Unveiling ochratoxin A and ochratoxigenic fungi in Brazilian artisanal Cheeses: Insights from production to consumption.

Ochratoxin A (OTA) is a toxin produced by several Aspergillus species, mainly those belonging to section Circumdati and section Nigri. The presence of OTA in cheese has been reported recently in cave cheese in Italy. As artisanal cheese production in Brazil has increased, the aim of this study was to investigate the presence of ochratoxin A and related fungi in artisanal cheese consumed in Brazil. A total of 130 samples of artisanal cheeses with natural moldy rind at different periods of maturation were collected. Of this total, 79 samples were collected from 6 producers from Canastra region in the state of Minas Gerais, since this is the largest artisanal cheese producer region; 13 samples from one producer in the Amparo region in the state of São Paulo and 36 samples from markets located in these 2 states. Aspergillus section Circumdati occurred in samples of three producers and some samples from the markets. A. section Circumdati colony counts varied from 102 to 106 CFU/g. Molecular analysis revealed Aspergillus westerdijkiae (67 %) as the most frequent species, followed by Aspergillus ostianus (22 %), and Aspergillus steynii (11 %). All of these isolates of A. section Circumdati were able to produce OTA in Yeast Extract Sucrose Agar (YESA) at 25 °C/7 days. OTA was found in 22 % of the artisanal cheese samples, ranging from 1.0 to above 1000 µg/kg, but only five samples had OTA higher than 1000 µg/kg. These findings emphasize the significance of ongoing monitoring and quality control in the artisanal cheese production process to minimize potential health risks linked to OTA contamination.

A green-footprint approach for parallel multiclass analysis of contaminants in roasted coffee via LC-HRMS.

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70-120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide.

Barcode-style lateral flow immunochromatographic strip for the semi-quantitative detection of ochratoxin A in coffee samples.

Ochratoxin A (OTA) is a mycotoxin contaminating agricultural products produced by fungi, associated with important toxic effects. Thus, the development of fast, sensitive, and economical approaches for OTA detection is crucial. In this study, a barcode-style lateral flow assay for the semi-quantitative detection of OTA in coffee samples was developed. To achieve this goal, a BSA-OTA complex was immobilized in three test zones to compete with OTA molecules in the sample for binding with anti-OTA antibodies labeled with gold nanoparticles. Different concentrations of OTA in the sample produced distinct colour patterns, allowing semi-quantification of the analyte. The assay exhibited high sensitivity, with a limit of detection of 2.5 µg.L-1, and high reproducibility, with variation coefficient values between 2% and 13%. Moreover, the colour patterns obtained in the analysis with coffee samples were similar to the results obtained with standard OTA solutions, demonstrating a reliable applicability in real samples.

Plasmonic immunosensors based on spoon-shaped waveguides for fast and on-site ultra-low detection of ochratoxin A in coffee samples.

The high toxicity and occurrence of ochratoxin A (OTA) in grains and foods has been a growing concern due to the impacts on health and the economy in many countries. In this sense, simplified devices with high sensitivity and specificity for local monitoring are enthusiastically pursued. In this work, we report for the first time the detection of ochratoxin A in coffee samples using a spoon-shaped waveguide immunosensor. The biosensor was built with the surface of the spoon-shaped waveguide covered by a 60 nm layer of gold to enable the SPR phenomenon. The measurements indicated a linear relationship between the change in the SPR phenomenon values and the OTA concentration in the range from 0.2 ppt to 5 ppt. When analyzed in coffee samples, the biosensor was highly selective and did not suffer matrix interference. The developed biosensor represents a promising analytical device for coffee quality analyses, as it is portable, simple, and suitable for onsite detection of target analytes.

Inhibition of Aspergillus carbonarius growth and Ochratoxin A production using lactic acid bacteria cultivated in an optimized medium.

