Reduction of Alternaria Toxins via the Extrusion Processing of Whole-Grain Red Sorghum Flour.

This study delved into the impact of two extrusion processing parameters-screw speed (SS at 400, 600, 800 RPM) and material moisture content in the extruder barrel (M at 12, 15, 18%) at constant feed rate (50 kg/h)-on reducing the content of alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) in whole-grain red sorghum flour. Ultra-performance liquid chromatography combined with a triple-quadrupole mass spectrometer (UPLC-MS/MS) was employed for the determination of Alternaria toxin levels. The extruder die temperature fluctuated between 136 and 177 °C, with die pressures ranging from 0.16 to 6.23 MPa. The specific mechanical energy spanned from 83.5 to 152.3 kWh/t, the torque varied between 88 and 162.8 Nm, and the average material retention time in the barrel ranged from 5.6 to 13 s. The optimal parameters for reducing the concentration of all Alternaria toxins with a satisfactory quality of the sorghum snacks were: SS = 400 RPM, M = 12%, with a reduction of 61.4, 76.4, 12.1, and 50.8% for AOH, AME, TeA, and TEN, respectively.

Incidence of Aflatoxins and Ochratoxin A in Wheat and Corn from Albania.

In this study, aflatoxins (AFs) and ochratoxin A (OTA) were analyzed in grains, specifically wheat and corn, from Albania. To summarize, 71 wheat and 45 corn samples from different growing areas were collected. The multi-toxin analytical procedure involved sample extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The incidence of AF was 18% in the analyzed wheat and 71% in the corn samples. The concentration of AFs was much higher in the corn samples than in the wheat samples. The maximum permitted levels for aflatoxin B1 (AFB1) and total AFs were not exceeded in the wheat samples, while they were exceeded in 36% of the corn samples. In the wheat samples, the AFB1 concentration varied between 0.2 and 0.4 µg kg-1. However, the highest concentrations in the corn samples were 2057, 2944, and 3550 µg kg-1. OTA was present in only three corn samples and one wheat sample. However, all contaminated samples exceeded the maximum permitted levels. This report reveals the presence of AFs and OTA in grain commodities, specifically wheat and corn, grown in Albania.

Effects of Coenzyme Q10 Supplementation on Oxidative Stress Markers, Inflammatory Markers, Lymphocyte Subpopulations, and Clinical Status in Dogs with Myxomatous Mitral Valve Disease.

Scarce data exist on the effects of coenzyme Q10 (CoQ10) supplementation in dogs with myxomatous mitral valve disease (MMVD). The purpose of this study was to investigate the effect of CoQ10 supplementation on oxidative stress markers (glutathione peroxidase, F2-isoprostanes), markers of inflammation (tumor necrosis factor-α, TNF soluble receptor II, leucocytes, and their subtypes), lymphocyte subpopulations (T helper and cytotoxic T lymphocytes, including activated T lymphocytes, and B lymphocytes), and echocardiographic and clinical parameters in dogs with MMVD. In this randomized, controlled, double-blind, longitudinal study, 43 MMVD dogs in stages ACVIM (American College of Veterinary Internal Medicine classification) B2 and ACVIM C and D (congestive heart failure (CHF)) received water-soluble coenzyme Q10 (100 mg twice daily) or placebo for 3 months, and 12 non-supplemented healthy dogs served as controls. All parameters were measured before and after supplementation in MMVD dogs and once in healthy dogs. CoQ10 supplementation had a positive impact on neutrophil percentage, lymphocyte percentage, and lymphocyte concentration in our cohort of dogs with CHF (ACVIM C and D). Conclusion: CoQ10 as an oral supplement may have benefits in terms of decreasing inflammation in dogs with MMVD and CHF.

Occurrence of Alternaria and Other Toxins in Cereal Grains Intended for Animal Feeding Collected in Slovenia: A Three-Year Study.

