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Article

Fusarium Fungi and Mycotoxins in Bee Pollen Collected in Lithuania

by
Jolanta Sinkevičienė
1,*,
Živilė Tarasevičienė
2 and
Vytautas Tamutis
3
1
Department of Agroecosystems and Soil Sciences, Faculty of Agronomy, Agriculture Academy, Vytautas Magnus University, Donelaičio Str. 58, LT-44248 Kaunas, Lithuania
2
Department of Plants Biology and Food Science, Faculty of Agronomy, Agriculture Academy, Vytautas Magnus University, Donelaičio Str. 58, LT-44248 Kaunas, Lithuania
3
Division of Dendrology and Phytopathology Sciences, Vytautas Magnus University Botanical Garden, Žiliberio Str. 6, LT-46324 Kaunas, Lithuania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(3), 1571; https://doi.org/10.3390/app13031571
Submission received: 18 October 2022 / Revised: 2 November 2022 / Accepted: 6 January 2023 / Published: 26 January 2023
(This article belongs to the Special Issue Advances in Bee Products and Its Processing Technology)
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Abstract

:
This paper presents the results of a study which was aimed at determining the concentration of Fusarium fungi and their mycotoxins in fresh bee pollen, stored for different periods. The analysed parameters included palynological analysis, moisture content, fungal counts, identification and toxigenic profiles. In this study, 45 bee pollen samples collected from the same apiary families were investigated. Palynological analysis determined six plant families, among which Brassicaceae prevailed. The number of detected isolates in the bee pollen during the study period ranged from 3.5 × 103 to 9.1 × 104 cfu g−1. During the study, the most prevalent fungal genera of Alternaria, Cladosporium and Yeasts were found in fresh bee pollen. The significantly highest amounts of fungal colonies were determined after 3 days of storage of undried pollen. Fusarium fungal genera were detected in 46% of all studied samples, with levels ranging from 101 cfu g−1. After 3 days of storage, the most significant Fusarium spp. increase (17.03%) was detected. F. graminearum and F. sporotrichioides prevailed during the whole period of the study. The highest concentrations of mycotoxins ZEN (280 µg kg−1) and DON (120 µg kg−1) were found after 3 days of pollen storage. The results of the present study report the importance of microbiological and mycotoxicological analyses in monitoring bee pollen from the initial stages of its production process.

1. Introduction

Fresh bee-collected pollen is a favourable substrate for the growth and development of microscopic fungi [1,2]. As a result of the presence of microscopic fungi, there exists a potential production of mycotoxins. The fungi that produce them are dangerous because the synthesis of mycotoxins is a part of their metabolic pathways [3]. Fungal toxins are secondary metabolites of different fungal species, and they can cause dangerous diseases—mycotoxicoses [4]. The mycotoxigenic fungi belong mainly to Aspergillus, Penicillium and Fusarium genera [3]. Aspergillus and Penicillium species are found in foods during storage. Fusarium species are plant pathogens producing mycotoxins before or immediately after harvest [5,6]. Fusarium is a widely distributed toxigenic fungal genus. It grows at 24–26 °C and at a minimum of 0.90 aw. The production of toxins largely mirrors growth conditions [3]. Fresh bee-collected pollen contains high levels of moisture and increased water activity, aw, which is conducive to the rapid development of fungi and enables the synthesis of mycotoxins [1,3].
Toxins produced by Fusarium spp. are receiving increasing attention. Almost all fusariotoxins are harmful for humans [7,8]. Trichothecenes and fumonisins are some of the most important mycotoxins produced by Fusarium species [9]. Trichothecenes are potential inhibitors of protein synthesis, and fumonisins are considered to be potentially carcinogenic [10]. Zearalenone (ZEN) is produced by various Fusarium species: F. equiseti, F. graminearum, F. culmorum, F. crookwellense, F. proliferatum, F. moniliforme and F. verticillioides are the most important producers of fumonisin (FBs) [7]. Deoxynivalenol (DON) is produced by F. culmorum and F. graminearum. The T-2 toxin is much more toxic and less common than DON. It is produced by F. tricinctum, F. sporotrichioides and F. poae [10,11].
Bee pollen is a valuable apitherapeutic product greatly appreciated in natural medicine because of its potential medical and nutritional applications [12]. Pollen can be a good food supplement, as it contains different polyphenolic compounds [13], carotenoids [14] and vitamins [15] and possesses substantial antioxidant properties [12]. As a nutritional enrichment product, bee pollen is used fresh, dried or frozen. In the literature [16,17,18,19], we usually find data on fungal contamination of already dried pollen. Microbiological testing of fresh bee pollen would help to clarify the initial mycological condition of bee pollen and allow further pollen quality control measures. As contamination of food raw materials with mycotoxins is difficult to avoid, data on the occurrence of mycotoxins are of major importance for food safety. However, data on the distribution of Fusarium mycotoxins in bee pollen are rather limited. Therefore, this study was aimed at determining the contamination of fresh bee pollen by microscopic fungi during a three-day storage period and the potential ability of Fusarium fungi to produce mycotoxins in fresh bee pollen.

