Mediterranean agro-industrial wastes as valuable substrates for lignocellulolytic enzymes and protein production by solid-state fermentation.

BACKGROUND: Mediterranean agro-food industries (such as wineries, breweries and olive mills) dispose of great amounts of waste. This generates environmental problems, and the waste has a low nutritional value for use as animal feed. In this sense, solid-state fermentation (SSF) can increase the nutritional value of these wastes and simultaneously produce lignocellulolytic enzymes. RESULTS: All fermented wastes were enriched in protein by the three fungi studied. Aspergillus ibericus was the fungus with the biggest increase of protein, which ranged from 1.4 times to 6.2 times with respect to unfermented wastes. Likewise, A. ibericus achieved the maximum cellulase and xylanase activities. The relationships among substrates composition, fungi used and SSF performance were evaluated by principal components analysis. The high content of cellulose and hemicellulose favoured lignocellulolytic enzymes production, and the phenolics content was negatively correlated with enzymes production and with the increase of protein by SSF. Furthermore, the scanning electron microscopy analysis showed the growth of fungi over solid wastes, the formation of conidiophores and the changes in their structures. CONCLUSION: The nutritional value of Mediterranean wastes was improved and other value-added products such as lignocellulolytic enzymes were produced in the same process, which could facilitate the efficient reuse of these wastes. © 2018 Society of Chemical Industry.

Optimization of lipase production by solid-state fermentation of olive pomace: from flask to laboratory-scale packed-bed bioreactor.

Lipases are versatile catalysts with many applications and can be produced by solid-state fermentation (SSF) using agro-industrial wastes. The aim of this work was to maximize the production of Aspergillus ibericus lipase under SSF of olive pomace (OP) and wheat bran (WB), evaluating the effect on lipase production of C/N ratio, lipids, phenols, content of sugars of substrates and nitrogen source addition. Moreover, the implementation of the SSF process in a packed-bed bioreactor and the improvement of lipase extraction conditions were assessed. Low C/N ratios and high content of lipids led to maximum lipase production. Optimum SSF conditions were achieved with a C/N mass ratio of 25.2 and 10.2% (w/w) lipids in substrate, by the mixture of OP:WB (1:1) and supplemented with 1.33% (w/w) (NH4)2SO4. Studies in a packed-bed bioreactor showed that the lower aeration rates tested prevented substrate dehydration, improving lipase production. In this work, the important role of Triton X-100 on lipase extraction from the fermented solid substrate has been shown. A final lipase activity of 223 ± 5 U g-1 (dry basis) was obtained after 7 days of fermentation.

A Review of Mycotoxins in Food and Feed Products in Portugal and Estimation of Probable Daily Intakes.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that occur naturally in agricultural commodities worldwide. Aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, trichothecenes, and ergot alkaloids are presently the most important for food and feed safety. These compounds are produced by several species that belong to the Aspergillus, Penicillium, Fusarium, and Claviceps genera and can be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic, and immunosuppressant. Human and animal exposure to mycotoxins is generally assessed by taking into account data on the occurrence of mycotoxins in food and feed as well as data on the consumption patterns of the concerned population. This evaluation is crucial to support measures to reduce consumer exposure to mycotoxins. This work reviews the occurrence and levels of mycotoxins in Portuguese food and feed to provide a global overview of this issue in Portugal. With the information collected, the exposure of the Portuguese population to those mycotoxins is assessed, and the estimated dietary intakes are presented.

Olive pomace valorization by Aspergillus species: lipase production using solid-state fermentation.