In recent years, the less-studied Alternaria mycotoxins have attracted increasing interest due to the lack of survey data and their ability to cause toxic effects in animals and humans. To fill the gap, the aim of this three-year survey was to investigate the presence and co-occurrence of Alternaria and other mycotoxins in a total of 433 cereal grain samples from Slovenian farms and agricultural cooperatives from 2014 to 2016. Using the multi-mycotoxin method, 14 mycotoxins were determined. In 53% of 433 analysed samples, contamination with at least one mycotoxin was found. Deoxynivalenol (DON) and tenuazonic acid (TeA) were present in 32% and 26% of cereal grain samples, respectively, whereas alternariol (AOH), tentoxin (TEN), alternariol monomethyl ether (AME), 3- and 15-acetyldeoxynivalenol (3- and 15-AcDON), and zearalenone (ZEN) were present in fewer than 15% of the samples. Ochratoxin A (OTA) was found in one rye sample, while diacetoxyscirpenol (DAS), HT-2 and T-2 toxin, and fumonisins B1 and B2 (FB1 and FB2) were not detected. The highest maximum and median concentrations of Alternaria toxins were determined in spelt in 2016 (TeA, 2277 µg/kg and 203 µg/kg, respectively), and those of Fusarium toxins in wheat in 2015 (DON, 4082 µg/kg and 387 µg/kg, respectively). The co-occurrence of two or more mycotoxins was found in 43% of the positive samples. The correlations between Alternaria toxins were very weak but statistically significant (r: 0.15-0.17, p: 0.0042-0.0165). A well-known correlation between Fusarium toxins DON and ZEN was weak and highly significant (r = 0.28, p < 0.0001).

Randomized, double-blinded, controlled trial of the effects of coenzyme Q10 supplementation on plasma coenzyme Q10 concentration in dogs with myxomatous mitral valve disease.

OBJECTIVE: To determine the dose of coenzyme Q10 (CoQ10) needed to achieve at least a 3-fold increase in plasma CoQ10 concentration in dogs with myxomatous mitral valve disease (MMVD) and congestive heart failure (CHF). ANIMALS: 18 dogs with CHF due to MMVD and 12 healthy dogs. PROCEDURES: In a randomized, double-blinded, controlled trial, dogs with MMVD were given 50 or 100 mg of water-soluble CoQ10 (ubiquinone; total daily dose, 100 mg [n = 5] or 200 mg [6]) or a placebo (7), PO, twice a day for 2 weeks in addition to regular cardiac treatment. Plasma CoQ10 concentration was measured in dogs with MMVD before (baseline) and at various time points after supplementation began and in healthy dogs once. Concentrations were compared among and within groups. RESULTS: No significant difference in median baseline plasma CoQ10 concentration was detected between healthy dogs and dogs with MMVD. Fold increases in plasma CoQ10 concentrations ranged from 1.7 to 4.7 and 3.2 to 6.8 for individual dogs in the 100-mg and 200-mg groups, respectively. The change in plasma CoQ10 concentration after supplementation began was significantly higher than in the placebo group at 4 hours and 1 and 2 weeks for dogs in the 200-mg group and at 1 and 2 weeks for dogs in the 100-mg group. CONCLUSIONS AND CLINICAL RELEVANCE: A daily CoQ10 dose of 200 mg was sufficient to achieve at least a 3-fold increase in plasma CoQ10 concentration and may be used in CoQ10 supplementation studies involving dogs with CHF due to MMVD.

Entomological Surveillance and Cantharidin Concentrations in Mylabris variabilis and Epicauta rufidorsum Blister Beetles in Slovenia.

True blister beetles (genus Epicauta, family Meloidae) produce cantharidin, which can cause toxicosis in humans and animals. Some recent reports suggest that poisoning by the blister beetle has occurred in the Mediterranean part of Slovenia, which has never been reported before. Drought and modern harvesting techniques are thought to increase the likelihood of blister beetle forage contamination and cantharidin intoxication in animals. A survey of fields associated with blister beetle contamination was conducted and the Meloid species present were identified. Entomological surveillance was conducted for Mylabris variabilis and Epicauta rufidorsum. Cantharidin concentrations were also measured in both blister beetle species. Cantharidin concentration in Mylabris variabilis (n = 17) ranged from 0.038 to 0.354 µg/mg (mean 0.151 µg/mg). Cantharidin concentration in Epicauta rufidorsum (n = 36) ranged from 0.055 to 0.341 µg/mg (mean 0.142 µg/mg). Both species exhibited variable concentrations of cantharidin that could not be associated with their biology, sex, age, size, and/or reproductive status. Epicauta rufidorsum have never previously been studied as a possible source of forage contamination, nor have cantharidin concentrations been determined in this species. It is the most likely source of forage contamination due to its abundance in the investigated fields, its swarming activity, and its tendency to reside in the green parts of plants immediately after cutting. Delaying the simultaneous processing and storage of forage after cutting would reduce the likelihood of forage contamination by blister beetles, as they can then retreat to the ground or fly away.