2. Materials and Methods

2.1. Experimental Site

The experiment was carried out during 2019–2020 at the Vytautas Magnus University Agriculture Academy (VMU AA) Open Access Joint Research Centre of Agriculture and Forestry (Lithuania).

2.2. Bee Pollen Samples

A total of 45 pollen samples (250 g) were supplied directly by a local beekeeper in the district Kaunas, Lithuania. Bees had collected pollen from different plant species. On the same day it was purified from different impurities (the plant particles, insect residues, etc., were removed). Secondly, moisture content (%) of undried pollen was determined. The collected undried bee pollen was stored for different durations (from 1 to 3 days) at 22–25 °C room temperature in plastic food containers.

2.3. Research Methods

Palynological analysis. Two grams of pollen samples were homogenized in 13 mL 70% ethanol, and after vortexing until homogenized they were centrifuged at 3500 rpm for 20 min. After centrifugation, the supernatant was discarded and 13 mL of 50% glycerine–water mixture (1:1) was added and centrifuged at 3500 rpm for 20 min. After centrifugation, the supernatant was poured, the tubes were inverted on blotter paper and the pellet was allowed to dry. The sediment of the tube was taken with the glycerine–gelatine matrix with the help of a dissecting needle and put on the slide, melted on the hot plate and covered with the coverslip. Three preparations were prepared for each sample [20]. Pollen was identified by optical microscopy, using previously published data [21,22].
Moisture content. Moisture content analysis was determined by drying 5 g of a sample in an oven at 105 °C temperature until constant weight.
Isolation and morphological characterization of fungi. The dilute plate method was used for isolation of fungi from bee pollen. Ten grams of each bee pollen sample were homogenized into 90 mL of physiological saline solution (NaCl, 8.5g L−1) and shaken on a horizontal shaker for ca. 15 min. at 400 rpm. For each sample, 1 mL aliquots of each of the four dilutions were inoculated (in triplicate) in the Potato Dextrose Agar (PDA) (Sigma-Aldrich) culture media.
The plates were incubated at 26 °C for 5 to 7 days and then fungi were counted and recorded as colony-forming units (cfu) per gram of sample. Separate fungal colonies were transferred onto appropriate identification media. The Czapek Dox agar (Biolife, Italy) and Potato Dextrose Agar (PDA) (Sigma-Aldrich) medium were used to isolate and identify individual Fusarium genera and species. The morphological characteristics of isolated colonies were identified based on macroscopic (colony appearance) and microscopic (spores forming) features in accordance with various descriptors [23,24,25,26,27].
Mycotoxins analysis. In this study, the following mycotoxins were investigated by enzyme-linked immunosorbent assay (ELISA): deoxynivalenol (DON), zearalenone (ZEN) and the T-2 toxin (T-2) [28]. The ELISA method is totally quantitative, simple, sensitive and fast; therefore, it can be used for the detection of mycotoxins in various foods [29]. The RIDASCREEN® ELISA kits produced by R-Biopharm AG (Darmstadt, Germany) approved by the AOAC Research Institute (Certificate No. 9001) were used for the analysis No R5906, No R5502. Mycotoxin analysis was performed following the manufacturer’s instructions.
For the preparation of the extracts, pollen samples were ground and then 5 g fraction were weighed. Three extracts were prepared: for the quantification of DON, extraction was carried out in 100 mL distilled water; in the others 100 mL of a methanol: water solution (70:30 v/v) was added for the quantification of ZEN and 50:50 v/v for T-2. Then, samples were stirred at 150 rpm at 25 °C in an orbital shaker (Certomat®, BS-1, Sartorius, Goettingen, Germany) for one hour and the samples were filtered using Whatman #1 filter. The filtrate was used directly for the quantification of T-2. For DON and ZEN, an additional dilution was required. Dilutions were 1:4 in distilled water for DON and 1:5 in 70% methanol for ZEN.
The method’s limits of detection (LoD) were the following: DON—18.5 μg kg−1, ZEN—17.0 μg kg−1 and T-2—5.0 μg kg−1. Samples with results lower than the LoD values were considered not contaminated.
The absorbance was determined using an ELISA reader (Stat Fax®303 Plus Microstrip Reader, Awareness Technologies, Westport, CT, USA) with a 450 nm absorbance filter. The optical densities (OD) of the samples were compared to the OD of standards through a linear regression, thus obtaining the concentration of mycotoxin present in each sample. Only calibration curves with R2 greater than 0.99 were considered. Analyses were performed in duplicate for each sample.

