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20 pages, 1431 KiB  
Article
Mapping Variability of Mycotoxins in Individual Oat Kernels from Batch Samples: Implications for Sampling and Food Safety
by Irene Teixido-Orries, Francisco Molino, Bianca Castro-Criado, Monika Jodkowska, Angel Medina, Sonia Marín and Carol Verheecke-Vaessen
Toxins 2025, 17(1), 34; https://doi.org/10.3390/toxins17010034 (registering DOI) - 11 Jan 2025
Abstract
Oats are susceptible to contamination by Fusarium mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and T-2/HT-2 toxins, posing food safety risks. This study analyses the variation in levels of 14 mycotoxins in 200 individual oat kernels from two DON-contaminated batch samples (mean = 3498 [...] Read more.
Oats are susceptible to contamination by Fusarium mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and T-2/HT-2 toxins, posing food safety risks. This study analyses the variation in levels of 14 mycotoxins in 200 individual oat kernels from two DON-contaminated batch samples (mean = 3498 µg/kg) using LC-MS/MS. The samples also contained deoxynivalenol-3-glucoside (DON-3G), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and ZEN. Contamination levels varied notably among individual kernels, with DON detected in 70% of them, followed by DON-3G (24.5%) and 3-ADON (20.5%). Importantly, 8% of kernels exceeded the EU legal limit for DON (1750 µg/kg), and some occasionally surpassed limits for ZEN and T-2/HT-2. Correlation analyses revealed strong associations between DON and its derivatives but weaker correlations with other toxins. Mycotoxin ratios varied widely, indicating that although they often co-occur, their concentrations differ between kernels. Contamination did not significantly impact kernel weight, though a slight trend toward lower weights in contaminated kernels was noted. Additionally, sampling statistics showed that as the percentage of selected kernels increased, the probability of batch sample rejection for DON contamination rose significantly. The study highlights the heterogeneity of mycotoxin contamination in oat batches, emphasising the importance of accurate detection and regulatory compliance to ensure safer oat-based products. Full article
(This article belongs to the Special Issue Occurrence, Toxicity, Metabolism, Analysis and Control of Mycotoxins)
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Figure 1
<p>Correlation heat map matrix (represented with correlation coefficients) of the 14 analysed mycotoxins (µg/kg) and grain weight (g) in individual oat kernels. DON = deoxynivalenol; DON-3G = deoxynivalenol-3-glucoside; 15-ADON = 15-acetyldeoxynivalenol; DAS = diacetoxyscirpenol; 15-AS = 15-acetoxyscirpenol; 3-ADON = 15-acetyldeoxynivalenol; ZEN = zearalenone; OTA = ochratoxin A.</p>
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<p>Co-occurrence (%) of the mycotoxins detected in individual oat kernels. Other co-occurrences were not found and, therefore, are not presented in this figure. DON = deoxynivalenol; DON-3G = deoxynivalenol-3-glucoside; 15-ADON = 15-acetyldeoxynivalenol; DAS = diacetoxyscirpenol; 15-AS = 15-acetoxyscirpenol; 3-ADON = 15-acetyldeoxynivalenol; ZEN = zearalenone; HT-2 = HT-2 toxin; T-2 = T-2 toxin.</p>
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<p>Kernel weight (mg) according to the contamination of the detected mycotoxins (above the LOD or below the LOD). LOD = limit of detection; DON = deoxynivalenol; DON-3G = deoxynivalenol-3-glucoside; 15-ADON = 15-acetyldeoxynivalenol; DAS = diacetoxyscirpenol; 15-AS = 15-acetoxyscirpenol; 3-ADON = 15-acetyldeoxynivalenol; ZEN = zearalenone.</p>
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<p>Distribution of deoxynivalenol (DON) content (mg/kg) in kernels from sample 1, sample 2, and combined samples, with an expanded view near the legal limit for DON (1.75 mg/kg).</p>
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17 pages, 5927 KiB  
Article
Pulsed Electric Field Induces Significant Changes in the Metabolome of Fusarium Species and Decreases Their Viability and Toxigenicity
by Adam Behner, Jana Palicova, Anna-Hirt Tobolkova, Nela Prusova and Milena Stranska
Toxins 2025, 17(1), 33; https://doi.org/10.3390/toxins17010033 (registering DOI) - 11 Jan 2025
Viewed by 37
Abstract
Fusarium fungi are widespread pathogens of food crops, primarily associated with the formation of mycotoxins. Therefore, effective mitigation strategies for these toxicogenic microorganisms are required. In this study, the potential of pulsed electric field (PEF) as an advanced technology of increasing use in [...] Read more.
