Effect of Long-Term Storage on Mycobiota of Barley Grain and Malt

Contamination of malting barley grain and malt with micromycetes sampled at various periods post-harvest (3rd, 6th, and 9th month of storage) and types of storage (storage silo and floor warehouse) was investigated. Each of these barley grain samples was malted. This article reports on the changes in the fungal microbiome composition and their overall count in barley grain and malt. From the surface-disinfected barley grain samples collected immediately after harvest, there were eight genera isolated, with a predominance of Alternaria. A small increase of isolated microfungi was detected in barley stored in silo for 3 and 6 months (from 142 isolates to 149) and decreased below the number of isolates in barley before storage (133 isolates). Fungal count during storage gradually decreased up to 9 month in barley stored in floor warehouse (from 142 isolates to 84). The initial total count of microscopic fungi in malt before storage was the highest (112 isolates) with 7 genera detected, compared to malts prepared from barley stored for longer time (54 isolates, 7 genera, 9th month of storage). Alternaria was the most abundant and frequent genus. Quantitative representation of the filamentous microscopic fungi was lower compared to yeasts especially in barley and malt prepared from barley stored at third month of storage in both type of storage. Yeasts were identified from all grain samples and malt samples with mass spectrometry. Most attention was given to the widely distributed fungus Penicillium, 79% of strains produced at least one mycotoxin detected under in vitro assays using the TLC method (97% of them produced griseofulvin, 94% CPA, 79% patulin, 14% roquefortin C, and penitrem A was produced by two screening strains under laboratory conditions). It is therefore important to monitor the microflora throughout the production cycle of “barley to beer”.


Introduction
Many factors can influence the microbiological contamination of cereal grains during cultivation, harvesting, drying, and their further storage [1]. Microorganisms can come from several sources, including air, dust, water, soil, insects, birds, and rodent feces. Contamination from harvesting equipment or grain processing and the conditions of storage are also important. The weather conditions during plant growth (such as, drought, rainfall, temperature, and sunlight) and even the growing region also influence the type of microbial contamination of the grain [2,3].
In general, the microbial community found on or in the barley seeds may contain numerous species from five groups-viruses, bacteria, fungi, micromycetes, and protozoa. Bacteria together with fungal organisms are the groups with the greatest perceived influence on the properties of barley grain. This is because they occur regularly in higher numbers and many of them are physiologically able to use the grain as nutrient source. The community of fungal species on or in barley seeds and malt grain is subject to change through the production process, starting from the developing ear and grain, through harvested grain, and ending with the kilned malt [4]. The presence of fungal pathogens and mycotoxins in grain is natural in character and the prevention of such occurrences is difficult even if good agricultural practices are followed [5]. The filamentous fungi occurring in grain can be divided into field fungi that occur on the grain until harvest, and storage fungi occurring after harvesting. Field fungi generally do not grow below the water activity (a w ) of 0.90, corresponding to 20 to 25% moisture, whereas storage fungi are often xerophilic and can grow at a w of 0.80 (18% moisture) or even 0.68 (14% moisture) [6,7]. Typical field fungi such as Alternaria spp., Aureobasidium spp., Cladosporium spp., Curvularia spp., Drechslera spp., Epicoccum nigrum, Fusarium spp., Microdochium spp., Nigrospora spp., Septoria spp., and Trichoderma spp. dominate the fungal community because they are able to use the developing grain as substrate without damaging or killing the embryo. However, many other fungal species representing all major taxonomic groups can be found upon plating of whole barley grains or dilutions of barley malt. Under storage, these fungi can survive for extended periods of time and will germinate and grow as the water content of barley exceeds 14 to 15% and CO 2 accumulates at elevated temperatures [8]. The spectrum of fungal species identified from a given barley sample may vary greatly in time, especially during the storage period. This is because many species in the genera Aspergillus and Penicillium, but also typical xerophiles such as Eurotium spp. or Wallemia sebi, only start to develop and multiply after harvest, when the water activity of grains decreases to low values during drying and storage [4].
Although the conditions in grain storage facilities are not conducive to the development of field fungi, these fungi can infect the grain while still in the field and continue to grow during storage [9]. Most species of Penicillium are xerophiles and are classified as storage fungi, but they can also infect the grain before harvesting. For example, P. oxalicum can infect damaged maize grains before harvesting [10]. According to Hill and Lacey [11], up to 85% of barley grains at harvest can be colonized by yeasts, including pink yeasts such as Sporobolomyces and Rhodotorula [12]. Other yeast species found on barley include Hansenula, Torulopsis, Candida, and Saccharomyces [12]. Harvesting insufficiently dry grain necessitates additional drying. When conducted incorrectly, that may cause spoilage of cereals, as it promotes the growth of microorganisms and may result in increased levels of mycotoxins [1]. If drying is delayed and the moisture content of the harvested grain is suitable, growth of the field fungi, e.g., Fusarium spp., may occur [12]. The mycobiota of stored grains predominantly consist of the ubiquitous mold genera Aspergillus (more common at ambient temperatures 20 to 25 • C), Alternaria, Cladosporium, Fusarium, Mucor, Rhizopus, and Penicillium (dominant at cooler temperatures) [1]. After harvesting, barley can be stored for some time before malting, but during storage time microbes may still develop. Storage conditions must be carefully monitored to ensure adequate low humidity and low temperature to minimize the microbial growth [13,14] that can have a very negative impact on the quality of beer [15]. Integrated management of mold spoilage risks in stored grain is based on five principles: prevention of mold development by keeping grain moisture below the critical limit of fungal growth; accurate monitoring of increase a w and temperature changes during the storage period, associated with the monitoring of early indicators of respiration activity of storage fungi; reduction of moistening trends in the bulk grain by physical intervention means; use of physical treatments (ozone, grain peeling, or abrasion) to limit mycotoxin contamination transfer to processed cereal products; and possible use of bio-competitive strains of fungi or bacteria to prevent the development of mycotoxigenic fungi in grain bulks [16].