BACKGROUND: Pollution by olive mill wastes is an important problem in the Mediterranean area and novel solutions for their proper management and valorization are needed. The aim of this work was to optimize a solid-state fermentation (SSF) process to produce lipase using olive pomace (OP) as the main source of nutrients by several Aspergillus spp. Optimized variables in two different designs were: ratio between olive pomace and wheat bran (OP:WB), NaNO3 , Czapek nutrients, fermentation time, moisture content (MC) and temperature. RESULTS: Results showed that the mixture OP:WB and MC were the most significant factors affecting lipase production for all fungi strains tested. With MC and temperature optimization, a 4.4-fold increase in A. ibericus lipase was achieved (90.5 ± 1.5 U g(-1) ), using a mixture of OP and WB at 1:1 ratio, 0.02 g NaNO3 g(-1) dry substrate, absence of Czapek nutrients, 60% of MC and incubation at 30 °C for 7 days. For A. niger and A. tubingensis, highest lipase activity obtained was 56.6 ± 5.4 and 7.6 ± 0.6 U g(-1) , respectively. CONCLUSION: Aspergillus ibericus was found to be the most promising microorganism for lipase production using mixtures of OP and WB. © 2015 Society of Chemical Industry.

Degradation of ochratoxins A and B by lipases: A kinetic study unraveled by molecular modeling.

Mycotoxins are toxic substances produced by fungi and, frequently, different mycotoxins cooccur in food commodities. Ochratoxin A (OTA) and Ochratoxin B (OTB) may co-occur in a variety of foods, like red wines and wheat, presenting a significant risk of population exposure. In this study, we investigated the potential of five lipases (Candida rugosa Lipase, Candida antarctica B Lipase, Thermomyces lanuginosus Lipase, Amano Lipase A from Aspergillus niger (ANL) and Porcine Pancreas Lipase (PPL)) to hydrolyze OTA and OTB into non-hazardous products. Only ANL and PPL degraded both substrates, however, with varying degrees of efficiency. PPL completely degraded OTB (9 h), but only 43% of OTA (25 h). Molecular simulations indicated a high binding energy of OTA to PPL, that can be explained by the impact of the chlorine group, impairing hydrolysis. ANL was able to completely degrade both mycotoxins, OTA in 3 h and OTB in 10 h. The ANL enzyme showed also high specificity to OTA, however, the activity of this enzyme is not affected by chlorine and hydrolyzes OTA faster than OTB. These two enzymes were found to be able to detoxify co-occurring ochratoxins A and B, making isolated enzymes an alternative to the direct use of microorganisms for mycotoxin mitigation in food.

Laccase-Catalyzed Synthesis of Added-Value Polymers from Cork and Grape Extracts.

The development of products from natural plant sources, including agriculture and food wastes, contributes significantly to the circular economy and global sustainability. Cork and grape wastes were employed as the primary sources in this study to obtain compounds of interest under mild extraction conditions. Laccase was applied to oxidize the cork and grape extracts, with the aim of producing value-added molecules with improved properties. Ultraviolet-visible (UV-vis) spectroscopy was assessed to monitor the oxidation process, and characterization of the end products was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectroscopy. The antioxidant and antiaging properties were evaluated by means of ABTS, DPPH, FRAP, and SPF testing. Overall, as compared to their monomeric counterparts, the polymeric compounds displayed remarkable antioxidant and antiaging characteristics after laccase oxidation, showing tremendous potential for applications in the food, pharmaceutical, cosmetic, and textile industries.

Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal.

Three new aflatoxin-producing species belonging to Aspergillus section Flavi are described. They are Aspergillus mottae, A. sergii and A. transmontanensis. These species were isolated from Portuguese almonds and maize. An investigation examined morphology, extrolite production and DNA sequence data to characterize these isolates and describe the new species. Phylogenetic analysis showed that A. transmontanensis and A. sergii form a clade with A. parasiticus whereas A. mottae shares a most recent common ancestor with the combined A. flavus and A. parasiticus clade.

The Potential of Fatty Acids and Their Derivatives as Antifungal Agents: A Review.