Fusarium fungi are widespread pathogens of food crops, primarily associated with the formation of mycotoxins. Therefore, effective mitigation strategies for these toxicogenic microorganisms are required. In this study, the potential of pulsed electric field (PEF) as an advanced technology of increasing use in the food processing industry was investigated to minimize the viability of Fusarium pathogens and to characterize the PEF-induced changes at the metabolomic level. Spores of four Fusarium species (Fusarium culmorum, Fusarium graminearum, Fusarium poae, and Fusarium sporotrichioides) were treated with PEF and cultured on potato dextrose agar (PDA) plates. The viability of the Fusarium species was assessed by counting the colony-forming units, and changes in the mycotoxin content and metabolomic fingerprints were evaluated by using UHPLC-HRMS/MS instrumental analysis. For metabolomic data processing and compound identification, the MS-DIAL (v. 4.80)–MS-CleanR–MS-Finder (v. 3.52) software platform was used. As we found out, both fungal viability and the ability to produce mycotoxins significantly decreased after the PEF treatment for all of the species tested. The metabolomes of the treated and untreated fungi showed statistically significant differences, and PEF-associated biomarkers from the classes oxidized fatty acid derivatives, cyclic hexapeptides, macrolides, pyranocoumarins, carbazoles, and guanidines were identified. Full article
(This article belongs to the Section Mycotoxins)
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<p>PCA (score scatter plot) including all samples (FC—<span class="html-italic">F. culmorum</span>; FG—<span class="html-italic">F. graminearum</span>; FP—<span class="html-italic">F. poae</span>; FS—<span class="html-italic">F. sporotrichioides</span>) colored according to the particular experimental conditions. Excluded PEF-treated samples are grouped with the “PDA blank” samples represented by gray dots in the left part of the plot.</p>
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<p>PCA (score scatter plots) of each <span class="html-italic">Fusarium</span> species dataset (FC—<span class="html-italic">F. culmorum</span>; FG—<span class="html-italic">F. graminearum</span>; FP—<span class="html-italic">F. poae</span>; FS—<span class="html-italic">F. sporotrichioides</span>) colored according to the particular experimental conditions.</p>
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<p>Selection of statistically significant features using VIP + ROC filters illustrated in the OPLS-DA S-plot.</p>
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<p>The extracted ion chromatograms (XICs) (<b>A</b>), match of experimental vs. in silico MS/MS spectra with chemical structures of fragments colored green (<b>B</b>), and boxplots before statistical analysis (<b>C</b>) of unique PEF-related biomarkers Compound<sup>1</sup> (<b>1</b>), Desotamide D (<b>2</b>), Megalomicin C1 (<b>3</b>), Compound<sup>2</sup> (<b>4</b>), Compound<sup>3</sup> (<b>5</b>), and Compound<sup>4</sup> (<b>6</b>). Tentative identifications and intensity trends of these biomarkers are presented in the FG and FP datasets. <sup>1</sup> 2-((3-(((2,3-dihydroxypropoxy)(hydroxy)phosphoryl)oxy)-2-hydroxypropoxy)(hydroxy)methyl)hexadecanoic acid. <sup>2</sup> 13-(hydroxymethyl)-19,19-dimethyl-3-(2-phenylethyl)-12-(propan-2-yl)-23-(propan-2-ylidene)-6,10,18,21-tetraoxapentacyclo[24.2.2.0<sup>7</sup>,<sup>20</sup>.0<sup>8</sup>,<sup>17</sup>.0<sup>9</sup>,<sup>14</sup>]triaconta-1(28),8,12,14,16,26,29-heptaene-5,11,22-trione. <sup>3</sup> [8-acetyl-12-(1-hydroxyethyl)-4,5-dimethoxy-14-methyl-17-oxo-8,14-diazatetracyclo[9.5.2.0¹,⁹.0²,⁷]octadeca-2(7),3,5,12-tetraen-10-yl]methyl acetate. <sup>4</sup> 1-[N′-[6-[[amino-[[N′-(2-hydroxyethyl)amidino]amino]methylene]amino]hexyl]amidino]-2-(2-hydroxyethyl)guanidine.</p>
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<p>Boxplots demonstrating the distribution of mycotoxin (diacetoxyscirpenol (DAS), deoxynivalenol (DON), neosolaniol (NEO), and zearalenone (ZEA)) abundance in PDA plates with fungi (FC—<span class="html-italic">F. culmorum</span>; FG—<span class="html-italic">F. graminearum</span>; FP—<span class="html-italic">F. poae</span>; FS—<span class="html-italic">F. sporotrichioides</span>) only with significant decreases after PEF treatment. Significance was statistically tested using the Wilcoxon rank-sum test (<span class="html-italic">p</span>-value &lt; 0.1). Red boxplots represent control samples, and green boxplots represent PEF-treated samples. The black dots in the boxplot represent each sample and the yellow square represents the average.</p>
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18 pages, 4143 KiB  
Article
Proteomic Analysis of the Murine Liver Response to Oral Exposure to Aflatoxin B1 and Ochratoxin A: The Protective Role to Bioactive Compounds
by Silvia Trombetti, Alessandra Cimbalo, Michela Grosso, Pilar Vila-Donat, Jordi Mañes and Lara Manyes
Toxins 2025, 17(1), 29; https://doi.org/10.3390/toxins17010029 - 9 Jan 2025
Viewed by 245
Abstract
Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of [...] Read more.
Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of fermented whey (FW) and pumpkin (P) as functional ingredients through a proteomic approach. For the experimental procedures, rats were fed AFB1 and OTA individually or in combination, with the addition of FW or a FW-P mixture during 28 days. For proteomics analysis, peptides were separated using a LC-MS/MS-QTOF system and differentially expressed proteins (DEPs) were statistically filtered (p < 0.05) distinguishing males from females. Gene ontology visualization allowed the identification of proteins involved in important biological processes such as the response to xenobiotic stimuli and liver development. Likewise, KEGG pathway analysis reported the metabolic routes as the most affected, followed by carbon metabolism and biosynthesis of amino acids. Overall, the results highlighted a strong downregulation of DEPs in the presence of AFB1 and OTA individually but not with the mixture of both, suggesting a synergistic effect. However, FW and P have helped in the mitigation of processes triggered by mycotoxins. Full article
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<p>Venn diagram representation of common DEPs for male (<b>A</b>) and female (<b>B</b>) rats exposed to mycotoxins versus the control. <span class="html-italic">p</span> &lt; 0.05 were significantly different from the control.</p>
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<p>Venn diagram representation of common DEPs for male (<b>A</b>) and female (<b>B</b>) rats exposed to FW and mycotoxins versus the corresponding mycotoxin. <span class="html-italic">p</span> &lt; 0.05 were significantly different from mycotoxins group.</p>
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<p>Venn diagram representation of common DEPs for male (<b>A</b>) and female (<b>B</b>) rats exposed to FW + P and mycotoxins versus the corresponding mycotoxin. <span class="html-italic">p</span> &lt; 0.05 were significantly different from mycotoxins group.</p>
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<p>Gene ontology (GO) functional annotation of differentially expressed proteins for biological processes and molecular functions of male (<b>A</b>,<b>C</b>) and female (<b>B</b>,<b>D</b>) rats exposed to FW + AFB1, FW + OTA, and FW + AFB1 + OTA compared with respective mycotoxins without functional ingredient.</p>
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<p>Gene ontology (GO) functional annotation of differentially expressed proteins for biological processes and molecular functions of male (<b>A</b>,<b>C</b>) and female (<b>B</b>,<b>D</b>) rats exposed to FW + P + AFB1, FW + P + OTA, and FW + P + AFB1 + OTA compared with respective mycotoxins without functional ingredients.</p>
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<p>Heatmap representation on the expression of DEPs involved in the main biological processes after AFB1, OTA, and the combination (AFB1 + OTA) exposure in presence of FW or FW + P in male (<b>A</b>) and female (<b>B</b>) rats compared to control. The red-to-green gradient represents the logarithmic fold change value for upregulation (Log2FC &gt; 0) and downregulation (Log2FC &lt; 0), respectively. Black box is log2FC = 0. <span class="html-italic">p</span> &lt; 0.05 significantly different from the mycotoxin groups.</p>
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<p>KEGG pathway visualization of significant signaling pathways in rats exposed to mycotoxins in combination with fermented whey (FW) (<b>A</b>) or fermented whey + pumpkin (FW + P) (<b>B</b>) feed related to the number of proteins involved compared with the exposure without functional ingredients.</p>
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<p>Heatmap representation of the expression of DEPs involved in the main signaling pathways after AFB1, OTA, and the combination (AFB1 + OTA) exposure in presence of FW or FW + P in male (<b>A</b>) and female (<b>B</b>) rat livers compared with the expression after exposure to mycotoxins without functional ingredients. The red-to-green gradient represents the logarithmic fold change value for upregulation (LogFC &gt; 0) and downregulation (LogFC &lt; 0), respectively. Black box is log2FC = 0. <span class="html-italic">p</span> &lt; 0.05 significantly different from the mycotoxin’s groups.</p>
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<p>KEGG pathway visualization showing key molecular events involved in the development of hepatocellular carcinoma (HCC). The diagram highlights the critical signaling pathways, including those related to cell cycle regulation, apoptosis, and metabolic alterations, which contribute to the initiation and progression of liver cancer. Red stars indicate DEPs found in this study after AFB1 and OTA exposure and bioactive ingredients.</p>
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19 pages, 3481 KiB  
Article
Holocellulose from a Winemaking By-Product to Develop a Biopolymeric System for Bacterial Immobilization: Adsorption of Ochratoxin A in Wine Model Solutions (Box–Behnken Design)
by Verónica Carrasco-Sánchez, V. Felipe Laurie, Marcelo Muñoz-Vera and Ricardo Ignacio Castro
Toxins 2025, 17(1), 26; https://doi.org/10.3390/toxins17010026 - 6 Jan 2025
Viewed by 402
Abstract
Significant agro-industrial waste is produced during the winemaking process, including grape stalks, which are a rich source of the valuable biopolymer holocellulose that can be utilized for biotechnological processes. The purpose of this study was to delignify grape stalks in order to extract [...] Read more.