Ergot and Ergot Alkaloids in Cereal Grains Intended for Animal Feeding Collected in Slovenia: Occurrence, Pattern and Correlations.

This four-year study reports the occurrence of ergot alkaloids (EAs) in cereals intended for animal feeding collected in Slovenia. A total of 517 samples of cereals were analysed using liquid chromatography-tandem mass spectrometry for the presence of EAs. The sample set included wheat, rye, triticale, oat, spelt and barley. The study revealed that 17% of the analysed cereal samples were contaminated with at least one ergot alkaloid. EAs have two epimeric forms: -ine and -inine. The incidence rates of the -ine and -inine forms in the analysed samples were 16% and 15%, respectively. The highest contamination rates were observed in rye (54%), oat (50%) and spelt (30%), where the highest mean concentrations of total EAs were also determined (502 µg/kg, 594 µg/kg and 715 µg/kg, respectively). However, the highest concentrations of total EAs were found in wheat and rye (4217 µg/kg and 4114 µg/kg, respectively). The predominant EAs were ergometrine, ergosine and ergocristinine. The occurrence of six or more ergot alkaloids was observed in 49% of the positive samples. A weak correlation (p = 0.284) in the positive samples was found between the mass of sclerotia and the total concentrations of EAs using the Spearman correlation coefficient.

Incidence of Fusarium Mycotoxins in Wheat and Maize from Albania.

In this study, ten Fusarium toxins were analysed in wheat and maize commodities from Albania. In total, 71 samples of wheat and 45 samples of maize were collected from different producing regions. The analytical procedure consisted of a simple one-step sample extraction followed by the determination of toxins using liquid chromatography coupled with tandem mass spectrometry. Fusarium toxins were found in 23% of the analysed wheat samples and in 78% of maize samples. In maize samples, most often fumonisins B1 (FB1) and B2 (FB2) were found. They were present in 76% of samples. They were detected in all positive samples except in one with concentrations ranging from 59.9 to 16,970 µg/kg. The sum of FB1 and FB2 exceeded the EU maximum permitted level (4000 µg/kg) in 31% of maize samples. In wheat samples, the only detected Fusarium mycotoxin was deoxynivalenol (DON), present in 23% of samples. In one sample with the concentration of 1916 µg/kg, the EU maximum permitted level (1250 µg/kg) was exceeded. This is the first report on the presence of Fusarium toxins in wheat and maize grains cultivated in Albania.

Mycotoxin Decontamination Efficacy of Atmospheric Pressure Air Plasma.

Mycotoxins, the toxic secondary metabolites of mould species, are a growing global concern, rendering almost 25% of all food produced unfit for human or animal consumption, thus placing immense pressure on the food supply chain. Cold Atmospheric pressure Plasma (CAP) represents a promising, low-cost, and environmentally friendly means to degrade mycotoxins with negligible effect on the quality of food products. Despite this promise, the study of CAP-mediated mycotoxin degradation has been limited to a small subset of the vast number of mycotoxins that plague the food supply chain. This study explores the degradation of aflatoxins, trichothecenes, fumonisins, and zearalenone using CAP generated in ambient air. CAP treatment was found to reduce aflatoxins by 93%, trichothecenes by 90%, fumonisins by 93%, and zearalenone by 100% after 8 minutes exposure. To demonstrate the potential of CAP-mediated mycotoxin degradation against more conventional methods, its efficiency was compared against ultraviolet C (UVC) light irradiation. In all cases, CAP was found to be considerably more efficient than UVC, with aflatoxin G1 and zearalenone being completely degraded, levels that could not be achieved using UVC irradiation.

Fungal and enzymatic treatment of mature municipal landfill leachate.

The aim of our study was to evaluate biotreatability of mature municipal landfill leachate by using white rot fungus and its extracellular enzymes. Leachates were collected in one active and one closed regional municipal landfill. Both chosen landfills were operating for many years and the leachates generated there were polluted by organic and inorganic compounds. The white rot fungus Dichomitus squalens was able to grow in the mature leachate from the closed landfill and as it utilizes present organic matter as a source of carbon, the results were showing 60% of DOC and COD removal and decreased toxicity to the bacterium Aliivibrio fischeri. On the other hand, growth of the fungus was inhibited in the presence of the leachate from the active landfill. However, when the leachate was introduced to a crude enzyme filtrate containing extracellular ligninolytic enzymes, removal levels of COD and DOC reached 61% and 44%, respectively. Furthermore, the treatment led to detoxification of the leachate to the bacterium Aliivibrio fischeri and to reduction of toxicity (42%) to the plant Sinapis alba. Fungal and enzymatic treatment seems to be a promising biological approach for treatment of mature landfill leachates and their application should be further investigated.

