genus includes plant pathogens which are of great concern to agricultural production and food/feed safety worldwide [1
], threatening yield and quality of cereals and producing mycotoxins, secondary metabolites that are toxic to humans and other animals [2
genus is capable of producing several mycotoxins, including fumonisins, trichothecenes and zearalenone. These are the most economically significant Fusarium
mycotoxins and are of particular concern in barley, maize and wheat [3
Trichothecenes have been classified into four groups: types A–D, according to their chemical structure [4
], the most important in cereals are types A and B [1
]. The B-trichothecenes include the mycotoxins deoxynivalenol (DON), its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) and nivalenol (NIV). DON is the most frequent type-B trichothecene and can be found worldwide. Furthermore, DON inhibits protein synthesis and it has been associated with intoxication of animals through consumption of contaminated feed [5
Zearalenone (ZEA) is a mycotoxin with estrogenic effects produced by several Fusarium
species and is usually found in cereal grains. Swine are especially sensitive to the estrogenic effects of ZEA. This toxin has been shown to bind to the estrogenic receptors and to inhibit ovulation. It is, therefore, often involved in hormonal disorders of farm animals and it is also related to hypoestrogenic syndromes in humans [6
]. ZEA has been classified into group 3 (non-classifiable due to its carcinogenicity to humans) by IARC (International Agency for Research on Cancer) [7
During recent years, mycotoxins have attracted international attention not only for their perceived impact on human health but also because of the economic losses accruing from contaminated foods. Considering barley matrix, several international studies have reported on Fusarium
and its mycotoxins contamination due to the beer gaining increased popularity [8
]. The main problem is the characteristics that these compounds have. Some mycotoxins such as trichothecenes, zearalenone and fumonisins are considered stable during the brewing process [9
] and can persist to the final product, the beer. Furthermore, Fusarium
infection causes a negative impact on barley germination rates which results in malting quality and yield reduction. Additionally, this produces gushing and changes in color and flavor of the beer [10
Barley is considered to have good characteristics for fungi contamination under favorable conditions. In addition, environmental factors associated with barley varieties and agronomic practices influence the Fusarium
infection and the mycotoxin production. The climate conditions during critical phases of barley plant growth could lead to noticeable difference between the mycotoxin diversity. Tropical conditions, such as those found in Brazil contribute to fungi dissemination and consequently mycotoxin production in barley. For example, 2015 demonstrated a high rainfall average and high levels of humidity, which prompted worse contamination levels found until now [11
]. It is necessary to mention that only two Fusarium
mycotoxins were analyzed. Other fungi mycotoxins should be evaluated along with their masked toxins.
All of the barley harvested in Brazil is destined for the brewing industry and the production continues to increase. Southern Brazil has the largest number of barley-producing regions, therefore, the quality of the grains needs to be monitored and studies about the fungi profile should be taken into account. The knowledge of the current contamination of barley in the region, as well as the constant monitoring, is necessary in order to evaluate if agronomic practices are being duly effective to control the contamination in grain production. The irrigation management, resistant cultivars, harvesting strategies, chemical and biological control and disease forecasting could minimize the loss of grain quality and avoid the disease caused by mycotoxigenic fungi [12
The current occurrence of mycotoxins in barley could lead to the necessity of developing new strategies or improving those currently in place for more effective management of mycotoxins in the future. This fact is even more relevant when taking into account that the Brazilian regulation for DON in barley will be updated by 2019 [13
] and therefore, the information on the occurrence of this toxin is still discussed in the country often.
The regulations have set maximum levels for mycotoxin contamination in grains, in order to avoid further accumulation of mycotoxins in processed food and to control mycotoxin of major concern in unprocessed cereals, such as brewing barley, with 1.250 µg/kg being the maximum permitted for DON and 100 µg/kg for ZEA. From January 2019, DON limits for brewing barley will be set at 1000 µg/kg [13
]. Similarly, the limits for DON and ZEA fixed by the European Commission [14
] are equal to 1.250 µg/kg and 100 µg/kg for unprocessed cereals.