Significant agro-industrial waste is produced during the winemaking process, including grape stalks, which are a rich source of the valuable biopolymer holocellulose that can be utilized for biotechnological processes. The purpose of this study was to delignify grape stalks in order to extract holocellulose. Then Lactobacillus plantarum (LP) was immobilized in the interstitial spaces of holocellulose and then coated with natural polymers (chitosan, Ch; and alginate, Al) to create the Holo-LP/Ch/Al complex. A physicochemical analysis of the system revealed strong bacterial immobilization and stability. The efficiency of the complex in adsorbing ochratoxin A (OTA) from wine model solutions was assessed using a Box–Behnken design under various pH, time, and concentration conditions. The results showed that at pH 3.0, 75.39 min, and a complex concentration of 43.82 mg mL−1, the best OTA removal (53.68%) took place. Because of its physicochemical interactions, the complex showed improved OTA adsorption in acidic environments. This study demonstrates the potential of biopolymeric systems based on holocellulose for reducing mycotoxin contamination in beverages and stabilizing bacterial cells. These results offer a viable way to increase food safety and value winemaking by-products. Full article
(This article belongs to the Special Issue Mitigation and Detoxification Strategies of Mycotoxins)
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<p>Development of a biopolymeric system for bacterial immobilization. (<b>A</b>) Lignin elimination from stalk grape; (<b>B</b>) Holo-LP complex formation; (<b>C</b>) Holo-LP/Ch/Al Complex Formation.</p>
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<p>Analysis of control and holocellulose: (<b>A</b>) TG curve; (<b>B</b>) DTG curves; (<b>C</b>) deconvolution curves of control; (<b>D</b>) deconvolution curves of holocellulose.</p>
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<p>(<b>A</b>) FTIR spectra, holocellulose (red line), and control (blue line); (<b>B</b>) range between 3800 at 2700 cm<sup>−1</sup>; (<b>C</b>) range between 1200 at 800 cm<sup>−1</sup>; (<b>D</b>) range between 1800 at 1300 cm<sup>−1</sup>.</p>
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<p>Scanning electronic microscopy (SEM); (<b>A</b>,<b>B</b>) waste from grape stalk of <span class="html-italic">Vitis vinifera</span> L. (control); (<b>C</b>,<b>D</b>) changes in waste due to mechanical; (<b>E</b>,<b>F</b>) Holocellulose; (<b>G</b>,<b>H</b>) representation of the formation of holocellulose.</p>
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<p>TGA of <span class="html-italic">Holo-LP/Ch/Al</span> complex and controls: (<b>A</b>) TG curve; (<b>B</b>) DTG curves relative loss mass.</p>
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<p>FT-IR spectra of different samples.</p>
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<p>(<b>A</b>) Pareto chart standardized effects; (<b>B</b>–<b>D</b>) three-dimensional response surface plot for the analytical response vs. the effect of A (pH), B (time; min.), and C (Concentrations; mg mL<sup>−1</sup>).</p>
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21 pages, 2044 KiB  
Review
Neurotoxicological Effects of Some Mycotoxins on Humans Health and Methods of Neuroprotection
by Aleksandra Kuć-Szymanek, Daria Kubik-Machura, Klaudia Kościelecka, Tomasz Męcik-Kronenberg and Lidia Radko
Toxins 2025, 17(1), 24; https://doi.org/10.3390/toxins17010024 - 6 Jan 2025
Viewed by 485
Abstract
Food contamination with mycotoxin-producing fungi increases the risk of many diseases, including neurological diseases closely related to the neurotoxicity of these toxins. Based on the latest literature data, we presented the association of common Fusarium mycotoxins with neurological diseases. Articles from 2001 to [...] Read more.