2.4. Statistical Analysis

The results were statistically processed using one-way analysis of variance. Statistical analysis of the experimental data was performed using the software ANOVA from the statistical analysis package SELEKCIJA [30]. Differences between averages of treatments marked with different letters are significant at 95% confidence level (p < 0.05). There are no significant differences when p > 0.05.
For the evaluation of the relationships between the bee pollen storage time, spread of different fungi species and amount of fungi, the principal component analysis (PCA) was performed using XLSTAT software version 2019.3.02 (Addinsoft, Paris, France).

3. Results

3.1. Palynological Analysis

During the study, different botanical origins of bee pollen samples were analysed (Table 1). Analysis of the floral sources of the samples revealed six pollen types belonging to six different botanical plant families. Pollen families are classified as dominant (≥45%), secondary (16–44%), minor (3–15%) and trace (<3%) according to their density [20].
The Brassicaceae type was the dominant class (54.65%), Rosaceae was secondary (16–44%), Fabaceae, Poaceae and Asteraceae were minor (3–15%) and Apiaceae was represented in small amounts (<3%). In terms of floral diversity, 55% of pollen showed only one floral source (in the class of dominant pollen), whereas the remaining presented a composition originating from five pollen types.

3.2. Levels of Fungal Contamination and Moisture Content of Fresh Bee Pollen

During the study, 10 genera of fungi were isolated from 45 samples in fresh pollen, which are presented in Table 2. Two genera of isolated fungi Mucor and Rhizopus according to their morphology were attributed to Phylum Zygomycota, class Zygomycetes; Phylum Ascomycota belonged to Chaetomium (class Pyrenomycetes, order Shaeriales) and Yeasts (class Archiascomycetes, order Schizosaccharomycetales). Acremonium, Alternaria, Aspergillus, Botrytis, Cladosporium, Epiccocum, Fusarium, Mycelia sterilia, Penicillium and Trichoderma belonged to anamorphic fungi (Deuteromycota, class Hyphomycetes, order Moniliales). Anamorphic fungi dominated among the isolated fungal taxa, and they comprised 73.57% from all isolated fungi.
After 5 h in fresh pollen Alternaria, Cladosporium and Yeasts prevailed. Alternaria fungi were common in 26.79% of the samples (32 samples out of 45) with levels ranging from 103–104 cfu g−1; Cladosporium fungi were isolated in 28 samples, which comprised 20.21%, with levels ranging from 102–103 cfu g−1. Yeasts comprised 12.14%, isolated from 12 samples, with levels ranging from 102–103 cfu g−1. Fungi of Fusarium genera were isolated from 46% of the studied samples (21 samples out of 45), with levels ranging from 101 cfu g−1. Fusarium frequency comprised 9.31%. According to frequency, they were the fourth most abundant fungi isolated.
After 1–3 days of storage, the genera of the predominant fungi quantity did not change, but the total amount of isolates increased by 0.7%, compared to 5 h. Alternaria fungi were predominant: their amount increased by 2.51% in the period of 1–3 days, and fungi were identified in 37 samples on average. The amount of Cladosporium decreased from 0.71%, but the number of positive samples remained unchanged (27 samples on average). Yeast frequency increased by 4.57% as the pollen storage time increased. Fusarium frequency increased by 1.18% after 3 days.
During the study period (5 h—3 days), the total amount of microscopic fungi in fresh pollen ranged from 3.5 × 103 cfu g−1 to 9.1 × 104 cfu g−1 (Table 3). On the day of pollen collection, fungal contamination averaged 6.4 × 102 cfu g−1, and after three days it comprised 7.2 × 103 cfu g−1. A statistically significant reduction in the amount of fungi was found in pollen stored for 1 day.
The highest moisture content (22.8%) in fresh pollen was found on the day of its collection—after 3 days the moisture content decreased significantly by 0.2%.