The Gram-positive bacteria lactic acid bacteria (LAB) are used in the food industry but are also known for inhibiting certain food spoilage microorganisms, especially fungi. Sources of nitrogen (N) for culture media are generally organic and expensive. Many attempts have been made to formulate economical culture media with alternative N sources obtained from agricultural and industrial byproducts. This study describes the design and optimization of an inexpensive culture medium for Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) MZ809351 strain B31. The culture medium was optimized using statistical experimental designs to identify the factors with the most significant effects on biomass concentration to reduce the overall cost, aiming to obtain a biomass concentration similar to that obtained with the reference LAB culture medium (de Man, Rogosa and Sharpe; MRS). Sodium acetate and magnesium sulfate were the most significant factors (p < 0.005), and their contents were reduced by 22 % and 40 %, respectively, without affecting biomass concentration. Malt germ extract (MGE) was used as an alternative nitrogen source to replace meat extract (ME) and proteose peptone (PP). Through these experiments, the composition of a culture medium that is less expensive than MRS broth was defined, which produced a biomass concentration (3.8 g/L) similar to that obtained with MRS medium. The inhibitory effects of two LAB strains isolated from the Ivory Coast and Mexico on the growth and production of ochratoxin A (OTA) in an ochratoxigenic fungus was tested. The minimum cellular concentration of the LAB to prevent the development of Aspergillus carbonarius Ac 089 and the production of OTA was determined in a model assay in Petri dishes. The conditions to inhibit the germination of A. carbonarius Ac 089 and the production of OTA were found. Using the optimized medium and a ratio of 2 × 104 LAB/spore (1 × 108 CFU/mL) strain B7 (L. plantarum MZ809351) and 2 × 103 LAB/spore (1 × 107 CFU/mL) strain B31 (L. plantarum MN922335) completely inhibited the growth of the fungus. A ratio of 2 × 105 LAB/spore (1 × 109 CFU/mL) was required to inhibit OTA production with strains B7 and B31. This study indicates the potential of cultivating LAB in an optimized and inexpensive culture medium for use as a biological control agent against ochratoxigenic fungi in food.

Polyurethane Foam as a Novel Material for Ochratoxin A Removal in Tea and Herbal Infusions-A Quantitative Approach.

A novel solid-phase extraction methodology followed by UHPLC-MS/MS has been developed for Ochratoxin A (OTA) analysis in herbal infusions. For this purpose, a commercial polyurethane foam (PUF) was used as sorbent, and the experimental conditions were fully optimized. The strategy was satisfactory for reducing the matrix effect and allowed for OTA quantification in black tea and herbal infusions, with suitable recoveries and quantitation limits in agreement with those required by the maximum levels allowed by current regulations. The achieved results demonstrated the unprecedented use of polyurethane foam as an effective alternative for OTA retention and quantification in herbal infusions with the advantages of simple preparation, time saving, sustainability, and low cost for routine analysis.

A Bacillus-based biofungicide agent prevents ochratoxins occurrence in grapes and impacts the volatile profile throughout the Chardonnay winemaking stages.

Bacillus-based biocontrol agents have emerged as a strategy to eliminate or reduce the use of synthetic fungicides that are detrimental to health and the environment. In vineyards, a special concern arises from the control of Aspergillus carbonarius, a fungus known for its potential to produce ochratoxins. Ochratoxin A (OTA) is the most toxic form among ochratoxins and its maximum limit in wine has been established in Europe and Brazil as 2 µg/kg. Wine quality, especially the volatile profile, may be influenced by the antifungal strategies, since fungicide residues are transferred from grapes to must during winemaking. The objective of this study was to evaluate, for the first time, the impact of a biocontrol strategy containing Bacillus velezensis P1 on the volatile profile and occurrence of ochratoxins when grapes infected with A. carbonarius were used in winemaking. The evaluation of ochratoxins was carried out by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS), and volatile compounds were analyzed using comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC × GC/qMS). Six ochratoxins were identified in must prepared with Chardonnay grapes inoculated with A. carbonarius (ochratoxin α, ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide and OTA). Although winemaking causes a decrease in the levels of all forms of ochratoxins, the co-occurrence of these mycotoxins was verified in wine made with grapes containing A. carbonarius. B. velezensis P1 prevented the occurrence of ochratoxins in must, ensuring the safety of wines. Regarding the volatile profile, a predominant presence of terpenic compounds was verified in samples treated with B. velezensis when compared with those not treated with the biocontrol strategy, whereas the presence of A. carbonarius resulted in a higher concentration of volatile compounds with an odor described as fatty/waxy, possibly compromising wine quality. Therefore, B. velezensis P1 is a new biofungicide possibility to produce ochratoxin-free grapes and high-quality wines.

In vitro and in vivo efficacy of poly(lactic acid) nanofiber packaging containing essential oils from Ocimum basilicum L. and Ocimum gratissimum L. against Aspergillus carbonarius and Aspergillus niger in table grapes.