Food contamination with mycotoxin-producing fungi increases the risk of many diseases, including neurological diseases closely related to the neurotoxicity of these toxins. Based on the latest literature data, we presented the association of common Fusarium mycotoxins with neurological diseases. Articles from 2001 to 2024 were analyzed. The mechanisms underlying the neurotoxicity of the described mycotoxins were presented. They are mainly related to the increase in oxidative stress in neuronal cells, which leads to higher levels of pro-inflammatory cytokines as IL-1β, IL-6 and TNF-α, enzymatic activity as GST, GPx, CAT and SOD and neurotransmitter dysfunction (5-HT, serotonin, dopamine and GABA). At the end of the article, based on the literature data, we attempted to present ways to mitigate mycotoxin neurotoxicity using mainly natural substances of plant origin. The data in this review focus on the Fusarium mycotoxins most frequently found in food and will be useful as comparative information for future studies. It is important to conduct further studies to mitigate the neurotoxic effects of Fusarium mycotoxins in order to reduce the development of diseases of the nervous system. Full article
(This article belongs to the Special Issue Mitigation and Detoxification Strategies of Mycotoxins)
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Graphical abstract
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<p>Chemical structure of ZEA.</p>
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<p>The general mechanisms affected by the mycotoxins ZEA, α-ZEL and β-ZEL are shown based on published data generated in neuronal cultures or collected in vivo by examination of the brain.</p>
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<p>Chemical structure of T-2 and HT-2.</p>
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<p>Chemical structure of DON.</p>
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<p>Chemical structure of BEA and ENN.</p>
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<p>Chemical structure of MPA.</p>
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<p>Chemical structure of MON.</p>
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<p>Chemical structure of STG and CPZ.</p>
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15 pages, 3054 KiB  
Article
Detection of Mycotoxins and Aflatoxigenic Fungi Associated with Compound Poultry Feedstuffs in Saudi Arabia
by Youssuf A. Gherbawy, Karima E. Abdel Fattah, Abdullah Altalhi, Pet Ioan and Mohamed A. Hussein
Microbiol. Res. 2025, 16(1), 11; https://doi.org/10.3390/microbiolres16010011 - 6 Jan 2025
Viewed by 277
Abstract
Poultry feeds with cereal grain-based constituents are vulnerable to fungal contamination during the processing and storage stages. A total of 100 samples of compound poultry feedstuffs were collected from the cities of Riyadh, Alhassa, Qassim, and Jeddah in Saudi Arabia. A quantitative enumeration [...] Read more.
Poultry feeds with cereal grain-based constituents are vulnerable to fungal contamination during the processing and storage stages. A total of 100 samples of compound poultry feedstuffs were collected from the cities of Riyadh, Alhassa, Qassim, and Jeddah in Saudi Arabia. A quantitative enumeration of fungal colony-forming units (CFUs) was performed on Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and Czapek Iprodione Dichloran Agar (CZID) media. Aspergillus flavus was the most predominant species, accounting for 18.714 × 103 and 3.956 × 103 CFU/g, with frequencies of 84 and 42% in the feed samples on DRBC and CZID media, respectively. The levels of different mycotoxins were estimated by the HPLC technique. One hundred percent of the compound poultry feedstuff samples were contaminated by mycotoxins such as AFB1, AFB2, AFG1, AFG2, FB1, DON, T2, OTA, and ZEN. Aflatoxins were recorded in 84% of the tested samples, of which 70 samples were contaminated by AFB1, ranging from 0.03 to 0.40 μg/kg. The aflatoxin analysis of the fungal species revealed that 89% and 100% of A. flavus and A. parasiticus isolates were aflatoxigenic, and all of them exhibited the presence of the aflR, omt-1, ver-1, and nor-1 genes. According to the PCR protocol based on FLA1, two primers were successful in directly and rapidly detecting A. flavus in the poultry feedstuff samples. Full article
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<p>Heatmap of CFUs, calculated per g dry feedstuff sample, of fungal species isolated from compound poultry feed samples (<span class="html-italic">n</span> = 100) collected from Riyadh, Alhassa, Qassim, and Jeddah cities on (<b>a</b>) DRBC and (<b>b</b>) CZID media at 27 °C.</p>
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<p>Number of cases of isolation (NCI) of fungal species isolated from compound poultry feedstuff (n = 100) gathered from Riyadh, Alhassa, Qassim, and Jeddah cities on (<b>a</b>) DRBC and (<b>b</b>) CZID media at 27 °C.</p>
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<p>Positive samples from mycotoxin analysis (μg/kg<sup>−1</sup>) of 100 compound poultry feedstuff samples collected from Riyadh, Alhassa, Qassim, and Jeddah areas.</p>
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<p>Agarose gel electrophoresis of PCR products of DNA fragments specific for <span class="html-italic">Aspergillus flavus</span> using FLA1 and FLA2 primers. Lane 1: representative sample collected from Riyadh; lane 2: representative sample from Alhassa; lane 3: positive control; lane 4: representative sample from Qassim; lane 5: representative sample from Jeddah; lane 6: <span class="html-italic">Aspergillus flavus</span>-free sample; lane 7: negative control. M: DNA marker.</p>
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13 pages, 866 KiB  
Article
In Vivo Effectiveness of Pleurotus ostreatus in Degradation of Toxic Metabolites of Filamentous Fungi Such as Aflatoxin B1 and Zearalenone
by Agnieszka Zapaśnik, Marcin Bryła, Adrian Wojtczak and Barbara Sokołowska
Metabolites 2025, 15(1), 20; https://doi.org/10.3390/metabo15010020 - 5 Jan 2025
Viewed by 451
Abstract
Background/Objectives: Mycotoxins, secondary metabolites synthesized by filamentous fungi, have been classified as dangerous substances and proven to be carcinogenic, as well as to have genotoxic, nephrotoxic, hepatotoxic, teratogenic, and mutagenic properties. Despite numerous trials to develop an effective and safe-for-human-health method of detoxification, [...] Read more.