Malting is a process in which a complex ecosystem evolves due to the prevailing moisture and temperature conditions, thus allowing the contaminating microorganisms to develop and to negatively influence the quality of malt [7,17,18]. During malting, the fungal community and other microorganisms growing compete with the grain metabolism for oxygen and may therefore considerably reduce grain germination [6]. Significant increases in levels of several mycotoxins can occur in addition to fungal growth during that same process [19,20]. Several Fusarium species were demonstrated to proliferate from steeping through germination until early stages of kilning [19,20] and also certain heat-resistant fungi, such as Rhizopus and Mucor, and some Ascomycetes continue to grow during the early hours of kilning [21].
The proliferation of these fungi is stimulated with higher grains moisture content, higher temperature during storage, long storage period, and intensive infection by fungi before mites. Therefore, it is important to identify the species of fungi in stored barley grains with special emphasis on mycotoxigenic species, which pose a potential risk to human and animal health. This work is a report on the evaluation of the mycobiota of barley and malt grains during storage under various conditions.

Endogenous Mycobiota of Barley
The filamentous fungi identified from surface disinfected barley grain before and during storage in silo are listed in Table 1, whereas the isolation frequency and relative density are presented in Table 2. The results indicate that mycological colonization of barley grain before and during storage may be diverse and a subject to change during storage. From the barley samples taken immediately after harvest, there were isolated 8 genera and Mycelia sterilia (isolates without sporulation) of specific microscopic fungi belonging to the field ecosystem. Of all the 142 strains found, species of Alternaria (58% RD) and Fusarium (13% RD) predominated, particularly in the Laudis variety. Higher genera representation (7) was obtained from the Kangoo variety, but the highest number of isolates was from the Laudis variety. Isolates of genera Alternaria, Arthrinium, and Epicoccum were found in all tested samples.   n  IF  RD  n  IF  RD  n  IF  RD  n  IF  RD  n  IF  RD   Alternaria  82  100  58  45  100  31  65  100  44  53  100  40  163  100  38  Arthrinium  17  100  12  16  100  11  11  100  7  9  100  7  36  100  8  Aspergillus  1  33  <1  2  33  2  4  67  3  4  67  3  Compared to the baseline of freshly harvested grain, a small increase of detected filamentous fungi was observed in barley stored for three or six months, except for the Laudis variety with the number of isolates decreased at that length of storage. A total of 146 strains from 10 genera and Mycelia sterilia were identified in barley stored for 3 months. There were negligible differences in the genera representation of filamentous fungi, but the highest number of isolates was found in the Laudis variety. Alternaria was the most frequently observed/isolated genus with 45 isolates and RD of 31% of all the isolates found. Cladosporium and Rhizopus were the second predominant genera with RD of 19%, each.
After 6 months of barley grain storage, the number of microfungi increased slightly to a total of 149 isolates with a comparable representation of 10 genera and Mycelia sterilia to those detected after 3 months of storage. In all samples, the genera Alternaria, Arthrinium, Epicoccum, Fusarium, Penicillium, and Rhizopus were detected. The RD of genus Alternaria was the highest (44%). From five distinct Penicillium species detected, the most frequent and the most abundant was P. griseofulvum. The numbers of genera of filamentous microscopic fungi detected in each barley variety reached their maximum at this storage period: 9 genera and Mycelia sterilia for the Wintmalt variety and 8 genera for the Laudis and Kangoo varieties, respectively.
The barley grain samples were contaminated comparably less with the filamentous microfungi at 9 months of storage (133 isolates). On the other hand, the most fungal genera (11) were detected in these samples. The highest RD was observed for the genus Alternaria (40%), followed by Cladosporium (19%), and Penicillium (10%). The highest amount of Alternaria isolates (24) was again isolated from the Laudis variety. Genera Al-ternaria, Arthrinium, Cladosporium, Epicoccum, and Penicillium were detected in all samples at 9 months of storage.
The data in Table 2 also show that, from the barley grains stored in silo for a period of 3 to 9 months, 13 fungal genera and Mycelia sterilia were identified (428 isolates). Alternaria, Arthrinium, and Penicillium were the main components of the mycobiota from 3 samples (100% IF), followed by Epicoccum (89%), Cladosporium, Fusarium, and Rhizopus (78%, each). Alternaria was the most abundantly detected genus (163 isolates) with RD of 38%. The isolated Penicillium consisted of eight species. Penicillium brevicompactum and P. griseofulvum showed the highest IF (33%) and RD (13%) of all the isolates from this genus.