Apergillus carbonarius and Aspergillus niger are the principal fungi that attack table grapes, and they are responsible for producing and contaminating these fruits with ochratoxin A. Packaging containing essential oils from Ocimum gratissimum L. and Ocimum basilicum L. encapsulated in poly(lactic acid) (PLA) nanofibers were produced, the antifungal and antiocratoxigenic activities against A. carbonarius and A. niger were evaluated in vitro and in vivo, and the effect of these packages on the quality of table grapes was determined. The nanofibers were produced by the Solution Blow Spinning technique and characterized by Scanning Electron Microscopy and Thermogravimetric Analysis. Fungal contamination and ochratoxin A production were significantly controlled by PLA nanofibers containing the essential oils and the physicochemical parameters of the grapes were preserved, preserving the quality and the shelf life of the fruit. Therefore, the active packaging developed herein has potential and may be suitable for application in fruits.

Occurrence of Aflatoxins and Ochratoxin A during Merkén Pepper Powder Production in Chile.

Berry fruits of Capsicum annuum L. cv. "Cacho de Cabra" are used for the manufacture of a traditional pepper powder known as Merkén. In the present study, aflatoxins (AFs) and ochratoxin A (OTA) contamination in berry fruits of C. annuum was determined at harvest, drying, and smoking stages of Merkén production, in cumin and coriander seeds used as Merkén ingredients, and in the final packaged Merkén produced by local farmers. Additionally, Merkén samples from local markets in the region of La Araucanía (Chile) were also evaluated. Chromatographic analysis was based on a qualitative method. AFs and OTA were not detected on pepper pods and seeds. There was no detection of AFs and OTA on cultured Aspergillus and Penicillium strains isolated from pepper pods, cumin and coriander seeds and Merkén. The lack of AFs/OTA-producers among the isolated fungal species can explain and support the absence of contamination in pepper pods. In contrast, the AFB1 was detected in 75% of Merkén obtained from farmers and 46% of Merkén samples purchased from local markets; while OTA was detected in 100% of Merkén samples obtained from farmers and local markets. In the Merkén production chain, the harvest and post-harvest are key stages for fungal growth while the commercialization stage is highly susceptible to AFs and OTA contamination.

Active packaging of poly(lactic acid) nanofibers and essential oils with antifungal action on table grapes.

The table grape is a non-climateric fruit that is very susceptible to fungal contamination, in addition to suffering an accelerated loss of quality during storage. The in vitro and in grape antifungal and antiocratoxigenic effects of the essential oils from Alpinia speciosa and Cymbopogon flexuosus against Aspergillus carbonarius and Aspergillus niger were studied. The oils were encapsulated in poly(lactic acid) (PLA) nanofibers as a potential active packaging to be applied to control the degradation of grapes stored during the post-harvest period. Fungal proliferation and ochratoxin A synthesis in A. carbonarius and A. niger decreased in the presence of the active packaging. However, the nanofiber containing the essential oil from C. flexuosus was more efficient in providing a fungicidal effect against A. carbonarius (10% and 20%) and A. niger (20%). In addition, weight loss and color changes were controlled and the parameters of acidity, °Brix, softening and the texture of the grape were maintained. A very small mass loss of the essential oils encapsulated in nanofibers was observed by thermogravimetric analysis, showing that the nanofiber was efficient in enabling the controlled release. The quality and safety of table grapes were maintained for longer periods of storage in the presence of active packaging, so the incorporation of these oils in nanofibers can be a promising way to increase the shelf life of grapes.

Acute intoxication with single oral dose of ochratoxin A (OTA) causes leukopenia, heteropenia, lymphopenia and lymphoid depletion in the bursa of Fabricius in broiler chicks / Intoxicação aguda com dose oral única de ocratoxina A (OTA) causa leucopenia, heteropenia, linfopenia e depleção linfoide na bolsa de Fabricius em pintainhos de corte