Background/Objectives: Mycotoxins, secondary metabolites synthesized by filamentous fungi, have been classified as dangerous substances and proven to be carcinogenic, as well as to have genotoxic, nephrotoxic, hepatotoxic, teratogenic, and mutagenic properties. Despite numerous trials to develop an effective and safe-for-human-health method of detoxification, there is still a high risk associated with the occurrence of these toxins in food and feed. Biological methods of food preservation are an alternative option to conventional chemical and physical methods, characterized by their less negative impact on human health as well as their high efficiency against filamentous fungi and other foodborne pathogens. Mycoremediation is a new biotechnique based on the capability of fungi to detoxify matrices from various pullulans. Ligninolytic enzymes produced by white rot fungi (WRF) characterize a high efficiency in the degradation of various mycotoxins. Methods: In our study, Pleurotus ostreatus, as a representative of WRF, was cultivated on a medium contaminated by AFB1 and ZEN (mushroom substrate and maize) in a few variants of concentration. After the cultivation, medium and fruiting bodies were collected and analyzed with the usage of HPLC and LC/MS methods. Results: The reduction oscillated between 53 and 87% (AFB1) and 73 and 97% (ZEN) depending on the initial concentration of toxins in the medium. Grown fruiting bodies contained insignificant amounts of both toxins. Conclusions: These findings confirm the potential of P. ostreatus as an effective biological agent for reducing mycotoxins in contaminated medium, highlighting its applicability in developing sustainable and safe methods for detoxification. Full article
(This article belongs to the Special Issue Metabolic Properties of Microbial Cells or Enzymes)
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<p>The reduction in toxins (AFB1 and ZEN) in spent mushroom medium after the cultivation. Medium 1 (100 μg/kg AFB1), Medium 2 (150 μg/kg AFB1), Medium 3 (300 μg/kg AFB1), Medium 4 (150 μg/kg ZEN), Medium 5 (300 μg/kg ZEN), and Medium 6 (1000 μg/kg ZEN). Groups labeled with different letters (a, b) indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between the control and the respective medium based on one-way ANOVA.</p>
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14 pages, 276 KiB  
Article
Exploring the Potential Effects of Soybean By-Product (Hulls) and Enzyme (Beta-Mannanase) on Laying Hens During Peak Production
by Muhammad Shuaib, Abdul Hafeez, Deependra Paneru, Woo Kyun Kim, Muhammad Tahir, Anthony Pokoo-Aikins, Obaid Ullah and Abubakar Sufyan
Animals 2025, 15(1), 98; https://doi.org/10.3390/ani15010098 - 4 Jan 2025
Viewed by 373
Abstract
This study determined the interaction between soybean hulls (SHs) and enzymes (β-mannanase) to improve the sustainability and efficacy of feeding programs for laying hens during peak production while ensuring the best health and efficiency. In a completely randomized design (CRD), 200 golden-brown hens [...] Read more.
This study determined the interaction between soybean hulls (SHs) and enzymes (β-mannanase) to improve the sustainability and efficacy of feeding programs for laying hens during peak production while ensuring the best health and efficiency. In a completely randomized design (CRD), 200 golden-brown hens were fed for four weeks (33 to 36 weeks) and randomly distributed into four groups, each containing four replicates of ten birds, with one group receiving a control diet (P0) and the others receiving diets that contained four combinations of SHs and enzymes (ENZs). e.g., 3% SHs and 0.02 g/kg ENZs (P1), 3% SHs and 0.03 g/kg ENZs (P2), 9% SHs and 0.02 g/kg ENZs (P3), and 9% SHs and 0.03 g/kg ENZs (P4). Although most egg quality measures remained similar, the P2 group showed enhanced (p = 0.630) egg weight, albumen weight, and height. Moreover, the P2 group improved gut (p < 0.05) shape by increasing villus width, height, crypt depth, and surface area throughout intestinal sections, while the P4 group markedly improved total cholesterol and LDL (p = 0.022) levels. The P1, P2, and P4 groups exhibited a significant enhancement in dry matter (p = 0.022) and crude fiber (p = 0.046) digestibility, while the P2 group demonstrated superior crude protein digestibility (p = 0.032), and the P1 and P2 groups showed increased crude fat digestibility compared to the other groups. In conclusion, adding 3% of SHs and 30 mg/kg of ENZs (β-mannanase) to the feed may help laying hens, enhance gut health and some egg quality indices, and decrease blood cholesterol and LDL levels without compromising nutrient digestibility. Full article
25 pages, 2852 KiB  
Article
Effects of Mixing Ratio and Lactic Acid Bacteria Preparation on the Quality of Whole-Plant Quinoa and Whole-Plant Corn or Stevia Powder Mixed Silage
by Chao He, Qian Li, Huaidong Xiao, Xuchun Sun, Zepeng Gao, Yuan Cai and Shengguo Zhao
Microorganisms 2025, 13(1), 78; https://doi.org/10.3390/microorganisms13010078 - 3 Jan 2025
Viewed by 357
Abstract
Quinoa is the only single plant that can meet all the nutritional needs of human, and its potential for feed utilization has been continuously explored, becoming a prosperous industry for poverty alleviation. In order to further tap the feeding value of whole quinoa, [...] Read more.