Analysis of the detected filamentous fungi contaminants of barley showed that the Laudis barley variety had slightly higher number of isolates (47) and of genera (9) in comparison with Kangoo or Wintmalt varieties (Table 3). Alternaria, Arthrinium, Cladosporium, Fusarium, and Penicillium were detected in all samples. The most abundant genus was Alternaria (39%), followed by Fusarium (15%), and Arthrinium (12%). During the survey, 7 isolates of 4 Penicillium species (P. crustosum, P. glabrum, P. griseofulvum, and P. chrysogenum) were isolated and identified. In the samples stored in floor warehouse, comparatively fewer filamentous fungi contaminants were detected than in the samples stored in grain storage silo. A total of 134 strains from 10 genera and Mycelia sterilia were identified at 3 months of storage (Table 4). A total of 89 isolates from 8 genera and Mycelia sterilia were isolated from samples stored for 6 months. Alternaria and Penicillium were detected in all samples. Alternaria was the most abundant genus (55%), especially in the Laudis variety. Compared with samples stored 6 months, a decrease of filamentous fungi detected was recorded at 9 months of storage in both types of storage. A total of 84 strains from 7 genera were identified from barley stored in floor warehouse for 9 months. Alternaria and Arthrinium were detected in all the samples examined. The occurrence of Penicillium spp. was generally low, but rich on species: P. griseofulvum, P. hordei, P. chrysogenum, and P. raistrickii. The lowest qualitative (4 genera) and quantitative (19 isolates) representation of microfungi was isolated from the Kangoo variety (Table 3). The most abundant genera were Alternaria and Arthrinium (42%, each).

Endogenous Mycobiota of Malt
Samples of barley grains fresh from the harvest and before storage, and of barley grains stored in silo for 3, 6, and 9 months were malted and these samples were examined for endogenous mycobiota by plating method with surface disinfection. The filamentous fungi identified in malt are indicated in Table 5, and their IF and RD are presented in Table 6. The total count of microscopic fungi isolated from grains malted directly after harvest was the highest (112) with 7 genera and Mycelia sterilia, compared to malts prepared from barley stored in silo. Alternaria, Epicoccum, and Fusarium were the dominant fungal genera detected in the control malt. Alternaria was the most abundant occurring genusthere, with RD of 45%, but their presence in the Laudis and Kangoo malt were lower compared to the non-malted barley grain samples. The second abundant genus was Epicoccum (17%), followed by Fusarium (16%). Interestingly, the Wintmalt variety had the highest number of isolates. We isolated toxicogenic Penicillium griseofulvum and P. chrysogenum from the Wintmalt variety only.  The endogenous mycobiome in malts prepared from barley stored for 3 months included 63 isolates from 8 genera and Mycelia sterilia. Alternaria was the most abundant occurring genus with 22 isolates and RD 35% of all the isolates found, followed by Mucor (32% RD). Their IF was the highest in the Wintmalt variety.
Isolation of fungi from malts from surface-sterilized barley grains stored for 6 months resulted in collecting of 94 fungal isolates from 8 genera and Mycelia sterilia. Alternaria, Cladosporium, Epicoccum, and Mucor were the predominant genera in all 3 samples of malt. Alternaria scored the highest RD (65%) and was the main component of Wintmalt and Laudis malt mycobiota.
The overall mycological colonization of 3 tested malts from barley stored for 9 months was the lowest. Within 54 isolates of filamentous fungi, the most abundant were Alternaria (35% RD) and Rhizopus (28% RD). Alternaria, Cladosporium, Epicoccum, Mucor, and Rhizopus were more frequently isolated from malts (67% IF), and the remaining 2 genera and Mycelia sterilia were found only in one sample of malt.
All the previous fungal genera except Absidia were also isolated from malted barley grain samples stored in floor warehouse (Table 7). Nevertheless, their relative abundances varied (Table 8). Isolates from malted barley stored in floor warehouse for 3 months included 93 strains from 7 genera of filamentous fungi and Mycelia sterilia with 100% isolation frequency for Alternaria and Mucor. Alternaria isolates predominated (55% RD), followed by Cladosporium (12% RD), Mucor, and Penicillium (10% RD, each). The incidence of Penicillium isolates revealed the occurrence of 4 different Penicillium species with maximum relative density for P. corylophilum (44% RD) of all the Penicillium isolates. Samples of malt prepared from barley grains stored for 6 months in floor warehouse had the lowest number (26) and genera (3) of filamentous fungi. The highest RD was again reached by the genus Alternaria (81%) which was present in all 3 samples. Microfungi in malt samples from barley grains stored in floor warehouse for 9 months included 73 isolates from 8 genera. The most common fungal genus was Epicoccum (100% IF) and the highest RD was reached by the genera Rhizopus (29%), followed by Alternaria and Penicillium (19% RD, each). Overall, 192 isolates from 10 genera and Mycelia sterillia were identified in malts prepared from barley stored in floor warehouse over a period of 3 to 9 months, very similar to the malt samples from grain stored in silo. The most frequent were Alternaria (78% IF), Epicoccum, Mucor (56% IF, each), Cladosporium, and Penicillium (44% IF, each). The most abundant genera found were Alternaria (45% RD), Penicillium, and Rhizopus (12% RD, each). From the 23 Penicillium isolates identified, the most frequent was P. corylophilum (22% IF) with the highest RD being 17% of all the Penicillium isolates.