Fungal contamination presents several problems: in humans, health issues arise from infections with opportunistic filamentous fungi and yeast, while in food, fungi cause spoilage and, in particular, in the case of mycotoxigenic fungi, can cause serious health issues. Several types of fatty acids and their derivatives, oxylipins, have been found to have inhibitory effect towards fungal growth and the production of mycotoxins. The use of fatty acids as antifungals could fulfil consumer's requests of more natural and environmentally friendly compounds, while being less likely to promote fungal resistance. In addition, due to their nature, fatty acids are easily used as food additives. In this work, we review the most relevant and recent studies on the antifungal ability of fatty acids. We focused on saturated fatty acids, unsaturated fatty acids, and oxylipins, their different impact on fungal inhibition, their proposed modes of action, and their ability to impair mycotoxin production. Applications of fatty acids as antifungals and their limitations are also addressed.

Ozone applications to prevent and degrade mycotoxins: a review.

Ozone, a powerful oxidant, may be used for the inactivation of various microorganisms and the degradation of chemical contaminants. Although there are not many reports on the use of ozone against filamentous fungi or their mycotoxins, promising results have been reported. With a short half-time, at neutral pH and ambient temperature, ozone is able to inactivate microorganisms and decompose their toxic metabolites, leaving no traces of ozone in the treated commodity. This fact makes the use of ozone safe in food applications. There has been relatively limited research in this topic, especially with the use of aqueous ozone. The best management strategy still remains to be developed, but initial studies have indicated that an application of ozone for a short period of exposure is capable of controlling the proliferation of filamentous fungi and of degrading many mycotoxins.

Detection Methods for Aflatoxin M1 in Dairy Products.

Mycotoxins are toxic compounds produced mainly by fungi of the genera Aspergillus, Fusarium and Penicillium. In the food chain, the original mycotoxin may be transformed in other toxic compounds, reaching the consumer. A good example is the occurrence of aflatoxin M1 (AFM1) in dairy products, which is due to the presence of aflatoxin B1 (AFB1) in the animal feed. Thus, milk-based foods, such as cheese and yogurts, may be contaminated with this toxin, which, although less toxic than AFB1, also exhibits hepatotoxic and carcinogenic effects and is relatively stable during pasteurization, storage and processing. For this reason, the establishment of allowed maximum limits in dairy products and the development of methodologies for its detection and quantification are of extreme importance. There are several methods for the detection of AFM1 in dairy products. Usually, the analytical procedures go through the following stages: sampling, extraction, clean-up, determination and quantification. For the extraction stage, the use of organic solvents (as acetonitrile and methanol) is still the most common, but recent advances include the use of the Quick, Easy, Cheap, Effective, Rugged, and Safe method (QuEChERS) and proteolytic enzymes, which have been demonstrated to be good alternatives. For the clean-up stage, the high selectivity of immunoaffinity columns is still a good option, but alternative and cheaper techniques are becoming more competitive. Regarding quantification of the toxin, screening strategies include the use of the enzyme-linked immunosorbent assay (ELISA) to select presumptive positive samples from a wider range of samples, and more reliable methods-high performance liquid chromatography with fluorescence detection or mass spectroscopy-for the separation, identification and quantification of the toxin.

Occurrence and Co-Occurrence of Mycotoxins in Cereal-Based Feed and Food.

Dietary (co)-exposure to mycotoxins is associated with human and animal health concerns as well as economic losses. This study aims to give a data-based insight from the scientific literature on the (co-)occurrence of mycotoxins (i.e., parent and modified forms) in European core cereals, and to estimate potential patterns of co-exposure in humans and animals. Mycotoxins were mainly reported in wheat and maize showing the highest concentrations of fumonisins (FBs), deoxynivalenol (DON), aflatoxins (AFs), and zearalenone (ZEN). The maximum concentrations of FB1+FB2 were reported in maize both in feed and food and were above legal maximum levels (MLs). Similar results were observed in DON-food, whose max concentrations in wheat, barley, maize, and oat exceeded the MLs. Co-occurrence was reported in 54.9% of total records, meaning that they were co-contaminated with at least two mycotoxins. In the context of parental mycotoxins, co-occurrence of DON was frequently observed with FBs in maize and ZEN in wheat; DON + NIV and DON + T2/HT2 were frequently reported in barley and oat, respectively. Apart from the occurrence of ZEN and its phase I and phase II modified forms, only a limited number of quantified data were available for other modified forms; i.e., mainly the acetyl derivatives of DON. Data gaps are highlighted together with the need for monitoring studies on multiple mycotoxins to identify co-occurrence patterns for parent mycotoxins, metabolites, and their modified forms.