Quinoa is the only single plant that can meet all the nutritional needs of human, and its potential for feed utilization has been continuously explored, becoming a prosperous industry for poverty alleviation. In order to further tap the feeding value of whole quinoa, develop quinoa as a feed substitute for conventional crops such as corn, and improve its comprehensive utilization rate, this experiment analyzed the silage quality and mycotoxin content of mixed silage of whole-plant quinoa (WPQ) with whole-plant corn (WPC) or stevia powder(SP) in different proportions, and further improved the silage quality of mixed silage by using two lactic acid bacteria preparations (Sila-Max and Sila-Mix). The quality, microbial population, and mycotoxin levels of quinoa and corn silage, as well as that of the mixed silage of quinoa and stevia, were evaluated using single-factor analysis of variance. The impact of various lactic acid bacteria preparations on the quality of whole-quinoa and whole-corn mixed silage was investigated through two-factor analysis of variance. WPQ and WPC were mixed at the ratio of 5:5 (QB5), 6:4 (QB6), 7:3 (QB7), 8:2 (QB8), 9:1 (QB9) and 10:0 (QB10). SP was mixed with WPQ at the supplemental levels of 0.2% (QB10S2), 0.4% (QB10S4), 0.6% (QB10S6), 0.8% (QB10S8) and 1.0% (QB10S10). After 60 days of silage, the silage indexes, the number of harmful microorganisms, and the mycotoxin levels were measured, to explore the appropriate ratio of mixed silage. The membership function analysis showed that the quality of mixed silage of WPQ with SP was better, and the optimal addition amount of SP was 0.6%. The results of Max and Mix on the quality improvement test of WPQ with WPC mixed silage showed that the two lactic acid bacteria formulations increased CP and AA content, and reduced NH3-N/TN; pH was significantly lower than the control group (p < 0.01), and LA was significantly higher than the control group (p < 0.01). The microbial count results showed that the addition of lactic acid bacteria preparation significantly reduced the number of molds and aerobic bacteria, and the effect of Mix was better than that of Max. When the mixing ratio was between QB7 and QB10, mold was not detected in the lactic-acid-bacteria preparation groups. Max and Mix significantly reduced the levels of mycotoxins, both of which were far below the range of feed safety testing, and 16S rRNA sequencing revealed that the silage microbiota varied with different mixing ratios and whether lactic acid bacteria preparations were used. Max and Mix increased the relative abundance of Firmicutes, with Mix having a more significant effect, especially in the QB6 (65.05%) and QB7 (63.61%) groups. The relative abundance of Lactobacillus was significantly higher than that of the control group (p < 0.05). The relative abundance of Enterobacteriaceae and Streptococcus were negatively and positively correlated with the addition level of quinoa, respectively. Comprehensive analysis showed that adding 0.6% SP to the WPQ and using Mix in mixed silage of WPQ and WPC with the proportion of WPQ no less than 70% had the best silage effect, and was more beneficial to animal health. Full article
(This article belongs to the Special Issue Gastrointestinal Fermentation and Microbiota)
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<p>The number of aerobic bacteria (<b>A</b>) and molds (<b>B</b>) in mixed silage (Test 1). The different lowercase letters (a–h) above the column indicate significant differences among treatments (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Changes in five mycotoxins’ content in two types of quinoa mixed silage (Test 1). The different lowercase letters (a–h) above the column indicate significant differences among treatments (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of additives on mycotoxins in mixed silage of WPQ and WPC (Test 2). The different lowercase letters (a–i) above the column indicate significant differences among treatments (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>(<b>A</b>) Multi-sample rarefaction curves. (<b>B</b>) Multi-sample shannon curves (Test 2).</p>
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<p>Beta diversity analysis. (<b>A</b>) Principal coordinates analysis (PCoA). (<b>B</b>) Unweighted Pair group Method with Arithmetic (UPGMA) Mean. (<b>C</b>) ANOSIM analysis box plot (Test 2).</p>
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<p>Column diagram of horizontal distribution between groups. (<b>A</b>) Composition of bacteria community at phylum level. (<b>B</b>) Composition of bacteria community at genus level (Test 2).</p>
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17 pages, 540 KiB  
Article
Mycotoxicological Assessment of Broiler Compound Feed: A Multi-Year Analysis of Five Mycotoxins in a Romanian Feed Mill
by Dragoș Mihai Lăpușneanu, Silvia-Ioana Petrescu, Cristina-Gabriela Radu-Rusu, Mădălina Matei and Ioan Mircea Pop
Agriculture 2025, 15(1), 84; https://doi.org/10.3390/agriculture15010084 - 2 Jan 2025
Viewed by 390
Abstract
Mycotoxins are secondary metabolites of filamentous fungi that cause massive agricultural losses worldwide and constitute a significant health problem for humans and animals. The aim of this five-year study was to investigate the contamination of compound feed for broiler chickens at all stages [...] Read more.