Thirteen different genera and Mycelia sterilia were isolated from 3 varieties of freshly harvested barley grains before storage ( Table 9). The total filamentous fungal count on control grains before storage ranged from 3.6 × 10 2 to 3.6 × 10 3 CFU/g and the total yeast count from 1.3 × 10 3 to 9.1 × 10 4 CFU/g.   Mean values with the same letters in columns were not significantly different (α = 0.05). The results for the indicated storage conditions and individual varieties are marked with small letters. The results for the varieties in the entire storage period, regardless of the conditions storage used, are marked with capital letters. Analyzing the data presented in Table 9, it is concluded that after 3 months of storage in silo, the count of the field fungi, respectively species of Fusarium and Mucor, decreased, and the development of stored grain specific fungi such as Penicillium and Aspergillus was observed. Altogether, 3 species of Penicillium were isolated from examined samples: 2 of them from the Wintmalt barley variety-P. citrinum and P. griseofulvum, and P. aurantiogriseum from the Kangoo variety. The quantitative representation of filamentous microscopic fungi was lower compared to yeasts, but higher compared to control grains, except the Wintmalt variety.
Gradually, at the 6th month of storage, the grains mycobiota increased in incidence of the Penicillium species, due to storage conditions such as high atmospheric humidity. The barley grains associated mycobiota consisted of various species of fungi such as Alternaria, Arthrinium, Aspergillus, Cladosporium, Fusarium, Penicillium, Ulocladium, Mycelia sterilia, Candida, Issatchenkia, Rhodotorula, Sporobolomyces, and Wickerhamomyces belonging with predominance to the Penicillium, Aspergillus, and Rhodotorula genera. Yeasts from the analyzed samples were identified using MALDI-TOF MS Biotyper. Penicillium strains were isolated from all samples. The genus Penicillium was represented by eight species: P. aurantiogriseum, P. canescens, P. citrinum, P. glabrum, P. griseofulvum, P. polonicum, and P. raistrickii. At the 6th month of storage, the yeast abundance reached its maximum in all monitored varieties. The highest increase was observed in the Laudis variety with the yeast population ranging from 1.3 × 10 3 CFU/g from the freshly harvested barley grain samples to 2.4 × 10 7 CFU/g. A slight decrease compared to this maximum count in yeasts and filamentous fungi was observed at the 9th month of storage. Cladosporium was the predominant genus, followed by Alternaria, Arthrinium, Aspergillus, Rhizopus, and Rhodotorula. Penicillium was comparably less frequently isolated. Rhodotorula isolates occurred in the barley varieties Laudis and Kangoo and Candida and Sporobolomyces in the variety Wintmalt throughout the storage period in both types of storage.
The studies of barley grains stored in floor warehouse revealed that the initial yeasts count increased at 3rd and 6th months of storage and slightly decreased at 9th months of storage. At the 6th month of storage, the yeast abundance reached maximum in all monitored varieties, and in the 9th month decreased. The yeast population isolated from barley grains ranged from 1.3 × 10 3 CFU/g from freshly harvested samples to 1.1 × 10 6 CFU/g in the Wintmalt variety stored for 6 months. Samples were less contaminated with fungi. At the 3rd month of storage, the microfungi abundance reached maximum and included Alternaria, Arthrinium, Aspergillus, Cladosporium, Epicoccum, Fusarium, Penicillium, and Mycelia sterilia. In the Kangoo variety stored for 3 months, fewer filamentous fungi were detected than from the freshly harvested samples. Saprophytic fungi such as Arthrinium and Aspergillus, isolated from samples at 3 months of storage, were not detected at all in the samples at 6th and 9th months of storage, and in these samples, only yeasts were observed. We did not isolate any microfungi from malting barley of the Wintmalt variety after 9 months of storage. Strains of Penicillium forming a dominant part of fungal isolates from barley stored in silo were less often isolated from samples stored in floor warehouse, mainly in the Wintmalt variety.
Analysis of the samples of malted barley grain revealed that malting conditions were favorable for fungal growth (Table 10). Changes in the abundance of fungi in malted barley grain were evident. In the malt from freshly harvested grains, the total yeast count was from 2.2 × 10 3 to 4.5 × 10 3 CFU/g and filamentous fungi from 1.1 × 10 3 to 2.2 × 10 3 CFU/g. We detected 6 genera of filamentous fungi and 3 genera of ascomycetous yeasts and 2 genera of basidiomycetous yeasts from the malting trials. Aspergillus and Cladosporium were the dominant genera in the samples of barley after malting.
In this work, the initial fungal counts during storage gradually increased in malt prepared from barley at 3rd, 6th, and 9th month under both types of storage. Alternaria formed a dominant part of filamentous fungal isolates from malted barley grains stored for 9 months in silo, followed by Penicillium. At the end of the 9th month, Mucor dominated in malted barley grains stored in floor warehouse, where Alternaria was the dominant genus at 3 months of storage; at 6th month of storage, there were Alternaria, Penicillium, and Mucor and at 9th month of storage, only Penicillium dominated. Rhodotorula spp. formed a dominant part of yeast isolates in malt prepared from the Laudis and Kangoo varieties and Candida saitoana and Sporobolomyces roseus dominated in malt prepared from the Wintmalt variety throughout the whole storage period in both types of storage.