For the reasons stated above, the aim of the present research was to characterize the Fusarium isolates and to assess DON and ZEA contamination in brewing barley grains. These findings provide new insights into the diversity of Fusarium species isolated from Brazilian barley and add information to the mycotoxin profile in a source (raw material) destined for the food industry.
This study has shown that the majority of the Fusarium species isolated from brewing barley grains belonged to the FSAMSC, whereas the main trichothecene producing species are clustered. Mycotoxins analysis demonstrated that most of the samples were contaminated with DON (90.6%) and ZEA (87.5%), highlighting the importance of this investigation.
species are found in cereal grains, such as barley, wheat, maize and rice worldwide, where mycotoxins can be found in high concentrations [15
]. This fact, may be worsened by weather conditions, such as high humidity and temperatures that tend to increase Fusarium
infection in plants [19
]. Humidity is an important environmental factor and it influences the water activity of the grains. This intrinsic factor is important for fungal growth and has considerable association with mycotoxin production [21
development as well as DON and ZEA production can be seen with higher levels of water activity (0.90) [22
]. Nevertheless, the current research showed a low variation of the water activity that was observed among the 64 brewing barley samples, with mean value of 0.622 ± 0.02. In this case, both the germination of fungal spores and the growth of storage fungi are inhibited. However, the grain analyses were carried out after the cleaning and drying stages, explaining the levels found. For safe storage of grain, the grain moisture content must be compatible with the period of time the grain will be stored in order to avoid the fungi growth and therefore, the water activity should be less. To associate Fusarium
mycotoxins found in this study it is necessary to assume that the DON and ZEA production could be correlated to the fungi presence and high water activity in the growing plant stages. This fact could be explained in our recent study with rice grains, where the levels of ZEA found are associated with the presence of Fusarium
during pre-harvest, in grains freshly harvested with high levels of moisture content and water activity. After the food processing steps were completed in the industry, there was not any Fusarium
growth in the grains, however, the ZEA levels remained in the parboiled rice (water activity: 0.64 ± 0.02), resisting the degradation [23
The phylogenetic study showed that the majority of the strains clustered with F. graminearum
, F. poae
and F. avenaceum
. Fusarium poae
can produce high levels of nivalenol; therefore, further analyses should be done to investigate the degree of nivalenol contamination in Brazilian barley. Previous studies carried out by [24
] in Russia and [25
] in Italy have shown a high incidence of F. avenaceum
in barley as well as other Fusarium
] observed a high incidence of F. avenaceum
, F. graminearum
and F. culmorum
in in Finnish barley grains between the years of 2005–14, with high levels of trichothecene contamination. The presence of F. avenaceum
in barley may indicate the presence of enniatins, moniliformin and beauvericin in the samples. In Brazil, another study in barley has demonstrated that the majority of the isolates belonged to the F. graminearum
lineage; however, mycotoxin analysis was not performed in that study [27
]. To our knowledge, this is the first report of phylogenetic identification of the Fusarium
species in Brazilian barley and the first correlation with DON and ZEA contamination.
With respect to DON contamination, the current study showed similarities with those found in a survey carried out in Spain with a mean level of 119.9 µg/kg [28
]. In addition, the highest results were obtained by [25
] reporting incidence and a maximum contamination level of 108.7 µg/kg. Also, [29
] in Italy, a study reported lower incidence and concentrations of DON with a maximum level of 35.5 µg/kg; and [30
] in Tunisia evaluated its presence with a maximum level of 6.1 µg/kg. In the current study, only one sample presented a high level of DON (1155.21 µg/kg) and was above the established maximum level set by the Brazilian regulation. The samples showed mean values of 147.65 µg/kg and median values of 98.68 µg/kg that also demonstrated low levels.
