Lyophilized alginate-based microspheres containing Lactobacillus fermentum D12, an exopolysaccharides producer, contribute to the strain's functionality in vitro.

Lactobacillus (Limosilactobacillus) fermentum D12 is an exopolysaccharide (EPS) producing strain whose genome contains a putative eps operon. Whole-genome analysis of D12 was performed to disclose the essential genes correlated with activation of precursor molecules, elongation and export of the polysaccharide chain, and regulation of EPS synthesis. These included the genes required for EPS biosynthesis such as epsA, B, C, D and E, also gt, wzx, and wzy and those involved in the activation of the precursor molecules galE, galT and galU. Both the biosynthesis and export mechanism of EPS were proposed based on functional annotation. When grown on MRS broth with an additional 2% w/v glucose, L. fermentum D12 secreted up to 200 mg/L of a mixture of EPSs, whose porous structure was visualized by scanning electron microscopy (SEM). Structural information obtained by 1HNMR spectroscopy together with composition and linkage analyses, suggested the presence of at least two different EPSs, a branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, and glycogen. Since recent reports showed that polysaccharides facilitate the probiotic-host interactions, we at first sought to evaluate the functional potential of L. fermentum D12. Strain D12 survived simulated gastrointestinal tract (GIT) conditions, exhibited antibacterial activity against enteropathogenic bacteria, adhered to Caco-2 cells in vitro, and as such showed potential for in vivo functionality. The EPS crude extract positively influenced D12 strain capacity to survive during freeze-drying and to adhere to extracellular matrix (ECM) proteins but did not interfere Caco-2 and mucin adherence when added at concentrations of 0.2, 0.5, and 1.0 mg/mL. Since the viable bacterial count of free D12 cells was 3 logarithmic units lower after the exposure to simulated GIT conditions than the initial count, the bacterial cells had been loaded into alginate for viability improvement. Microspheres of D12 cells, which were previously analyzed at SEM, significantly influenced their survival during freeze-drying and in simulated GIT conditions. Furthermore, the addition of the prebiotic substrates mannitol and lactulose improved the viability of L. fermentum D12 in freeze-dried alginate microspheres during 1-year storage at 4 °C compared to the control.

Properties and Fermentation Activity of Industrial Yeasts Saccharomyces cerevisiae, S. uvarum, Candida utilis and Kluyveromyces marxianus Exposed to AFB1, OTA and ZEA.

In this paper the effect of aflatoxin B1, ochratoxin A and zearalenon on morphology, growth parameters and metabolic activity of yeasts Saccharomyces cerevisiae, Saccharomyces uvarum, Candida utilis and Kluyveromyces marxianus was determined. The results showed that the three mycotoxins affected the morphology of all these yeasts, primarily the cell diameter, but not their final cell count. Fourier transform infrared spectroscopy showed that the yeast membranes bound the mycotoxins, C. utilis in particular. The cell membranes of most yeasts underwent denaturation, except S. uvarum exposed to ochratoxin A and zearalenone. In the early stage of fermentation, all mycotoxin-exposed yeasts had lower metabolic activity and biomass growth than controls, but fermentation products and biomass concentrations reached the control levels by the end of the fermentation, except for C. utilis exposed to 20 µg/mL of zearalenone. The adaptive response to mycotoxins suggests that certain yeasts could be used to control mycotoxin concentrations in the production of fermented food and beverages.

Biocontrol of Occurrence Ochratoxin A in Wine: A Review.

Viticulture has been an important economic sector for centuries. In recent decades, global wine production has fluctuated between 250 and almost 300 million hectoliters, and in 2022, the value of wine exports reached EUR 37.6 billion. Climate change and the associated higher temperatures could favor the occurrence of ochratoxin A (OTA) in wine. OTA is a mycotoxin produced by some species of the genera Aspergillus and Penicillium and has nephrotoxic, immunotoxic, teratogenic, hepatotoxic, and carcinogenic effects on animals and humans. The presence of this toxin in wine is related to the type of wine-red wines are more frequently contaminated with OTA-and the geographical location of the vineyard. In Europe, the lower the latitude, the greater the risk of OTA contamination in wine. However, climate change could increase the risk of OTA contamination in wine in other regions. Due to their toxic effects, the development of effective and environmentally friendly methods to prevent, decontaminate, and degrade OTA is essential. This review summarises the available research on biological aspects of OTA prevention, removal, and degradation.