3.3. Fusarium Fungi and Mycotoxins

The total amount of Fusarium fungi in pollen stored for 3 days with moisture content at 22.6%, as compared to the control, significantly increased on an average up to 17.03%. The diversity of Fusarium species in fresh pollen was not high. Six Fusarium species were identified (Table 4). After 5 h in the pollen studied, F. graminearum and F. sporotrichioides dominated, which were identified among 30.06% and 27.07% of all isolated Fusarium genera, respectively. F. culmorum (14.38%) and F. oxysporum (13.11%) were the most common species and F. poae (7.69%) and F. verticilioides (7.69%) were classified as the least common species. During the study period, the number of isolates of most Fusarium species were increasing with a longer time of pollen storage. After 3 days, increases in F. graminearum (19.23%) and F. sporotrichioides (15.92%) isolates were observed compared to the control. These species dominated throughout the study period (Figure 1). A positive strong correlation was found (r = 0.99, p < 0.05) between F. graminerum and pollen storage time, and a weak (r = 0.95, p < 0.05) correlation was found between F. sporotrichioides and storage time. The amounts of F. poae, F. oxysporum and F. verticilioides species increased by 4.82%, 2.17% and 0.8%, respectively. A very strong correlation (r= 0.95, p < 0.01) was found between F. culmorum and storage time.
The results of mycotoxins (Table 5) indicate that the samples of bee pollen were contaminated by ZEN and DON mycotoxins. The average concentration of the toxins zearalenone (ZEN) and deoxynivalenol (DON) varied considerably, ranging from 65 to 280 µg kg−1 and from 47 up to 120 µg kg−1, respectively. A storage duration of 3 days influenced the highest concentrations of mycotoxins ZEN and DON. Contamination by the T-2 toxin in all bee pollen samples was found to be below the detection limit.
On the day of pollen collection, 67 µg kg−1 of mycotoxin zearalenone (ZEN) was detected. The pollen was most heavily contaminated with zearalenone (ZEN) after 3 days of storage, at 280 µg kg−1, and it was the highest concentration of this mycotoxin found in all study variants.
Mycotoxin deoxynivalenol (DON) at 47 µg kg−1 in the pollen was found on the day of pollen collection. A significant increase in the concentration of deoxynivalenol (DON) was found after 3 days, comprising 120 µg kg−1.
A positive correlation (r = 0.55, p < 0.05) was ascertained between moisture content of the pollen and ZEN concentration. No correlation was found between ZEN and storage duration or the total amount of fungi. No correlation was found between DON and storage period or the total amount of fungi with prolonged storage period.
PCA was used to determine the grouping possibilities of bee pollen samples, considering their storage duration and fungal and mycotoxin contamination (Figure 2). The first and second PCA components were composed of 87.28%.
All bee pollen samples were completely separated depending on their storage duration. The samples before storage were situated at negative PC2 values, while longer storage duration samples were situated at positive PC2 values.
One storage day samples were closely related to the F. verticilioides, while three-day samples were related to F. poae, F. graminearum, zearalenone and deoxivalenol.