Ochratoxin A (OTA) is a mycotoxin produced by species of Penicillium and Aspergillus, agricultural product contaminants. Chronic and sub-chronic OTA intoxication by chickens ingesting contaminated feed, leads to health damages due to its hepatotoxic, nephrotoxic, cytotoxic, immunotoxic, gastrotoxic, and possibly carcinogenic effects. As there are few data on acute intoxication, the present study evaluated the effects of a single acute OTA intoxication dose on immunological and hematological parameters in chicks. Sixteen one-day-old chicks were used, separated into two groups (n=8). A single dose of OTA (1400 µg kg-1 body weight) was administered, via gavage, for the OTA group and one dose of sterile PBS for the control group. On the 13th day, blood samples were collected to assess hematological and biochemical parameters, and on the 14th day, euthanasia and collection of lymphoid organs were performed. The animals of the OTA group demonstrated a significant decrease in total circulating leukocytes (p<0.001) with heteropenia (p<0.001) and lymphopenia (p=0.023), decrease hematocrit (p=0.020), hemoglobin (p=0.032), and plasma IgA (p =0.044), and increased plasma uric acid level (p=0.045), in relation to the control group. In addition, the animals intoxicated with OTA showed depletion of lymphoid cells in the bursa of Fabricius (p=0.016), but not in the thymus or spleen (p>0.05), compared to the control. For the other parameters: total plasma proteins, plasma IgY levels, and anti-Newcastle Disease Virus (NDV) vaccine titers from matrices, there were no significant differences between the analyzed groups (p>0.05), although there was a worsening tendency in contaminated animals. In conclusion, even a single acute OTA intoxication at a high dose, leads to the suppression of the systemic immune response, also affecting some hematological or biochemical parameters in chicks.

Ocratoxina A (OTA) é uma micotoxina produzida por espécies de Penicillium e Aspergillus, contaminantes de produtos agrícolas. Intoxicação crônica e subcrônica por OTA em frangos que ingerem ração contaminada, levam à danos à saúde devido aos seus efeitos hepatotóxicos, nefrotóxicos, citotóxicos, imunotóxicos, gastrotóxicos e possivelmente carcinogênicos. Como há poucos dados sobre intoxicação aguda, o presente estudo avaliou os efeitos de uma dose única de intoxicação aguda por OTA sobre parâmetros imunológicos e hematológicos em pintainhos. Foram utilizados 16 pintainhos de um dia de idade, separados em dois grupos (n=8). Uma dose única de OTA (1400 µg kg-1 de peso corporal) foi administrada, via gavagem, para o grupo OTA e uma dose de PBS estéril para o grupo controle. No 13º dia foram coletadas amostras de sangue para avaliação dos parâmetros hematológicos e bioquímicos, e no 14º dia foi realizada a eutanásia e coleta de órgãos linfoides. Os animais do grupo OTA demonstraram diminuição significativa do total de leucócitos circulantes (p<0,001) com heteropenia (p<0,001) e linfopenia (p=0,023), diminuição do hematócrito (p=0,020), hemoglobina (p=0,032) e IgA plasmática (p=0,044) e aumento do nível plasmático de ácido úrico (p=0,045), em relação ao grupo controle. Além disso, os animais intoxicados com OTA apresentaram depleção de células linfóides na bolsa de Fabricius (p=0,016), mas não no timo ou baço (p>0,05), em relação ao controle. Para os demais parâmetros: proteínas totais do plasma, níveis plasmáticos de IgY e títulos de vacinas contra o Vírus da Doença de Newcastle (NDV) das matrizes, não houve diferenças significativas entre os grupos analisados (p>0,05), embora tenha havido uma tendência de piora nos animais contaminados. Em conclusão, mesmo uma intoxicação única aguda por OTA em alta dose, leva à supressão da resposta imune sistêmica, afetando também alguns parâmetros hematológicos ou bioquímicos em pintainhos.

Inhibitory Effect of GRAS Essential Oils and Plant Extracts on the Growth of Aspergillus westerdijkiae and Aspergillus carbonarius Strains.

The effect of essential oils (obtained using hydrodistillation) and plant extracts (ethanolic, aqueous, and hexanic extractions) of 10 different plants cultivated in Brazil were tested using the diffusion agar method, with the objective of evaluating the inhibitory effect of the oils and extracts on the mycelial growth of Aspergillus westerdijkiae NRRL 3174 and A. carbonarius RC 2054 (UNRC). Of the 40 essential oils and plant extracts analyzed, oregano essential oil and plant extract, rosemary essential oil, and the clove ethanolic extract were the best choice to obtain the growth parameters (radial growth rates (mm day-1) and lag phase (h)) due the good results presented and the volume of oil/extract obtained. Comparing all the essential oils and plant extracts that were tested for growth parameters, the best results were obtained for the clove ethanolic extract for both strains assayed. These results demonstrated an outstanding potential use of some of these products in prevention of fungal contamination in food. However, further studies need to be conducted to determine the ability of these oils and extracts to inhibit or reduce ochratoxin A production.