Optimization of ligninolytic enzyme activity and production rate with Ceriporiopsis subvermispora for application in bioremediation by varying submerged media composition and growth immobilization support.

Response surface methodology (central composite design of experiments) was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid media (OLM), where the highest experimentally obtained activities and productivities were 41 U L(-1) and 16 U L(-1) day(-1) for laccase (Lac), and 193 U L(-1) and 80 U L(-1) day(-1) for manganese peroxidase (MnP). Considering culture growth in OLM on various types of immobilization support, the best results were obtained with 1 cm beech wood cubes (BWCM). Enzyme activities in culture filtrate were 152 U L(-1) for Lac and 58 U L(-1) for MnP, since the chemical composition of this immobilization material induced higher Lac activity. Lower enzyme activities were obtained with polyurethane foam. Culture filtrates of OLM and BWCM were applied for dye decolorization. Remazol Brilliant Blue R (RBBR) was decolorized faster and more efficiently than Copper(II)phthalocyanine (CuP) with BWCM (80% and 60%), since Lac played a crucial role. Decolorization of CuP was initially faster than that of RBBR, due to higher MnP activities in OLM. The extent of decolorization after 14 h was 60% for both dyes.

Enhanced enzyme production with the pelleted form of D. squalens in laboratory bioreactors using added natural lignin inducer.

White-rot fungi are extensively used in various submerged biotechnology processes to produce ligninolytic enzymes. Transfer of the process from the laboratory to the industrial level requires optimization of the cultivation conditions on the laboratory scale. An interesting area of optimization is pellet growth since this morphological form solves problems such as the decreased oxygen concentration, limited heat, and nutrient transport, which usually occur in dispersed mycelium cultures. Many submerged fermentations with basidiomycetes in pellet form were done with Phanerochaete, Trametes, and Bjerkandera species, among others. In our study, another promising basidiomycete, D. squalens, was used for ligninolytic enzyme production. With the addition of wood particles (sawdust) as a natural inducer and optimization of mixing and aeration conditions in laboratory stirred tank (STR) and bubble column (BCR) reactors on pellet growth and morphology, the secretion of laccase and the manganese-dependent peroxidase into the medium was substantially enhanced. The maximum mean pellet radius was achieved after 10 days in the BCR (5.1 mm) where pellets were fluffy and 5 days in the STR (3.5 mm) where they were round and smooth. The maximum Lac activity (1,882 U l(-1)) was obtained after 12 days in the STR, while maximum MnP activity (449.8 U l(-1)) occurred after 18 days in the BCR. The pellet size and morphology depended on the agitation and aeration conditions and consequently influenced a particular enzyme synthesis. The enzyme activities were high and comparable with the activities found for other investigations in reactors with basidiomycetes in the form of pellets.

Preparation and characterisation of ribonuclease monolithic bioreactor.

In gene therapy and DNA vaccination, RNA removal from DNA preparations is vital and is typically achieved by the addition of ribonuclease into the sample. Removal of ribonuclease from DNA samples requires an additional purification step. An alternative is the implementation of immobilized ribonuclease. In our work, ribonuclease was covalently coupled onto the surface of methacrylate monoliths via epoxy or imidazole carbamate groups. Various immobilization conditions were tested by changing immobilization pH. Ribonuclease immobilized on the monolith via imidazole carbamate groups at pH 9 was found to be six times more active than the ribonuclease immobilized on the monolith via epoxy groups. Under optimal immobilization conditions the Michaelis-Menten constant, Km, for cytidine-2,3-cyclic monophosphate, and turnover number, k3 were 0.52 mM and 4.6s(-1), respectively, and mirrored properties of free enzyme. Enzyme reactor was found to efficiently eliminate RNA contaminants from DNA samples. It was active for several weeks of operation and processed 300 column volumes of sample. Required residence time to eliminate RNA was estimated to be around 0.5 min enabling flow rates above 1 column volume per min.