplement (or as medicament), there is a great chance for the development of some toxigenic fungi [41]. Their presence may indicate mycotoxin production in pollen with or without their quantification. In that sense, this review also gives information on pollen investigations concerning the presence of mycotoxin producing fungi [41] made without further mycotoxicological analysis. The results of a cross-check of the available literature data, with appropriate comments and information, are given in Table 3.  The results show that in all analyzed samples of pollen 21 fungal species of 13 genera of microscopic fungi were detected. The dominant identified species, over 62% of the isolates belonged to following genera: Mucor, Rhizopus, Aspergillus, Alternaria, and Paecilomyces. [65] 28 samples (fresh and dried bee pollen) Cuba Mold/Yeast Not specified Total mold and yeast count: 10 4 -1.5 × 10 5 cfu/g All samples had quantified number of mold and yeast above proposed limits (10 4 cfu/g for the fresh and 10 2 cfu/g for dried pollen). Nevertheless, in the dry pollen, a smaller number of high contaminated samples were recorded. Drying could not be used as reliable method for obtaining pollen with acceptable microbiological quality. Not specified Individually identified yeast Total mold and yeast count: <10 to 9.4 × 10 2 cfu/g All samples were contaminated with yeast and mold. Further, yeast species were identified, and results indicated the presence of five different genus of yeast which can influence the risk of food-borne illness and spoilage or can serve as an indicator of a lack of hygiene standards.

Legislations of Mycotoxins Level in Food and Pollen
In order to prevent undesirable consequences and to protect consumers health, the European Commission, as well as some other international agencies, have proposed maximum permissible concentrations (MPC) for several mycotoxins in different types of food [33,81]. Maximum permissible concentrations vary due to differences in food origin and greater/less possibility of contamination with mycotoxins, as well as because of smaller or larger intake in meals. For instance, the MPC for AFB 1 alters from 0 to 8 µg/kg [33]. Zero tolerance is established for milk and dairy products due to regular daily consumption while the maximal value has been proposed for groundnut-based food. Furthermore, for sensitive groups (such as infants and children), special lower limits have been usually established. The proposed limits are subject to corrections as a result of the development of new, more precise, and sensitive analytical methods for determining the content of mycotoxins [81]. In Table 4 current EU MPC values for some food types are given. Cereals based products OTA 3 µg/kg [33] Instant coffee OTA 10 µg/kg [33] Roasted coffee OTA 5 µg/kg [33] The Scientific Committee of Food requested and obtained from the European Food Safety Authority (EFSA) current data for Tolerable Weekly Intake (TWI) for OTA-0.12 µg/kg of body weight (bw) [82]. Recently, EFSA published new information about the potential increase of maximum allowable level (from 4 to 10 µg/kg) for total AFs in peanuts and processed products, requested by EU Commission [83]. The CONTAM panel (EFSA Panel on Contaminants in the Food Chain) strongly opposed this request due to the significant increase of cancer risk (factor value = 1.6-1.8). For other mycotoxins proposed Tolerable Daily Intake (TDI) values are: 2 µg/kg bw for nivalenol, 0.25 µg/kg bw for ZEN [84], 2 µg/kg (provisional maximum TDI) for FBs [85], 1 µg/kg bw for DON [86], 0.1 µg/kg bw for the sum of T-2 and HT-2 toxins [87], 0.06 µg/kg for combined trichothecenes mycotoxins group [33]. In these legislations, there is no information about proposed limits for mycotoxins in pollen. In 2008 Campos et al. [2] proposed that in the case of AFB 1 occurrence in pollen the MPC value should be set at 2 µg/kg i.e., 4.2 µg/kg for total AFs. To the best of our knowledge, this is the only proposal which defines the level of some mycotoxins in pollen. Since this paper gives an overview about the presence of different mycotoxins in pollen samples originating from various locations around the world, it will be of great importance to define some tolerable levels for other fungi-produced toxins in pollen, especially for OTA. Moreover, current values for AFB 1 and AFs should be reconsidered and checked due to an increasingly frequent aflatoxin contamination caused by climatic changes. Special concerns exist due to mixed (cross) contamination of pollen samples as confirmed by the presented data. Previously, several authors [32,88,89] confirmed that some combined mycotoxins have a more distinct detrimental effect on human health. Furthermore, Manafi et al. [90] have shown that AFs and T-2 toxin synergistically influenced the decrease of total serum protein and albumin levels in broiler chickens as well as decreased antibody titers. It is therefore of the utmost importance to evaluate the toxicological impact of mycotoxin combinations on animal and human health risks.

Conclusions and Future Perspectives
Pollen could be used as a food supplement which can be attributed to its appropriate chemical composition. The microbiological quality of pollen is equally important as its nutritional characteristics. The fungal contamination of different feed/food, including pollen will be more frequent as a result of intensive climatic changes. The quality of pollen can be significantly influenced by the presence of toxigenic fungi. Since it has been proved that the absence of microbial contamination in pollen does not exclude the presence of mycotoxins, mycotoxicological analyses should also be included as a regular control measure together with microbiological tests. Since aflatoxins and ochratoxins are proven as carcinogenic substances, their presence in pollen is extremely undesirable. Therefore, it is important to monitor mold and mycotoxin levels in feed/food in order to avoid adverse health effects. The incorporation of pollen as a food supplement in current legislation will be useful. Proposed quality parameters need to cover tolerable daily/weekly intake for different mycotoxins as well as their sum. In order to obtain reliable and accurate recommendations for pollen quality control, further studies on the toxicological impact of mycotoxin combinations should be conducted.