Toxigenicity of Penicillium Species
The toxigenic profile of the Penicillium isolates representing P. crustosum, P. expansum, P. griseofulvum, P. hordei, P. chrysogenum, and P. raistrickii from barley grains and malt is shown in Table 11. In total, 14 isolates representing 3 potentially toxigenic species were isolated from barley and tested for their toxigenic ability. Positive toxigenicity was detected in P. chrysogenum and P. raistrickii. Penicillium griseofulvum produced griseofulvin (G), cyclopiazonic acid (CPA), and patulin (P, 5 out of 6 isolates screened) but did not produce roquefortin C (RC). In total, 25 endogenous isolates representing 6 potentially toxigenic species from barley were tested for their toxigenic ability. Positive toxigenicity was detected in P. hordei and P. raistrickii. Penicillium crustosum produced penitrem A, but did not produce RC. Penicillium expansum produced P, G, but not RC. RC production was also tested in 8 isolates of P. griseofulvum, but it was not detected. On the other hand, Penicillium griseofulvum produced P (6 out of 8 isolates tested), G (7 out of 8 isolates), and CPA (7 out of 8 isolates). No toxigenicity was detected in P. chrysogenum isolated from malt or the endogenous mycobiota of malt. Penicillium griseofulvum from the endogenous mycobiota of malt produced CPA, G, and P (1 out of 2) and did not produce RC. Out of 47 isolates tested, 79% produced at least one mycotoxin as revealed by the method used here. *-number of isolates with ability to produce mycotoxins, **-number of tested isolates, P-patulin, Ggriseofulvin, RC-roquefortin C, CPA-cyclopiazonic acid, PA-penitrem A.

Discussion
Papadopoulou et al. [22] reported that the fungi found most frequently on barley include Fusarium, Aspergillus, Penicillium, Alternaria, and Cladosporium. All but Penicillium were isolated from our samples, with Aspergillus and Cladosporium with low incidence. The most frequently isolated fungal species on 43 malting barley samples from Italy belonged to the genera Alternaria and Fusarium [23]. The Fusarium species belongs to the most toxigenic fungi in the northern temperate regions [24]. Barley contaminated with these pathogenic fungi is negatively influenced during vegetation, can show reduced germination capacity and quality of malting grain, can show losses in yield, decrease in the quality of malt and beer, and result in non-safe products [20,25]. One known side effect of Fusarium fungi presence is gushing, with occasionally great economic losses for breweries from the over-foaming of beer. As such, one of the consequences of barley grain contamination can be the loss of customers [26]. Biliková and Hudec [27] found out that the use of corn as a pre-crop could increase the existence of Fusarium head blight of barley as well as in the manufactured beer. The production of harmful metabolitesmycotoxins that have great impact on human and animal health-poses an even greater risk [20]. Krasauskas [24] reported that some species of Alternaria spp. on barley grain samples were dominant before storage in Lithuania, which is in line with our results. Alternaria is one of the major mycotoxigenic fungal genera with more than 70 reported toxic metabolites. Alternaria is a fungal genus encompassing both saprophytic and pathogenic species, widespread in nature. The highest levels of Alternaria mycotoxin tenuazonic acid were detected in cereals, followed by beer; the highest levels of alternariol and alternariol methyl ether were found in lentils, oilseeds, tomatoes, carrots, juices, wines, and cereals [28]. Several other fungi were isolated from the Lithuanian samples of barley grain: Aspergillus niger, A. flavus, Cladosporium herbarum, Fusarium culmorum, F. poae, F. equiseti, and Rhizopus nigricans. These fungi were also isolated from our samples. Krasauskas [24] mentioned other microfungi not isolated in our experiment: Acremonium strictum, Botrytis cinerea, Drechslera sorokiniana, Mucor spp., Nigrospora sphaerica, Penicillium spp., Rhizoctonia spp., Stemphylium spp., Trichoderma spp., and Verticillium alboatrum.
Birck et al. [29] analyzed fungal contamination in wheat grain during storage for 180 days. Fusarium spp. was the most prevalent fungus after harvest as well as after 30 days of storage, and the counts decreased gradually until the end of the storage period. Aspergillus, Fusarium, and Penicillium were found in 96.7%, 46.7%, and 80.0% of wheat samples, respectively, after 180 days of storage. These results are similar to those that we accrued in our study. Fusarium genera were one of the most abundant genera after harvest, but their count gradually decreased. The genera Fusarium, Alternaria, Cladosporium, and Epicoccum belong to common fungi on cereal grains in the field [30]. The micromycetes of Fusarium spp. are described as "field fungi", but in some cases, they can grow during storage as well [20,31]. Alternaria was the most abundant occurring genus (38% RD) in our samples. Krnjaja et al. [32] focused on mycobiota of maize from Serbia. Mycological analyses found the presence of Aspergillus, Fusarium, and Penicillium on both freshly harvested and stored grains. The predominant species were different in each stage of processing. A similar observation was made for maize from various locations in Romania. The samples analyzed immediately after the harvest showed predominance species of Alternaria, followed by species of Cladosporium and Penicillium. The maize grains after 120 days of storage contained mycobiota composed of various fungi with predominance of Fusarium, Aspegillus, and Penicillum [33]. Fusarium does not survive on dry stored barley as long as Alternaria does; the spores of Alternaria may remain viable for a number of years [6]. This explains the fact that if barley is properly stored, mainly Alternaria species are detected.