In contrast, the ZEA contamination found in the present study were of significance, considering the maximum levels established by the Brazilian and international regulation, where 86% of the samples were above [13
]. The samples showed mean values of 123.24 µg/kg and median values of 119.26 µg/kg. In a study performed by [32
] in the Czech Republic, a few samples were contaminated with ZEA with values ranging from 181.2 to 204.4 µg/kg, which is quite similar to the results of our study. Furthermore, high levels (max 985.9 µg/kg) of ZEA were found in another survey carried out in the Czech Republic with samples from the 2011 crop [33
Two other studies were conducted in Brazil by the current author and can be compared to this study. The first one was from the 2014 crop and low levels of DON were found ranging from 200 µg/kg to 15.000 µg/kg [34
]. The other one was from the 2015 crop and higher levels were found for both toxins, DON and ZEA. The last study was considered an issue for the industry due to the levels exceeding the regulation levels established. The mean levels ranged from 1700 to 7500 µg/kg and from 300 to 630 µg/kg for DON and ZEA, respectively.
It is necessary to mention that not only the large-scale brewing industry is increasing but that the craft breweries in Brazil are expanding. These small brewing groups always look for the best sources and have the characteristic of “German beer purity law,” meaning that just barley is used for beer production. For these reasons, barley needs to be of higher quality. On the other hand, the large-scale industry in Brazil uses other grains such as corn, rice and sorghum for beer production which are considered low quality grains [35
]. Some studies were carried out and they showed contamination with fumonisin B1
] that is commonly found in corn and its derivatives [37
In the last years the stability of these metabolites has been studied. Deoxynivalenol showed to be a mycotoxin that persists through the process and demonstrated stability in some industry processes, such as cleaning, milling, brewing and extrusion [9
]. Zearalenone has had some studies published about it which showed lower stability. However, its levels are high when they are found. A research conducted by [38
] showed a considerable reduction of ZEA levels in the presence of Saccharomyces cerevisiae
yeast. The main point is even if there is a significant reduction of these metabolites, sometimes they still offer a risk, especially when in beer, which is considered one of the most consumed beverages in the world.
Another aspect that should be taken into account is the masked toxins. ZEN-14-sulfate and DON-3-glucoside are most commonly observed in grains. They could be present in the matrix, however masked toxins are either bound to carbohydrates or proteins and, therefore, are not extractable with existing protocols aimed at the extraction of the toxin, or they are not detectable using established chromatography routines; hence their name “masked” mycotoxins [39
]. Further studies are being planned to gain more knowledge on these metabolites in barley and also in beer.
mycotoxins found in barley grains and the toxigenic potential analysis of the F. graminearum
strains isolated in this study reinforce the importance of these genera in this relevant commodity. Furthermore, the identification of F. poae
, F. avenaceum
and the genus Alternaria
in barley samples highlights the importance of further research on other mycotoxins in barley and its by-products. Alternaria
species were recovered from 28% of the barley samples. This may have important implications on other mycotoxins that may be found in high concentrations, such as tenuazonic acid. Further studies should be conducted in order to evaluate the co-occurrence of the mycotoxins produced by this genus [25
In this study, the toxigenic potential of the F. graminearum
strains isolated in the brewing barley was evaluated and both toxins detected in the samples were produced by them. In total, 80% and 20% of the strains produced DON (mean: 297.02 µg/kg and median: 268.74 µg/kg) and ZEA (mean: 79.7 µg/kg and median: 64.86 µg/kg), respectively. The toxins levels found were lower when compared to that of a study performed by Wu et al. (2017), in which the strains exhibited a production of 1405.05 µg/kg for DON and 4118.31 µg/kg for ZEA. The parameter temperature is crucial for DON production and some studies showed a variation of the optimal value, that varies between 20 and 28 °C [19
]. On the other hand, temperature negatively affected ZEA production. The optimized condition for ZEA production was cultivation at 15 °C [41
]. The toxin combination can be related to several mycotoxigenic fungi that contaminate barley in the field. However, the mycotoxin occurrence in Brazilian barley suggests high prevalence of toxigenic F. graminearum
and related species, which could explain the DON and ZEA levels in almost 85% of the samples.