Effect of Gamma-Radiation on Zearalenone-Degradation, Cytotoxicity and Estrogenicity.

Zearalenone (ZEA) is produced in cereals by different species of Fusarium, being a non-steroidal estrogenic mycotoxin. Despite having a low acute toxicity, ZEA strongly interferes with estrogen receptors. Gamma-radiation has been investigated to eliminate mycotoxins from food and feed, showing promising results. The present study aims to investigate the gamma-radiation effect on ZEA at different moisture conditions and to evaluate the cytotoxicity and estrogenicity of the irradiated ZEA. Different concentrations of dehydrated ZEA and aqueous solutions of ZEA were exposed to gamma-radiation doses ranging from 0.4 to 8.6 kGy and the mycotoxin concentration determined after exposure by high performance liquid chromatography (HPLC) with fluorescence detection. Following this, the cytotoxicity of irradiated samples was assessed in HepG2 cells, by measuring alterations of metabolic activity, plasma membrane integrity and lysosomal function, and their estrogenicity by measuring luciferase activity in HeLa 9903 cells. Gamma-radiation was found to be effective in reducing ZEA, with significant increases in degradation with increased moisture content. Furthermore, a reduction of cytotoxicity with irradiation was observed. ZEA estrogenicity was also increasingly reduced with increasing radiation doses, but mainly in aqueous solutions. These results suggest reduction of ZEA levels and of its toxicity in food and feed commodities may be achieved by irradiation.

BSA-based sample clean-up columns for ochratoxin A determination in wine: Method development and validation.

Analytical chromatographic techniques for mycotoxins control are well established, but they often depend on costly immunoaffinity sample clean-up. Serum albumins, particularly that from bovine origin (BSA), have stable binding affinity towards some mycotoxins, and can be cheaper alternative receptors for sample clean-up due to their wide availability. Thus, this work used BSA immobilized in agarose beads as a novel solid-phase extraction method for quantification of ochratoxin A (OTA) in wine. Constructed BSA-agarose columns could extract OTA efficiently from red wine after its dilution (4-fold) in 0.1 M Tris pH 8.0. The method was linear (R2 = 0.9999) in the OTA concentration range studied (0.05 to 3.0 µg L-1), with recovery rates above 98%. It also showed low detection (0.017 µg L-1) and quantification (0.051 µg L-1) limits. The efficacy of the BSA-based method was further validated by direct comparison with commercial immunoaffinity columns. Portuguese wines analyzed by both methods had agreeing results.

Mycobiota and mycotoxins in Portuguese pork, goat and sheep dry-cured hams.

The objectives of the present work were to survey, for the first time, the contamination of Portuguese fresh and dry-cured meat products with ochratoxin A (OTA) and aflatoxin B1 (AFB1), and to determine the fungi potentially responsible for this contamination. A total of 128 samples including pork fresh legs, dry-cured legs and shoulders, as well as goat and sheep dry-cured legs were analysed. Mycological analysis of these samples yielded a total of 630 fungal isolates. Penicillium sp. was the dominant fungal genus in all products (66% of all isolates). Penicillium nordicum and Aspergillus westerdijkiae were only rarely isolated from pork ham samples. In fresh pork meat, 40% of the samples were contaminated with OTA at levels below 1 µg/kg. In pork dry-cured legs with 20 to 25 months of ripening, 43% of the samples showed detectable contamination, while 18% of the shoulder hams were contaminated. OTA was not detected in any of the goat and sheep samples. OTA contamination does not seem to be a risk in small-piece and short-ripe products like goat and sheep legs, but affects longer ripe products like pork legs and shoulders. Although aflatoxigenic fungi were identified, AFB1 was not detected in any sample, and it should not be considered a risk in dry-cured hams.