Chemical analysis, antifungal and antimycotoxigenic activity of tetradenia riparia essential oil and crude extract.

Tetradenia riparia is known for its richness in essential oil which has been widely investigated due to its biological activities such as antimicrobial, insecticidal, trypanocidal, antimalarial and antioxidant. The objective of this work was to chemically analyze and evaluate the antifungal and antimycotoxigenic activity of the essential oil and the crude extract of leaves, flower buds and stems of T. riparia from the northwest region of the state of Paraná. The essential oil was obtained by hydrodistillation using a Clevenger-type apparatus. To obtain the crude extract, the leaves, flower buds and stems were pulverized and subjected to a dynamic maceration process using 70% v v-1 ethyl alcohol. Chemical analysis of the essential oil was performed by GC/MS, and chemical identification of the crude extract by UHPLC-ESI/qTOF. Antifungal activity (Rhizopus oryzae, Aspergillus flavus, Aspergillus ochraceus, Penicillium verrucosum and Fusarium graminearum) was performed by broth microdilution and the antimycotoxigenic assay was performed with A. ochraceus and P. verrucosum. Ochratoxin A was extracted by partition with chloroform and quantified by HPLC-FL. The oil yield was 0.29% for leaves, 0.34% for stems and 0.38% for flower buds, and the major compounds were fenchone, ß-caryophyllene, α-cadinol, 14-hydroxy-9- epi-caryophyllene, 9ß,13ß-epoxy-7-abietene, α-cadinol and 6-7-dehydroroyleanone. The main chemical compounds identified in the crude extract were terpenes, anthocyanins, flavonoids, tannins and phenolic acids. The minimum inhibitory concentration (MIC) of oils from leaves, flower buds and stems for the strains tested ranged from 0.87 mg mL-1 to 33.3 mg mL-1, while the minimum fungicidal concentration (MFC) ranged from 6.94 mg mL-1 and 33.3 mg mL-1. The MIC and MFC for ketoconazole, tebuconazole, sorbate and nitrite ranged from 0.05 to 33.3 mg mL-1. The oil and crude extract of leaves, stems and flower buds showed an inhibition of ochratoxin A production for P. verrucosum of approximately 100%.

Mycotoxin Exposure in Children through Breakfast Cereal Consumption in Chile.

Mycotoxins are unavoidable contaminants produced by fungi in food, especially grains. This study aimed to measure the occurrence and levels of total aflatoxins (AFs); ochratoxin A (OTA); zearalenone (ZEN); fumonisins B1, B2, and B3 (FUM); deoxynivalenol (DON); and T-2/HT-2 toxins in the four most commonly consumed breakfast cereals in Chile and to assess mycotoxin exposure and risk in children aged 2 to 13 years due to cereal consumption. In this study, a total of 110 batches with three subsamples of the four brands were sampled in supermarkets from November 2019 to June 2021. Samples were analyzed by Veratox® ELISA (Neogen). Exposure was assessed by estimated daily intake (EDI) considering the levels found in a modified lower bound (mLB) and upper bound (UB). Risk was estimated by margin of exposure (MOE) in the case of OTA and AFs and hazard quotient (HQ) for the rest of the mycotoxins. No T2/HT2 toxins were detected. Few samples had quantifiable levels of ZEN, FUM, and DON except for brand 1, with a mean (standard deviation, SD) of 54 (20), 1552 (351), and 706 (218) ng/g, respectively. In addition, three FUM samples and one DON sample had values over the Chilean regulation. Brands 2, 3, and 4 had quantifiable levels of AFs, with mean (SD) values of 1.3 (0.1), 2.1 (0.6), and 1.9 (0.4) ng/g, respectively. Brand 3 had quantifiable levels of OTA, with a mean (SD) of 2.3 (0.4) ng/g. Estimated exposure indicated a risk of AFs in all scenarios, and of FUM for brand 1 consumption, OTA and DON for brand 3 consumption, and OTA for brand 4 consumption in the mLB worst-case scenario. In general, mycotoxin levels were below the Chilean regulatory limits, but most of them were above the EU regulation for processed cereal-based food in young children. Because the risk was higher in the 2- to 5-year-old children, we recommend special regulations for this group in Chile.

