Adam Behner

The Pulsed Electric Field (PEF) technique represents a modern technology for treating and processing food and agricultural raw materials. The application of high-voltage electric pulses has been shown to modify macrostructure, improve extractability, and enhance the microbial safety of the treated matrix. In this study, we investigated metabolomic changes occurring during the individual technological steps of malting following PEF treatment. Methanolic extracts of technological intermediates of malting barley were analyzed using metabolomic fingerprinting performed with UHPLC-HRMS/MS. For data processing and interpretation, the freely available MS-DIAL—MS-CleanR—MS-Finder software platform was used. The metabolomes of the treated and untreated barley samples revealed significant changes. Tentatively identified PEF-related biomarkers included 1,2-diacylglycerol-3-phosphates, triacylglycerols, linoleic acids and their derivatives, octadecanoids, N-acylserotonins, and very long-chain fatty acids, and probably reflect abiotic stress response. Monitoring of the profiles of selected biomarkers in PEF malting batch indirectly revealed a potential enhancement of enzymatic activity after the PEF treatment. These results contribute to fundamental knowledge regarding the influence of PEF on final malt from a metabolomic perspective.

Pulsed electric field (PEF) has previously been recognized as a method of gentle food processing, and its use has been shown to be helpful in reducing the levels of toxigenic Fusarium micromycetes developed during malting. The aim of this study was to describe the effects of PEF on gene expression and metabolite production at the pre-finishing stage of barley malting by using a novel multi-omics data-driven approach. The study helps to uncover the processes occurring in the germinated grain and discusses the up-/downregulation of genes and metabolites in relation to fungal infection and/or PEF-induced abiotic stress. Among the factors upregulated by PEF and previously described as supportive against Fusarium diseases, we identified the increased expression of genes encoding vegetative gp1-like protein, which positively correlated with flavonoids, (methylsulfanyl)prop-2-enoates, triterpenoid glycosides, and indole alkaloids. On the other hand, some genes associated with barley resistance to fungal infection were also overexpressed in the untreated control (in particular, genes encoding ethylene response factor 3-like, putrescine hydroxycinnamoyltransferase 3-like, and dirigent protein 21-like). This study provides the first ‘data-driven’ basic research results that contribute to the understanding of the role of PEF as an effective fungal decontamination strategy and allows the formulation of new hypotheses related to Fusarium pathogen crosstalk.

Fusarium fungi are widespread pathogens of food crops, primarily associated with the formation of mycotoxins. Therefore, effective mitigation strategies for these toxicogenic microorganisms are required. In this study, the potential of pulsed electric field (PEF) as an advanced technology of increasing use in the food processing industry was investigated to minimize the viability of Fusarium pathogens and to characterize the PEF-induced changes at the metabolomic level. Spores of four Fusarium species (Fusarium culmorum, Fusarium graminearum, Fusarium poae, and Fusarium sporotrichioides) were treated with PEF and cultured on potato dextrose agar (PDA) plates. The viability of the Fusarium species was assessed by counting the colony-forming units, and changes in the mycotoxin content and metabolomic fingerprints were evaluated by using UHPLC-HRMS/MS instrumental analysis. For metabolomic data processing and compound identification, the MS-DIAL (v. 4.80)–MS-CleanR–MS-Finder (v. 3.52) software platform was used. As we found out, both fungal viability and the ability to produce mycotoxins significantly decreased after the PEF treatment for all of the species tested. The metabolomes of the treated and untreated fungi showed statistically significant differences, and PEF-associated biomarkers from the classes oxidized fatty acid derivatives, cyclic hexapeptides, macrolides, pyranocoumarins, carbazoles, and guanidines were identified.

The increasing contamination of cereals by micromycetes and mycotoxins during malting still poses an unresolved food safety problem. This study characterises the potential of the novel, rapidly developing food production technology of Pulsed Electric Field (PEF) to reduce the viability of Fusarium fungi and the production of mycotoxins during malting. Barley, artificially inoculated with four Fusarium species, was treated by PEF with two different intensities and then malted using a standard Pilsner-type technology. Concentrations of fungi were quantified by RT-PCR, expression of fungal growth-related genes was assessed using mRNA sequencing, and mycotoxin levels were analysed by U-HPLC-HRMS/MS. Despite the different trends for micromycetes and mycotoxins after application of variously intense PEF conditions, significant reductions were generally observed. The greatest decrease was for F. sporotrichioides and F. poae, where up to six fold lower levels were achieved for malts produced from the PEF-treated barley when compared to the control. For F. culmorum and F. graminearum, up to a two-fold reduction in the PEF-generated malts was observed. These reductions mostly correlated with a decrease in relevant mycotoxins, specifically type A trichothecenes.