4. Discussion

Microbiological contamination of bee pollen arises from different sources. The primary sources of contamination of bee pollen by microscopic fungi are very difficult to control, and they can come from plants, from the digestive tract of bees or enter with dust from the air or soil. Possible sources of contamination may be the type of collector or the impurity of pollen traps [4,19]. The botanical origin of the pollen is revealed by floral species, which varies depending on the climatic conditions during flowering and the region where the apiary is located [31]. In this study, the pollen of Brassicaceae plants dominated, as bees collected the pollen from Rosaceae Fabaceae, Poaceae and Asteraceae family plants.
Some fungi can spread together with pollen [32]. Reports of fungi associated with pollen grains on plants are rare. The intricacy of the surface of many pollen grains could provide ample opportunities for the attachment of fungal spores [33]. During the flowering and pollination process, spores can germinate on female flower parts. The pollen from some Asteraceae species is an optimal substrate for fungal growth due to its high lipid content [18]. The dominant endophytes such as Alternaria, Cladosporium and Epicoccum seem not to be host-specific and are opportunistic colonizers of many plants [34]. According to researchers [35,36], Alternaria, Cladosporium and Fusarium are some of the dominant mycobiota in fodder legumes. If bees visit these plants, the contaminated pollen grains will be transferred into the hives [3]. Fusarium fungi producing trichothecenes were found in all species of leguminous grasses, the duration of vegetation of which is directly related to the amount of Fusarium fungi, and with increasing duration increases the amount of Fusarium fungi [37].
In the study, the moisture content of fresh bee pollen 5 h after collection was high (22.8%). Other researchers also obtained similar results: 14.6–37.7% [6], 21–30% [38] and 20% [39]. The moisture content of fresh bee pollen in this study varies slightly—between 0.01 and 0.02%—and was sufficient for fungi growth. The high moisture content can lead to an increase in microscopic fungal contamination which causes the production of mycotoxins [15,40].
Fungi belonging to 14 genera were detected in the samples of fresh bee pollen. The highest number of colonies among fungi comprised Alternaria, Cladosporium and Yeasts. Fungi of this genera are classified as saprotrophic field fungi derived from soil or plant residues [2]. Fungi of the genera Alternaria and Cladosporium are sufficiently resistant to the negative effects of the environment due to the accumulation of melanin [41], and their development and growth require high relative air humidity and low availability of food resources [2]. Studies conducted by Gonzales et al. [16] have revealed that the amount of prevailing Alternaria fungi in bee pollen comprised 86.6%. According to Deveza et al. [31], Cladosporium in bee pollen was singled out as the second most common fungi. Kačaniová et al. [17] found that Alternaria alternata and Cladosporium cladosporoides were the most frequent isolates found in Slovakia bee pollen samples.
Yeasts are not classified as microorganisms dangerous to human health, and they usually indicate the sanitary condition of hives and hygienic conditions during pollen collection and transportation [42]. Twelve analysed samples were contaminated with Yeasts, and their amount was abundant. The most abundant Yeasts were isolated in the pollen with the highest moisture content (22.8%). The abundance of Yeasts decreases rapidly in the first days of fresh pollen storage [43]. This is partly reflected by the data of our study.
The total amount of microscopic fungi in bee pollen on the day of collection (after 5 h) was similar to the previous results of studies by Sinkevičienė and Amšiejus [44], in which the contamination of fresh pollen after 9 h was from 4.5 × 103 to 9.5 × 103 cfu g−1. Beev et al. [45] also performed a fungal colony count in fresh bee pollen, which varied from 560 to 37,000 cfu g−1 and was significantly (p < 0.01) higher than the fungal colony count in dried pollen. In our study, the amount of fungi in different periods varied depending on storage duration time. Fresh bee pollen stored for different time periods showed that the amount of fungi started to increase with longer duration of pollen storage. The highest amount of fungi (7.2 × 103 cfu g−1) was determined after 3 days of storage duration of fresh pollen, however, according to scientists from the International Honey Commission (IHC), this amount of fungi failed to exceed the limits of recommended quality criteria (<5 × 104 cfu g−1) [46].