The Effect of Mushroom Culture Filtrates on the Inhibition of Mycotoxins Produced by Aspergillus flavus and Aspergillus carbonarius.

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B1 (AFB1), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB1 or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB1 in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB1 by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan®, by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis.

Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus.

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the "oxidative message" to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the ApyapA gene, a homologue of the yeast Yap-1, disables the fungus's capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer AoloxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Aoyap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus.

Oligosaccharides Derived from Tramesan: Their Structure and Activity on Mycotoxin Inhibition in Aspergillus flavus and Aspergillus carbonarius.

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, "green", approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition; heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

Natural functions of mycotoxins and control of their biosynthesis in fungi.

Mycotoxins are harmful secondary metabolites produced by a range of widespread fungi belonging in the main to Fusarium, Aspergillus and Penicillium genera. But why should fungi produce toxins? And how is the biosynthesis of these toxins regulated? Several separate factors are now known to be capable of modulating mycotoxin synthesis; however, in this study, focussing just on mycotoxins whose regulatory mechanisms have already been established, we introduce a further factor based on a novel consideration. Various different mycotoxin biosynthetic pathways appear to share a common factor in that they are all susceptible to the influence of reactive oxygen species. In fact, when a fungus receives an external stimulus, it reacts by activating, through a well-defined signal cascade, a profound change in its lifestyle. This change usually leads to the activation of global gene regulators and, in particular, of transcription factors which modulate mycotoxin gene cluster expression. Some mycotoxins have a clear-cut role both in generating a pathogenetic process, i.e. fumonisins and some trichothecenes, and in competing with other organisms, i.e. patulin. In other cases, such as aflatoxins, more than one role can be hypothesised. In this review, we suggest an "oxidative stress theory of mycotoxin biosynthesis" to explain the role and the regulation of some of the above mentioned toxins.

Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat seeds.

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

Tramesan Elicits Durum Wheat Defense against the Septoria Disease Complex.

The Septoria Leaf Blotch Complex (SLBC), caused by the two ascomycetes Zymoseptoria tritici and Parastagonospora nodorum, can reduce wheat global yearly yield by up to 50%. In the last decade, SLBC incidence has increased in Italy; notably, durum wheat has proven to be more susceptible than common wheat. Field fungicide treatment can efficiently control these pathogens, but it leads to the emergence of resistant strains and adversely affects human and animal health and the environment. Our previous studies indicated that active compounds produced by Trametes versicolor can restrict the growth of mycotoxigenic fungi and the biosynthesis of their secondary metabolites (e.g., mycotoxins). Specifically, we identified Tramesan: a 23 kDa α-heteropolysaccharide secreted by T. versicolor that acts as a pro-antioxidant molecule in animal cells, fungi, and plants. Foliar-spray of Tramesan (3.3 µM) on SLBC-susceptible durum wheat cultivars, before inoculation of causal agents of Stagonospora Nodorum Blotch (SNB) and Septoria Tritici Blotch (STB), significantly decreased disease incidence both in controlled conditions (SNB: -99%, STB: -75%) and field assays (SNB: -25%, STB: -30%). We conducted these tests were conducted under controlled conditions as well as in field. We showed that Tramesan increased the levels of jasmonic acid (JA), a plant defense-related hormone. Tramesan also increased the early expression (24 hours after inoculation - hai) of plant defense genes such as PR4 for SNB infected plants, and RBOH, PR1, and PR9 for STB infected plants. These results suggest that Tramesan protects wheat by eliciting plant defenses, since it has no direct fungicidal activity. In field experiments, the yield of durum wheat plants treated with Tramesan was similar to that of healthy untreated plots. These results encourage the use of Tramesan to protect durum wheat against SLBC.

Modulation of antioxidant defense in Aspergillus parasiticus is involved in aflatoxin biosynthesis: a role for the ApyapA gene.

Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the beta-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus. To better understand the role of ROS formation in toxin production, we generated a mutant (Delta ApyapA) having the ApyapA gene deleted, given that ApyapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the Delta ApyapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of ApyapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.