Pulsed electric field (PEF) has previously been recognized as a method of gentle food processing and its use has been shown to be helpful in reducing the levels of toxigenic Fusarium micromycetes developed during malting. The aim of this study was to describe the effect of PEF on gene expression and metabolite production at the pre-finishing stage of barley malting by using a novel multi-omics data-driven approach. The study helps to uncover the processes occurring in the germinated grain, and discusses the up-/downregulation of genes and metabolites in relation with fungal infection and/or PEF-induced abiotic stress. Among the factors upregulated by PEF and previously described as supportive against Fusarium diseases, we identified increased expression of gene encoding vegetative gp1-like protein which positively correlated with flavonoids, (methylsulfanyl)prop-2-enoates, triterpenoid glycosides and indole alkaloids. On the other hand, some genes associated with barley resistance to fungal infection were also overexpressed in the untreated control (in particular genes encoding ethylene response factor 3-like, putrescine hydroxycinnamoyltransferase 3-like and dirigent protein 21-like). The study provides the first 'data-driven' basic research results that contribute to the understanding of the role of PEF as an effective fungal decontamination strategy, and allows the formulation of new hypotheses related to Fusarium-pathogen cross-talk.

Figure: Graphical abstract Although anammox-based processes have been widely applied in mesophilic conditions of reject water and recently in mainstream conditions, the potential of their implementation in high-temperature wastewaters remains largely unexplored. Therefore, this study investigated the operation parameters for the successful adaptation of anammox bacteria and the mechanisms involved on the proteomic and cellular level including unique ladderane lipids. For this purpose, the enrichment of ‘ Candidatus Brocadia’ was cultivated in two fed-batch reactors (FBRs) at a lab scale. The temperature of one FBR was gradually increased from 30 to 40 °C while the other FBR was maintained at 30 °C with four consecutive replicates of this experiment. For this adaptation to be successful, the original loading rate had to be at least halved, or ideally maintained below half the value of the specific anammox activity at the time. The most notable adaptation mechanisms included: (1) upregulation of chaperones and (2) doubled ladderane cyclization via the replacement of non-ladderane fatty acid by a ladderane fatty acid in ladderane lipids (p-value 0.005). To our best knowledge, this is the first study to describe the novel mechanism of ladderane cyclization which together with other adaptation strategies presents crucial indicators in anammox adaptation to high-temperature wastewaters.

Fungal endophytes occurring in grapevine (Vitis vinifera L.) are usually important sources of various compounds with biological activities with great potential for use in agriculture. Nevertheless, many species isolated from this plant belong to the genera Fusarium, Alternaria, or Aspergillus, all of which are well-known to produce mycotoxins. Our study is focused on the assessment of the toxinogenic potential of fungal endophytes isolated from vineyards in the Czech Republic. In total, 20 endophytic fungal species were cultivated in wine must, and 57 mycotoxins of different classes were analysed by liquid chromatography coupled with mass spectrometry. As a result, alternariol, tentoxin, meleagrin, roquefortine C, gliotoxin, and verruculogen were detected in the culture medium, of which verruculogen followed by gliotoxin were the most frequent (present in 90 and 40% of samples, respectively) and most concentrated (up to thousands ng/mL). The alternaria mycotoxins alternariol and tentoxin were detected not only in Alternaria sp. cultures, but traces of these mycotoxins were also quantified in the Diatripe and Epicoccum cultures. Meleagrin and roquefortine C were detected in Didymella sancta and Penicillium crustosum, gliotoxin was detected in Alternaria sp., Didymella sp., Aureobasidium pullulans, Cladosporium herbarum, Penicillium crustosum and Pleurophoma ossicola, and verruculogen was quantified in 99% of endophytic isolates investigated. The potential of endophytes to produce mycotoxins should be carefully checked, specifically in cases where they are intended for the purpose of V. vinifera growing.