Multilocus sequence identification of Penicillium species in cork bark during plank preparation for the manufacture of stoppers.

Despite several studies reporting Penicillium as one of the most frequent fungal genera in cork planks, the isolates were rarely identified to species level. We conducted a detailed study to identify Penicillium species from the field to the factory environment prior to and after boiling the cork planks. A total of 84 samples were analyzed. Of the 486 Penicillium isolates phenotypically identified, 32 representative or unusual strains were selected for identification by multilocus DNA sequence type. Cork proved to be a rich source of Penicillium biodiversity. A total of 30 taxa were recognized from cork including rarely seen species and 6 phylogenetically unique groups. Spores of some species lodged deep in cork can survive the boiling process. P. glabrum, P. glandicola and P. toxicarium, species with high CFU numbers in the field, are still frequently present in cork after boiling. Other species are killed by the boiling treatment and replaced by Penicillium species originating from the factory environment. Species known to contribute to cork taint were isolated at all stages. Good manufacturing practices are necessary at all stages in the preparation of cork planks to minimize the load of Penicillium species that produce cork taint.

Antifungal effect of organic acids from lactic acid bacteria on Penicillium nordicum.

The control of fungal contamination is particularly important to avoid both spoilage of food and feed products and the occurrence of toxic compounds, known as mycotoxins. Some lactic acid bacteria (LAB) strains have shown the capacity to inhibit fungal growth and the production of mycotoxins. In this work, cell-free supernatants (CFS) of Lactobacillus plantarum UM55 and Lactobacillus buchneri UTAD104 were tested against Penicillium nordicum radial growth and OTA production. When CFS of these strains were used, the radial growth of the fungus was inhibited by less than 20%, but the production of OTA was reduced by approx. 60%. These antifungal effects resulted from organic acids produced by LAB. The CFS of L. plantarum UM55 contained lactic acid, phenyllactic acid (PLA), hydroxyphenyllactic acid (OH-PLA) and indole lactic acid (ILA), while L. buchneri UTAD104 CFS contained acetic acid, lactic acid and PLA. These organic acids were further tested individually for their inhibitory capacity. Calculation of the inhibitory concentrations (ICs) showed that acetic acid, ILA and PLA were the most effective in inhibiting P. nordicum growth and OTA production. When the inhibitory activity of LAB cells incorporated into the culture medium was tested, L. buchneri UTAD104 inhibited the production of OTA entirely in all conditions tested, but fungal growth was only inhibited completely by the highest concentrations of cells. Acetic acid production was primarily responsible for this effect. In conclusion, the ability of LAB to inhibit mycotoxigenic fungi depends on strain capability to produce specific organic acids, and those acids may differ from strain to strain. Also, the use of LAB cells, especially from L. buchneri, in food products prone to contamination with P. nordicum (e.g. dry-cured meats and cheeses) may be an alternative solution to control fungal growth and OTA production.

Gamma irradiation effects on ochratoxin A: Degradation, cytotoxicity and application in food.

Ochratoxin A (OTA) is one of the main mycotoxins that can be found in food. The use of gamma radiation is a technique for preserving food that may exert some effects on mycotoxins. OTA was irradiated in its dry form, in aqueous and in methanolic solutions, and in wheat flour, grape juice and wine. Additionally, the toxicity of OTA irradiated in water was tested. In aqueous solutions, more than 90% of the OTA was degraded by γ-radiation doses ≥2.5kGy, and a 2-fold reduction in OTA cytotoxicity was observed. In food matrices, the elimination of OTA by γ-radiation was found more difficult, as radiation doses of 30kGy eliminate at most 24% of the OTA. Higher moisture content of food matrices did not substantially increase OTA elimination. It is concluded that OTA is very sensitive to irradiation in water solutions but resistant in its dry form and in food matrices.

Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum.