In this study, the genus Fusarium was detected in less than half of fresh pollen samples tested. Fusarium fungi can survive as saprotrophs and require more than 20% substrate moisture to develop and multiply [5]. According to frequency, among all identified fungi Fusarium spp. was the fourth. Beev et al. [45] found Fusarium spp. to be the second most frequently encountered microscopic fungi in dried bee pollen, but no Fusarium fungi were detected in fresh bee pollen. According to the data by Gonzalez et al. [16], the Fusarium spp. was rarely detected in dried bee pollen. In the study by Brindza et al. [2], Fusarium occurred mostly in dried bee pollen.
Fusarium is the main toxin-producing genus among fungi [10]. Many toxic species of the genus Fusarium are widespread in all European cereal growing areas, including Lithuania [47]. Isolates of Fusarium species were isolated in the pollen in order to elucidate the producers of fusariotoxins. Six species of the genus Fusarium were detected in 21 samples. Similar results (five species) were obtained by other researchers [17].
In this study, F. graminearum was clearly predominant in the period of all studies (1–3 days). F. graminearum is known as the most common Fusarium species and the producer of deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) [19,48]. The next dominant species were F. sporotrichioides. [4], F. sporotrichioides and F. verticilioides, which dominated in the study of Kačaniová et al. [17]. However, in our study the contamination level of F. verticilioides was generally low, especially at the end of the storage (Figure 1 and Figure 2). Based on other research [17], F. oxysporum was one of the prevailing species, comprising 55.6%. In our study, the amount of F. oxysporum isolates was low, and on average comprised 14.19%. Other species, F. culmorum (11.18%) and F.poae (10.78%), were also less common. In the PCA it was found that F. poae was closely related to three storage days duration when at that time the amount of F. poae increased. (Figure 2).
The production of mycotoxins depends on a number of factors, such as temperature, humidity, substrate composition and the quantity of fungal inoculum. A long period between collection and drying of pollen can also affect the growth of toxigenic fungi and the production of mycotoxins [49]. The increase in mycotoxins after 3 days showed that the temperature of 22–25 °C and the moisture of the substrate may be favourable for the growth and development of Fusarium fungi. The increases found in F. graminearum, F. sporotrichioides and F. culmorum during this period are possibly related to the highest levels of zearalenone (ZEN) and deoxynivalenol (DON) released. PCA analysis indicates the strongest correlation between the colony forming units of F. graminearum the concentration of DON and ZEN at the end of the storage (Figure 2). Our previous investigation [50] examined dried pollen samples contaminated with ZEN and DON; the highest DON concentration (185 µg kg−1) was ascertained after 4 months of storage at 8–9 °C temperature, and the highest ZEN concentration (830 µg kg−1) was found after 1 month of storage at 20–22 °C temperature. It was these research results that encouraged us to investigate the mycological and mycotoxicological contamination of fresh pollen collected only from plants. The fungal and toxigenic load detected in bee pollen raises questions on how contamination occurs during pollen production and collection. Kostic et al. [18] states that the source of pollen pollution with mycotoxins can be the hive, and they also mention that the growth of toxin-producing fungi can be stimulated by infected flowering plants. Trichoderma, Myrothecium, Phomopsis, etc., have been also mentioned as DON sources. Fusarium spp. are field fungi and produce mycotoxins pre-harvest [47]. Therefore, the detection of Fusarium toxins is only a retrospective indicator for the conditions on field and is not directly related to the toxin content during storage [51].
Due to climatic changes, which extensively influence weather conditions in Europe, the presence of toxins is becoming more frequent. DON and ZEN mycotoxins were the most dominant quantified mycotoxins in the pollen samples from Spain [52]. DON, ZEN were the most dominant quantified mycotoxins in the bee pollen samples from Slovakia [3]. Prevailing in pollen, F. sporotrichioides and F. poae are co-producers of the T-2 mycotoxin [7], but this mycotoxin has not been detected in pollen. However, the amount of mycotoxins may not depend on the number of fungi present [53]. The production of toxins largely mirrors growth conditions [19]. Therefore, according to Petrovic et al. [19] and Kačaniova et al. [17], fresh bee pollen is recommended to be stored at low temperatures for no more than 48 h, and mycotoxin levels should be monitored regularly [4].