Penicillium is characterized as xerophilic with a short time span of the asexual reproduction. Xerophilic species Penicillium corylophilum takes advantage of the possibility to disperse to open and available habitats where rapid asexual life cycle takes place. The competition advantage for P. corylophilum probably does not persist if the relative humidity increases. On the contrary, under a relatively higher humidity, probably more species combating for the same open niche could be found. The presence of these species poses did not confirm inhalation hazards and dermal inoculation hazard as well [34]. Penicillium corylophilum was the most frequent species (67% IF) isolated with high relative density (44%) of all the Penicillium isolates of endogenous colonization from malted barley grains stored for 3 months.
The total filamentous fungal count on control grains (freshly harvested, before storage) ranged from 3.6 × 10 2 to 3.6 × 10 3 CFU/g and the total yeast count from 1.3 × 10 3 to 9.1 × 10 4 CFU/g. The number of fungi propagules was comparatively lower, from 6.2 up to 9.0 CFU/g in barley grain before storage from Lithuania [24]. The malting barley grains collected from Southern Brazil also presented a low count of fungal colonies, with values ranging from 10.5 to 50 CFU/g (mean: 28 ± 25.5 CFU/g) [35]. In total, 9 fungal isolates were identified from 4 distinct genera: Fusarium (6), Penicillium (1), Alternaria (1), and Rhizopus (1). Climatic conditions (temperature and high humidity) in Southern Brazil may influence the contamination by F. graminearum and, consequently, maximize the production of deoxynivalenol. Samples colonized by Fusarium, especially F. graminearum and F. verticillioides, were predominant in the 19 isolates, with 13 belonging to F. graminearum and 6 belonging to F. verticillioides. The incidence of both species was 26% and 12%, respectively. There was high contamination of barley with this fungi found in other experiments [31,35], especially for the species F. graminearum. Their results are in line with our study and confirmed that Fusarium was detected from all 3 barley varieties analyzed. The majority (61%) of brewer's grain samples collected from a major Argentinean brewery and 83% of the malted barley (malt) had a total fungal count >1 × 10 4 CFU/g [36]. In our study, it was comparatively lower, with 48% of brewer's grain samples and 79% of the malt samples that had this count. Yeasts were isolated from all brewer's grain and malt samples [36], as in our study. Genera containing some of the most important toxicogenic species-Fusarium spp., Aspergillus spp., Penicillium spp., and Alternaria spp.-were isolated from the analyzed samples, along with other environmental saprophytic fungi such as Geotrichum spp., Mucorales, and Cladosporium spp. All mentioned fungal genera were isolated from our samples as well.
The main factor responsible for limiting the development of microscopic fungi is low humidity in grain. In the temperate climate zone, the majority of micromycetes recorded on grain are mesophiles. The most suitable temperature for their development is between 15 to 30 • C [12], although some micromycetes species are able to develop at temperatures below 0 • C (Cladosporium herbarum, Fusarium nivale, Fusarium avenaceum) [37]. Humidity of our grain was below 15% and the air temperature during storage fluctuated below 25 • C. The number of fungi propagules increased under the storage conditions and fluctuated from 8.3 up to 15.2 CFU/g [24]. The number of fungi propagules was comparatively higher than in barley ears before the harvest (the dust rising from the soil was carried into the tank of the combine harvester together with grain during the harvest). Fungi isolated from dried and stored barley grain in storage silo included Arthrobotrys oligospora, Aspergillus niger, A flavus, Aspergillus oryzae, Fusarium sporotrichioides, F. poae, Penicillium expansum, P. verrucosum, P. viridicatum, and Scopulariopsis brevicaulis. The dominant species on samples of barley grain from a storage silo were Aspergillus spp. and Scopulariopsis spp. [24]. Przybylska-Balcerek et al. [38] reported that the content of microscopic fungi in all 44 barley grain samples from cereal silo was very low from October to November and it ranged from 1.81 to 2.19 log CFU/g. Fungi from the genus Fusarium were identified among three most frequently found genera of microscopic fungi, which is in contrast to our results.
The initial yeasts count increased at the 3rd and 6th month of both types of storage and slightly decreased at the 9th month of storage. The quantitative representation of filamentous microscopic fungi was lower compared to yeasts. Some encapsulated yeasts were able to survive during the long-term storage of barley. On the other hand, the number of bacteria and filamentous fungi associated with kernels decreased [21,39].
The most important group on samples of barley after malting was genus Fusarium, which was quite frequent. Krasauskas [24] reported the intensive growth of Fusarium during the steeping, even when the barley from the storage silo initially had only a low level of Fusarium contamination. Geotrichum spp. and other yeasts, Mucor spp. and Fusarium spp., were dominant species on samples of barley after malting.