Buckwheat Hull Extracts Inhibit Aspergillus flavus Growth and AFB1 Biosynthesis.

Fungal contamination poses at risk the whole food production chain - from farm to fork - with potential negative impact on human health. So far, the insurgence of pathogens has been restrained by the use of chemical compounds, whose residues have gradually accumulated determining toxic effects in the environment. Modern innovative techniques imply the use of natural and eco-sustainable bioactive plant molecules as pathogens and pests-control agents. These may be profitably recovered in large amounts at the end of industrial milling processes. This is the case of the non-digestible hull of common buckwheat (Fagopyrum esculentum Moench), a natural source of polyphenols, tocopherols, phytosterols and fatty acids. We extract these compounds from the hull of buckwheat; apply them to Aspergillus flavus - aflatoxin producer - under in vitro conditions, checking their ability to inhibit fungal growth and aflatoxin biosynthesis. Moreover, a solvent free method implying the adoption of supercritical CO2 as solvent was set up to extract lipophilic molecules from the buckwheat' hulls. Positive results in controlling fungal growth and aflatoxin biosynthesis let infer that the extracts could be further tested also under in vivo conditions.

Tramesan, a novel polysaccharide from Trametes versicolor. Structural characterization and biological effects.

Mushrooms represent a formidable source of bioactive compounds. Some of these may be considered as biological response modifiers; these include compounds with a specific biological function: antibiotics (e.g. plectasin), immune system stimulator (e,g, lentinan), antitumor agents (e.g. krestin, PSK) and hypolipidemic agents (e.g. lovastatin) inter alia. In this study, we focused on the Chinese medicinal mushroom "yun zhi", Trametes versicolor, traditionally used for (cit.) "replenish essence and qi (vital energy)". Previous studies indicated the potential activity of extracts from culture filtrate of asexual mycelia of T. versicolor in controlling the growth and secondary metabolism (e.g. mycotoxins) of plant pathogenic fungi. The quest of active principles produced by T. versicolor, allowed us characterising an exo-polysaccharide released in its culture filtrate and naming it Tramesan. Herein we evaluate the biological activity of Tramesan in different organisms: plants, mammals and plant pathogenic fungi. We suggest that the bioactivity of Tramesan relies mostly on its ability to act as pro antioxidant molecule regardless the biological system on which it was applied.

Trametes versicolor: a possible tool for aflatoxin control.

The genotoxic, mutagenic and cancerogenic aflatoxins produced by Aspergillus parasiticus are not yet efficiently controlled besides the increasing researches on this topic. Aflatoxin production by A. parasiticus is related to oxidative stress and some antioxidants can inhibit their production. Some basidiomycetes as Trametes versicolor used as "healing mushrooms" present beta-glucans and glycoproteins which are responsible for the stimulation of the host immune response. In this work T. versicolor culture filtrates, from different isolates, have been tested on A. parasiticus cultures to assay their inhibiting effect on aflatoxin production. Filtrates from T. versicolor were lyophilised and exopolysaccharides and glycoproteins were extracted by subsequent steps and added (2% w/v) to liquid cultures of a toxigenic A. parasiticus strain. Fungal growth and aflatoxins production by A. parasiticus were analysed both in filtrates and in mycelia and no interference on the output of toxins from mycelia was evidenced. Furthermore antioxidant capacity (by crocin test) of the T. versicolor extracts was analysed. All the strains assayed are able to inhibit the toxin production from 40% to above 90% in liquid cultures as well as in maize and wheat seeds inoculated with A. parasiticus. Antioxidant activity and beta-glucans amount in T. versicolor extracts showed a close relationship with aflatoxin inhibition ability and demonstrated that beta-glucans could be involved in aflatoxin inhibition. Molecular data indicate the almost complete inhibition of norA mRNA expression and a delay of aflR mRNA transcription. Filtrates and fractions from T. versicolor could be promising agents in the challenge against aflatoxins.

Relationship among lipoperoxides, jasmonates and indole-3-acetic acid formation in potato tuber after wounding.

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0-30?min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC-MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.

Aflatoxin control in maize by Trametes versicolor.

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

How peroxisomes affect aflatoxin biosynthesis in Aspergillus flavus.

In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids ß-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal ß-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal ß-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids ß-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.