Lactic acid bacteria (LAB), which are commonly used in the production of fermented foods, have been gaining attention for their antifungal and antimycotoxin properties. In this work, the strain Lactobacillus plantarum UM55 was selected among other LAB for inhibiting the growth of Aspergillus flavus. Further, it is shown that cell-free supernatant (CFS) of this strain inhibits the production of aflatoxins (AFLs) by 91%. This inhibition was dependent on CFS pH, increased with increasing concentrations of CFS, and was independent of fungal growth, which was inhibited only by 32%. CFS was also effective in inhibiting the growth and AFLs production in A. parasiticus, A. arachidicola, A. nomius and A. minisclerotigenes. Further, L. plantarum UM55 CFS was analysed for the presence of organic acids and the main differences compared to controls were found in the levels of lactic acid, phenyllactic acid (PLA), hydroxyphenyllactic acid (OH-PLA), and indole lactic acid (ILA). These compounds were individually tested against A. flavus, with all of the compounds showing an inhibiting effect on fungal growth and AFLs production. PLA showed the stronger effects, and the obtained IC90 for the inhibition of growth and AFLs was of 11.9 and 0.87mg/mL, respectively. AFLs IC90 for ILA, OH-PLA and lactic acid were of 1.47, 1.80, and 3.92mg/mL, respectively. The antiaflatoxigenic properties of LAB depend on strain's capability to produce lactic acid, PLA, OH-PLA and ILA.

Predominant mycotoxins, mycotoxigenic fungi and climate change related to wine.

Wine is a significant contributor to the economies of many countries. However, the commodity can become contaminated with mycotoxins produced by certain fungi. Most information on mycotoxins in wine is from Spain, Italy and France. Grapes can be infected by mycotoxigenic fungi, of which Aspergillus carbonarius producing ochratoxin A (OTA) is of highest concern. Climate is the most important factor in determining contamination once the fungi are established, with high temperatures being a major factor for OTA contamination: OTA in wine is at higher concentrations in warmer southern Europe than northern. Contamination by fumonisins is a particular concern, related to Aspergillus niger producing these compounds and the fungus being isolated frequently from grapes. Aflatoxins can be present in wine, but patulin is seldom detected. Alternaria mycotoxins (e.g. alternariol) have been frequently observed. There are indications that T-2 toxin may be common. Also, the combined effects of mycotoxins in wine require consideration. No other mycotoxins are currently of concern. Accurate fungal identifications and mycotoxin detection from the fungi are important and a consideration of practical methods are required. There is a diversity of wines that can be contaminated (e.g. red, white, sweet, dry and fortified). The occurrence of OTA is higher in red and sweet than white wines. Steps to control mycotoxins in wine involve good agriculture practices. The effect of climate change on vines and mycotoxins in wine needs urgent consideration by well-constructed modelling studies and expert interpretation of existing data. Reliable models of the effect of climate change on vines is a priority: the health of vines affects mycotoxin contamination. A modelling study of OTA in grapes at higher temperatures over 100years is required. Progress has been made in reducing OTA in wine. The other mycotoxins require consideration and the effects of climate change will become crucial.

A practical approach for identifications based on mycotoxin characters of Penicillium.

The taxonomy of the penicillia is unstable particularly in the important antibiotic and mycotoxin-producing subgenus Penicillium. There are difficulties relating identifications to mycotoxin production. Also, the validity of dual nomenclature for pleomorphic fungi is under discussion increasingly. Patulin is an important mycotoxin produced by various fungi and has strict limits in the European Union. The mycotoxin and/or the isoepoxydon dehydrogenase (IDH) gene of the metabolic pathway have been assessed in 318 strains predominately of subgenus Penicillium. These data were used to classify the isolates. Subgenus Penicillium contained most of the IDH and patulin positives. The species and varieties in subgenus Penicillium which were associated with patulin detection can be reduced to one name, viz. Penicillium Pen p+ (p = patulin). This has been extended to other mycotoxin producing penicillia to indicate the scope of the scheme. The classification will lead to the number of taxa being reduced, while avoiding species names and hence dual nomenclature. Culture independent analysis of environmental samples is mentioned. The scheme could be used with advantage for other fungi.