5. Conclusions

This study found high levels of contamination of fresh bee pollen by microscopic fungi. The most prevalent fungal genera of Alternaria, Cladosporium and Yeasts were found in fresh bee pollen. Among them, six Fusarium species and ZEN and DON mycotoxins produced by them were isolated, the amounts of which were increasing with a longer storage period. The study found that the pollen on the day of collection was already contaminated by Fusarium mycotoxins. Therefore, in controlling the contamination of fresh bee pollen by toxigenic fungi and mycotoxins, it is very important that the pollen is dried as quickly as possible to reduce the levels of fungal contamination. The obtained results emphasize the importance of microbiological analysis in monitoring bee pollen from the initial stages of their production process. Information on the presence of mycotoxins in bee pollen can be very important for risk assessment on human health.

Author Contributions

Conceptualization, J.S. and Ž.T.; methodology, J.S.; validation, V.T.; formal analysis, J.S.; investigation, J.S. and V.T.; resources, Ž.T.; data curation, J.S., Ž.T. and V.T.; writing—original draft preparation, J.S., Ž.T. and V.T.; writing—review and editing, J.S.; visualization, J.S.; supervision, J.S.; project administration, J.S.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 01571 g001 550
Figure 1. The dynamics of fungi in bee pollen during storage, %.
Figure 1. The dynamics of fungi in bee pollen during storage, %.
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Figure 2. Principal component analysis (PCA) for bee pollen stored for three days (DON—deoxivalenol; ZEN—zearalenone).
Figure 2. Principal component analysis (PCA) for bee pollen stored for three days (DON—deoxivalenol; ZEN—zearalenone).
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Table
Table 1. Origins of bee pollen.
Table 1. Origins of bee pollen.
Plant Sources (Family)PercentagesFrequency of Pollen Types
Brassicaceae54.65Dominant (≥45%)
Rosaceae15.83Secondary (16–44%)
Fabaceae11.24Minor (3–15%)
Poaceae10.18Minor (3–15%)
Asteraceae5.41Minor (3–15%)
Apiaceae2.69Trace (<3%)
Table
Table 2. Occurrence of fungi in bee pollen.
Table 2. Occurrence of fungi in bee pollen.
GenusFrequency of Occurrence (%)
5 h1 Day2 Day3 Day
Acremonium2.141.331.691.41
Alternaria26.7929.529.8829.3
Aspergillus1.790.490.731.22
Botrytis4.293.83.513.2
Chaetomium0.710.110.350.29
Cladosporium13.2114.649.8912.5
Epiccocum1.7911.20.85
Fusarium9.317.9310.9110.49
Yeast12.1414.216.516.71
Mucor6.795.916.737.12
Mycelia sterilia6.436.796.947.33
Penicillium7.57.349.6110.5
Rhizopus1.431.92.22.61
Trichoderma3.934.52.953.75
Table
Table 3. The levels of fungal contamination and moisture content of bee pollen.
Table 3. The levels of fungal contamination and moisture content of bee pollen.
Storage DurationFungiMoisture Content (%)
Levels of Fungal
Contamination (cfu g−1)		Average (cfu g−1)
Control (after 5 h)3.9 × 101–7.7 × 1036.4 × 102 b22.8a
After 1 day3.5 × 101–7.8 × 1036.2 × 102 b22.7ab
After 2 days3.8 × 101–8.4 × 1036.6 × 102 b22.7b
After 3 days4.5 × 101–9.1 × 1047.2 × 103 a22.6b
Note: cfu g−1—colony forming units per gram in bee pollen; the differences between the averages of treatments, marked by different letter (a, b) are significant (p < 0.05).
Table
Table 4. The levels of Fusarium species in bee pollen samples (cfu g-1).
Table 4. The levels of Fusarium species in bee pollen samples (cfu g-1).
FungiStorage Duration
After 5 hAfter 1 DayAfter 2 DaysAfter 3 Days
F. culmorum1.1 × 101A b1 × 101A bc1 × 101A b1.1 × 101A b
F. gramineraum1.6 × 102B a1.6 × 102B a2 × 102A a2.3 × 102A a
F. oxysporum1 × 101A b1.2 × 101A b1.3 × 101A b1.2 × 101A b
F. poae1 × 101B b1 × 101C c1.3 × 101AB b1.3 × 101A b
F. sporotrichioides1.3 × 102B a1.4 × 102B a1.8 × 102AB a2 × 102A a
F. verticilioides1 × 101A b1 × 101A bc1 × 101A b1 × 101A b
Note: cfu g−1—colony forming units per gram in bee pollen; the averages of the variants in the uppercase (rows, a, b, c) and lowercase (columns, A, B, C) are significant (p < 0.05).
Table
Table 5. The concentration of mycotoxins (µg kg−1).
Table 5. The concentration of mycotoxins (µg kg−1).
Storage DurationNumber of Positive SamplesZENDONT-2
5 h2167b47b<LoD
After 1 day1965b<LoDc<LoD
After 2 days2467b48b<LoD
After 3 days26280a120a<LoD
Note: <LoD—below limit of detection; means not sharing common letters are significant (p < 0.05).
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Sinkevičienė, J.; Tarasevičienė, Ž.; Tamutis, V. Fusarium Fungi and Mycotoxins in Bee Pollen Collected in Lithuania. Appl. Sci. 2023, 13, 1571. https://doi.org/10.3390/app13031571

AMA Style

Sinkevičienė J, Tarasevičienė Ž, Tamutis V. Fusarium Fungi and Mycotoxins in Bee Pollen Collected in Lithuania. Applied Sciences. 2023; 13(3):1571. https://doi.org/10.3390/app13031571

Chicago/Turabian Style

Sinkevičienė, Jolanta, Živilė Tarasevičienė, and Vytautas Tamutis. 2023. "Fusarium Fungi and Mycotoxins in Bee Pollen Collected in Lithuania" Applied Sciences 13, no. 3: 1571. https://doi.org/10.3390/app13031571

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