Removal of Ochratoxin A from Red Wine Using Alginate-PVA-L. plantarum (APLP) Complexes: A Preliminary Study.

The presence of ochratoxin A (OTA) in wines is a problem mainly due to the health damage it can cause to frequent drinkers. A method for removing these toxic substances from wine is the use of lactic acid bacteria with mycotoxin-adsorption capacities; however, their use is limited since a matrix in which they can be immobilized, to remove them after use, is needed. In this study, L. plantarum (LP) was encapsulated in a polymeric matrix composed of polyvinyl alcohol (PVA) and alginate, forming alginate-PVA-LP (APLP) complexes. Then, these complexes were characterized, and assays of OTA and phenol removal from wines were performed. As a result, it was observed that the APLP complexes at a concentration of 0.5 g mL-1 removed over 50% of the OTA without substantially affecting the concentration of total phenols. In addition, it was determined that the presence of L. plantarum directly affected the ability to adsorb OTA from wines and did not decrease the total phenols. In conclusion, an alginate-PVA matrix allows immobilizing LP, and the complexes formed are an alternative for removing ochratoxin from contaminated wines.

PCV2 infection aggravates OTA-induced immunotoxicity in vivo and in vitro.

Ochratoxin A (OTA), frequently existing in the food and feeds, could induce immunotoxicity. Porcine circovirus type 2 (PCV2), as a primary causative agent of porcine circovirus-associated disease, also could induce immunosuppression. However, it is still unknown whether PCV2 infection impacts OTA-induced immunotoxicity. The pigs and porcine alveolar macrophages (PAMs) were used as the model in the present experiment. The results in vivo indicated that PCV2 infection exacerbated OTA-induced immunotoxicity, NF-κB p65 phosphorylation, and TLR4 and MyD88 mRNA and protein expression in spleen. The results in vitro showed that OTA at 7.0 and 9.0 µM decreased cell viability and increased LDH release of PAMs without PCV2 infection. However, with PCV2 infection, OTA at 5.0, 7.0 and 9.0 µM significantly decreased cell viability and increased LDH release compared with absence of PCV2 infection. In addition, OTA at 5.0 and 7.0 µM significantly increased Annexin V/PI-positive rate, apoptosis of nuclear, γ-H2AX foci, IL-1α and TNF-α expression in PAMs with PCV2 infection compared with absence of PCV2 infection. In addition, PCV2 infection enhanced OTA-induced TLR4 and MyD88 mRNA and protein expression and NF-κB p65 phosphorylation. Knockdown of TLR4 alleviated the exacerbating effects of PCV2 infection on OTA-induced cytotoxicity, apoptosis and DNA damage in PAMs. These results indicated that PCV2 infection aggravated OTA-induced immunotoxicity and reduced the dose of OTA-induced immunotoxicity via TLR4/NF-κB p65 signaling pathway, which could provide basis for establishing limits for OTA.

Antifungal and antiocratoxigenic potential of Alpinia speciosa and Cymbopogon flexuosus essential oils encapsulated in poly(lactic acid) nanofibres against Aspergillus fungi.

Essential oils encapsulated in a polymeric matrix can be used as an alternative method to control fungi and mycotoxins. The essential oils were extracted by hydrodistillation and characterized by gas chromatography. The nanofibres were produced from poly (acid lactic) (PLA) containing essential oils by the Solution Blow Spinning method. The antifungal and antimicotoxygenic properties were evaluated against Aspergillus ochraceus and Aspergillus westerdijkiae by the fumigation method. Terpinen-4-ol (20·23%), sabinene (20·18%), 1·8-cineole (16·69%) and γ-terpinene (11·03%) were the principal compounds present in the essential oil from Alpinia speciosa, whereas citral (97·67%) was dominant from Cymbopogon flexuosus. Microscopy images showed that the addition of essential oils caused an increase in the diameter of the nanofibres. The infrared spectroscopy results indicated the presence of essential oils in the PLA nanofibres. Differential scanning calorimetry curves also indicated the existence of interactions between the essential oils and polymeric macromolecules through their plasticizing action. The hydrophobic character of nanofibres was revealed by the contact angle technique. An antifungal effect was observed, the mycelial growths (3·25-100%) and the synthesis of ochratoxin A (25·94-100%) were inhibited by the presence of the nanofibres. The results suggest that bioactive nanofibres hold promise for application to control toxigenic fungi.