The most significant mycotoxins found in cereals are produced by Fusarium (trichothecenes, zearalenone, fumonisins), Aspergillus (aflatoxins, ochratoxin A), and Penicillium (ochratoxin A). The range of the mycotoxins present depends on the locality, weather, crop variety, as well as the growing year [40]. A recent multi-annual global survey [41] concluded that deoxynivalenol was the abundant mycotoxin in the barley samples collected from different countries, followed by zearalenone and T-2/HT-2 toxins. The presence of the potentially toxigenic fungus Aspergillus was very low (<1%). The genus Fusarium occurred mainly in the field, and we recorded its decreased frequency during storage. The occurrence of the genus Penicillium was the highest in warehouses, but its RD did not exceed 7%. The mycotoxin levels declined during malting; however, their production can increase during germination as the warm, humid environment of a malthouse is a suitable environment for mold growth. The further mycotoxin production is possible during kilning. High temperature can cause the elimination of fungal growth, but an increase in temperature during initial stages of kilning can stimulate an increased mycotoxin production in some fungal species [19]. We observed an increase only in the genus Penicillium in malt during the storage of barley. Ochratoxin A (OTA) is one of the most important mycotoxin in cereals. There was no isolated Penicillium verrucosum, as a potential producer of OTA. Our results showed that the Penicillium species commonly isolated from barley and malt could be a potential source of the mycotoxins patulin, griseofulvin, and cyclopiazonic acid under in vitro conditions.

Samples
Contamination of barley grains with micromycetes during different periods of storage was investigated from 2018 to 2019. Grain samples of barley were taken for mycological analysis both directly from the fields of the farmers who grew it for malt producers and from the grains stored in floor warehouse and grain silo in Maltery Heineken Slovensko Sladovne in Hurbanovo, Slovak Republic.
Silos-Each silo consists of several cells; in our case, it was 21 hexagonal square chambers with a capacity of 1000 tons and with a conical bottom (45 • ) to facilitate emptying. Each cell in the silo was equipped with 10 thermometers placed along its entire height. The dedicated program then calculated an average value from 10 recorded temperatures. The program works on the principle of a traffic light controller and according to the value of the recorded average temperature, it is marked either in green (suitable temperature up to 20 • C), yellow (20 to 25 • C), or red (above 25 • C), in which case it is necessary to intervene. In the event of deteriorating conditions or as part of regular relocations, every 2 to 3 months, transfer of the stored grain to another free cell is performed. The emptied cell is then cleaned and gassed before refilling to limit the multiplication of microbial contaminants and pests.
Floor warehouse-The grain was stored on the floor in layers of 4 to 5 m, with temperatures 15 to 22 • C. The storage facility was built of concrete halls, usually with recessed grate ducts enabling air distribution during active ventilation. This type of storage has a system of perforated pipes placed on the floor, which use fans to regulate the aeration in order to remove heat, carbon dioxide, and thus reduce humidity. Parts with different humidity should not be mixed during storage, because in places with higher humidity, a quicker heat formation might occur accompanied with material losses and also with possible microbial contamination.
Barley samples (15 kg each) were placed in perforated bags that were stored in grain silo cells, along with other stored barley. With this procedure, we were able to imitate the process of mass grain storage. Once every 3 months (until the 9th month), we removed these bags and took out the required amount of barley for analysis (3 kg). Then, we returned the bags back into the cells. Temperatures in floor warehouse during the storage were in the range 15 to 22 • C and in the silos from 15 to 25 • C. Humidity of grain was below 15%. The study was conducted using 3 types of malting barley Laudis and Kangoo (two-row spring malting barley variety), grown in the Levice region, and Wintmalt (two-row winter malting barley variety) grown in the Zlaté Moravce region in Slovak Republic. Samples were sent to the laboratory and 1 kg from each sample was malted.

Micro-Malting Procedure
Malt processing was carried out in the center of AgroBioTech in Slovak University of Agriculture in Nitra, Slovakia. Samples (1 kg each) of barley grain were malted in laboratory micromalting plant 4 times, from freshly harvested barley (0 weeks of storage), 3 months, 6 months, and 9 months after start of the storage trial.
Steeping-1st day water for 5 h, 2nd day water for 4 h, full steeped on water content 45.5%. Between the two water phases, air rests were performed, where barley samples were aerated with fresh air in the steeping box.
Germination was performed at a malt temperature of 15 to 16 • C and proceeded at continuous aerating with fresh conditioned air. Total time of steeping and germination was six days.
The kilning process was performed on an electrically heated one-floor kiln, with a gentle and gradual increase in temperature up to 80 • C for 4 h.

Mycological Analysis of Barley and Malt
A total of 50 grains from each sample were surface disinfected in 1% NaClO for 1 min according methods of Magnoli et al. [42] and rinsed 3 times by submersion in sterile distilled water (total amount 1 L), dried, plated in DRBC (Dichloran Rose Bengal Chloramphenicol agar medium; MERCK, Germany), and incubated at 25 ± 1 • C in the dark for 7 days. Endogenous mycobiota was determined in this way.
Dilution plating method (surface-spread method) was used for colony counting. Ten grams of each grain sample were individually homogenized in 90 mL of physiological saline solution (0.89%, MERCK, Germany) for 30 min. Serial decimal dilutions up to 10 −5 were made and 0.1 mL were inoculated in triplicates onto Petri dishes with Dichloran Rose-Bengal Chloramphenicol (DRBC) medium. The dishes were incubated in the dark in thermostat (BT 120, Praha, Czech Republic) for 7 days, at the temperature of 25 ± 1 • C.