Antifungal and physicochemical properties of Ocimum essential oil loaded in poly(lactic acid) nanofibers.

Poly(lactic acid) (PLA) nanofibres containing different proportions of the essential oils from Ocimum basilicum L. and Ocimum gratissimum L. were prepared by solution blow spinning method. The essential oils were extracted by hydrodistillation and characterized by gas chromatography. MEV, contact angle, DSC and FTIR were used to characterize the nanofibres. The effect of bioative nanofibres on the growth of the fungus and on the production of ochratoxin A were evaluated using the fumigation test. Linalool, 1·8-cineole and camphor were the principal components of the essential oil from O. basilicum, and eugenol was the principal constituent in the oil from O. gratissimum. An increase in the average diameter of the nanofibres was observed with the addition of the essential oils. The essential oils acted as a plasticizer, resulting in a reduction in the crystallinity of the PLA. The encapsulation of essential oils in PLA nanofibres was verified by FTIR. An effective antifungal and antimicotoxygenic activity against Aspergillus ochraceus and Aspergillus westerdjikiae was observed for the bioative nanofibres. These results confirm the potential of PLA nanofibres containing the essential oils for the control of toxigenic fungi that cause the deterioration of food and are harmful to human health.

Aspergillus carbonarius-derived ochratoxins are inhibited by Amazonian Bacillus spp. used as a biocontrol agent in grapes.

Bacillus spp. have been used as a biocontrol strategy to eliminate/reduce toxic fungicides in viticulture. Furthermore, the presence of fungi that are resistant to commonly used products is frequent, highlighting the need for new biocontrol strains. Aspergillus carbonarius can produce ochratoxins, including ochratoxin A (OTA), which has a regulatory maximum allowable limit for grape products. The purpose of this study was to assess the ability of four Amazonian strains of Bacillus (P1, P7, P11, and P45) to biocontrol A. carbonarius and various forms of ochratoxins in grapes. Berries treated with strain P1 presented no fungal colonies (100% reduction), while P7, P11 and P45 strains caused a reduction of 95, 95 and 61% on fungal counts, respectively. Six forms of ochratoxin were found in the grapes inoculated with A. carbonarius, including ochratoxin α, ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide, and OTA. Four of these ochratoxin forms (ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide) are reported for the first time in grapes. These ochratoxins were identified using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS). All Bacillus strains inhibited the synthesis of OTA, which is the most toxic form of ochratoxin. No ochratoxin form was found when P1 and P7 were used. Although some forms of ochratoxin were detected in grapes treated with Bacillus spp. P11 and P45, the levels decreased by 97%. To our knowledge, this is the first report on the inhibition of Aspergillus carbonarius-derived ochratoxin by Bacillus species. P1 strain, identified as Bacillus velezensis, was found to be the most promising for completely inhibiting fungal growth and production of all ochratoxins.

Effect of Modified Clinoptilolite to Counteract the Deleterious Effects of Ochratoxin A on Egg Production and Quality

The objective of this study was to evaluate the efficacy of modified clinoptilolite as mycotoxins adsorbent in preventing negative effects of ochratoxin A (OTA) on egg production and egg quality of laying hens exposed to this mycotoxin. Forty-eight (n=48) laying hens (27 weeks old) were used in this study. The hens were randomly divided into six equal groups and were fed for 7 weeks with a standard diet in addition to: E-I group - 1 mg/kg OTA; E-II group 0.25 mg/kg OTA; E-III group 1mg/kg OTA + 0.2% of MZ; E-IV group 0.25 mg/kg OTA + 0.2% of MZ. MZ group of hens was fed with standard diets containing 0.2% of the adsorbent (MZ). The control group of hens was fed with standard diet, without any addition of OTA or MZ. The present study showed that laying hens fed with 1 mg/kg of OTA (E-I), had a significant decrease (p<0.05) of all performance parameters during the trial, while group fed with 0.25 mg/kg OTA have shown no adverse effects on egg production and egg quality. Addition of modified clinoptilolite (0.2%) to the diet containing the OTA, minimized these effects bringing values not significantly different from the control diet for most of the parameters. These findings clearly indicate the protective potential of modified clinoptilolite against the toxic effects of OTA in laying hens.(AU)