The results were expressed as isolation frequency of the fungal genera (IF; defined as the percentage of samples in which each genus or species was present in relation to the total number of samples) and relative density (RD; the relative percentage of isolates of each genus or species, occurring in the analyzed sample) [46]. These values were calculated according to González et al. [47] as follows: IF (%) = (ns/N) × 100 RD (%) = (ni/Ni) × 100 ns-number of samples with a given species or genus; N-total number of samples; ninumber of isolates of a given species or genus; Ni-total number of isolated fungi.

Mycotoxins Detection
Toxigenicity assays of selected isolates were performed under in vitro conditions using thin layer chromatography (TLC) [48], modified by Labuda and Tančinová [49]-Griseofulvin and patulin assays were carried out on YES agar as extracellular metabolites and roquefortin C, cyclopiazonic acid, and penitrem A on CYA agar as intracellular metabolites. A few pieces of mycelium with approximate size 5 × 5 mm were cut from colonies and placed in an Eppendorf tube with 500 µL of chloroform:methanol (2:1; v/v; Reachem, Bratislava-Petržalka, Slovak Republic). The content of the tubes was stirred for 5 min by Vortex G-560E (Scientific Industries, Bohemia, NY, USA). The extracts were screened for mycotoxins contamination by applying small spots of 30 µL of each extract and 10 µL standard solutions (Sigma, Taufkirchen, Germany) on a silica gel 60 TLC aluminum sheet (20 × 20 cm; Alugram ® SIL G, Macherey-Nagel, Düren, Germany) and developed with TEF solvent mobile phase (toluene:ethyl acetate:formic acid, 5:4:1 v/v/v; toluene-Mikrochem, Pezinok, Slovak Republic; ethyl acetate and formic acid-Slavus, Bratislava, Slovak Republic). Thereafter, the plate was air-dried. Roquefortin C was visible after spraying with Ce(SO 4 ) 2 × 4 H 2 O as an orange spot, patulin by spraying with 0.5% methyl-benzothiazolone hydrochloride (MBTH), (Merck, Darmstadt, Germany) in methanol and heating at 130 • C for 8 min as a yellow-orange spot. Cyclopiazonic acid was visible directly in daylight after spraying with the Ehrlich reagent as a violet-tailed spot. Penitrem A after spraying with 20% AlCl 3 in 60% ethanol and heating at 130 • C for 8 min as a dark blue spot. Griseofulvin was visualized directly under UV light (Krüss Optronic, Hamburg, Germany) with a wavelength of 365 nm as a blue spot.

Mass Spectrometry Identification of Yeast Isolates
Qualitative determination of yeasts was carried out using the MALDI-TOF Mass Spectrometry device (Bruker Daltonics, Bremen, Germany). The isolates of yeasts were subcultured on Tryptone soya agar (TSA, Oxoid, Basingstoke, UK) at 30 • C for 24 h. The criteria for reliable identification were a score of ≥2.0 at the species level [50]. Isolates were put in 300 µL of distilled water and 900 µL of ethanol, and the suspension centrifuged for 2 min at 14,000 rpm. The pellet was centrifuged repeatedly and allowed to dry. Then, 30 µL of 70% formic acid and 30 µL of acetonitrile were added to the pellet. Tubes were centrifuged for 2 min at 14,000 rpm and 1 µL of the supernatant was used for MALDI identification. Once dry, every spot was overlaid with 1 µL of an HCCA (α-Cyano-4-hydroxycinnamic acid) matrix and left to dry at room temperature before analysis. Generated spectra were analyzed on a MALDI-TOF Microflex LT (Bruker Daltonics, Bremen, Germany) instrument using Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC-specific software.

Statistical Analysis
For every test, three independent repeated measurements were done, unless stated otherwise. All measurements were compared using one-way analysis of variance independently for each dependent variable. Post-hoc Tukey HSD (honest significant difference) multiple comparison tests were used to identify statistically homogeneous subsets at α = 0.05. Statistical analysis of the data was performed with the Statistica 13 (Dell Software Inc., Round Rock, TX, USA) software.

Conclusions
The present study contributes to the knowledge of mycobiota in Slovakian malting barley and malt, and to the presence of toxigenic fungi during the storage period. Based on the obtained results, from the potentially toxigenic fungi Alternaria, Aspergillus, Fusarium, and Penicillium, only the field fungi Alternaria and Fusarium were present in the harvested barley grain samples and all of them were present in stored grain, with the highest incident for Alternaria, followed by Penicillium, Fusarium, and Aspergillus. This study also provided a clear indication of the yeast diversity in the malt ecosystem. In this work, Rhodotorula spp. was the most prevalent genus isolated from pre-and post-stored malt samples. The quantity of fungi in grain conversion technology from the storage to the malting process was increasing. Species of the genus Penicillium had higher incidence in stored samples compared to malt. This study also provides new insights into the diversity of Penicillium species isolated from Slovakian barley: P. crustosum, P. expansum, P. griseofulvum, P. hordei, P. chrysogenum, and P. raistrickii, with the predominant species Penicillium griseofulvum, were all detected. Fungi were isolated from healthy grain up to 9 months of storage. However, the number of samples from which the fungi were isolated generally decreased with increasing storage time. For this reason, constant supervision and monitoring of potential toxigenic fungi or mycotoxins occurrence in barley and malt pre-and post-harvest, as well as application of preventive measures are very important to reduce the related risks to consumer health.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.