Mycotoxins are secondary metabolites of filamentous fungi that cause massive agricultural losses worldwide and constitute a significant health problem for humans and animals. The aim of this five-year study was to investigate the contamination of compound feed for broiler chickens at all stages (starter, grower and finisher) from a feed mill in Romania with mycotoxins such as total aflatoxins (AFT), deoxynivalenol (DON), fumonisins (FUMs), ochratoxin A (OTA) and zearalenone (ZEN). AFT was detected in 49.3–72.2% of the samples with concentrations ranging from 0.01 to 5.2 µg/kg. DON was detected in 77.6–98.9% of the samples, with maximum concentrations ranging from 330 to 1740 µg/kg. FUM contamination ranged from 42.7% to 87.2%, with maximum levels between 460 and 1400 µg/kg. OTA was present in 44.2–87.9% of the samples, with maximum concentrations reaching 21.4 µg/kg. ZEN was consistently elevated at all feeding stages, being detected in 86.5–97.4% of the samples, with maximum levels of 89.4 µg/kg. Mycotoxin co-occurrence was common in the samples, with the most common combination of four mycotoxins occurring in 38.51% of the samples. Samples were collected from storage bunkers, homogenised and analysed in certified laboratories, with sampling procedures varying according to batch size to ensure representativeness. Investigation of the transfer of mycotoxins into animal products and the combined effects of mycotoxins on animal health, including potential synergistic or antagonistic interactions, is particularly relevant. This study emphasises the essential role of comprehensive and continuous monitoring of mycotoxins in protecting animal health and food safety. Full article
(This article belongs to the Special Issue Farming Factors’ Influence on Animal Productions)
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<p>Co-occurrence of mycotoxins in broiler feed over the five-year study period.</p>
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26 pages, 6972 KiB  
Article
Exposure to Subclinical Doses of Fumonisins, Deoxynivalenol, and Zearalenone Affects Immune Response, Amino Acid Digestibility, and Intestinal Morphology in Broiler Chickens
by Revathi Shanmugasundaram, Laharika Kappari, Mohammad Pilewar, Matthew K. Jones, Oluyinka A. Olukosi, Anthony Pokoo-Aikins, Todd J. Applegate and Anthony E. Glenn
Toxins 2025, 17(1), 16; https://doi.org/10.3390/toxins17010016 - 1 Jan 2025
Viewed by 638
Abstract
Fusarium mycotoxins often co-occur in broiler feed, and their presence negatively impacts health even at subclinical concentrations, so there is a need to identify the concentrations of these toxins that do not adversely affect chickens health and performance. The study was conducted to [...] Read more.
Fusarium mycotoxins often co-occur in broiler feed, and their presence negatively impacts health even at subclinical concentrations, so there is a need to identify the concentrations of these toxins that do not adversely affect chickens health and performance. The study was conducted to evaluate the least toxic effects of combined mycotoxins fumonisins (FUM), deoxynivalenol (DON), and zearalenone (ZEA) on the production performance, immune response, intestinal morphology, and nutrient digestibility of broiler chickens. A total of 960 one-day-old broilers were distributed into eight dietary treatments: T1 (Control); T2: 33.0 FUM + 3.0 DON + 0.8 ZEA; T3: 14.0 FUM + 3.5 DON + 0.7 ZEA; T4: 26.0 FUM + 1.0 DON + 0.2 ZEA; T5: 7.7 FUM + 0.4 DON + 0.1 ZEA; T6: 3.6 FUM + 2.5 DON + 0.9 ZEA; T7: 0.8 FUM + 1.0 DON + 0.3 ZEA; T8: 1.0 FUM + 0.5 DON + 0.1 ZEA, all in mg/kg diet. The results showed that exposure to higher mycotoxin concentrations, T2 and T3, had significantly reduced body weight gain (BWG) by 17% on d35 (p < 0.05). The T2, T3, and T4 groups had a significant decrease in villi length in the jejunum and ileum (p < 0.05) and disruption of tight junction proteins, occludin, and claudin-4 (p < 0.05). Higher mycotoxin groups T2 to T6 had a reduction in the digestibility of amino acids methionine (p < 0.05), aspartate (p < 0.05), and serine (p < 0.05); a reduction in CD4+, CD8+ T-cell populations (p < 0.05) and an increase in T regulatory cell percentages in the spleen (p < 0.05); a decrease in splenic macrophage nitric oxide production and total IgA production (p < 0.05); and upregulated cytochrome P450-1A1 and 1A4 gene expression (p < 0.05). Birds fed the lower mycotoxin concentration groups, T7 and T8, did not have a significant effect on performance, intestinal health, and immune responses, suggesting that these concentrations pose the least negative effects in broiler chickens. These findings are essential for developing acceptable thresholds for combined mycotoxin exposure and efficient feed management strategies to improve broiler performance. Full article
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<p>Representative hematoxylin and eosin-stained images of jejunum and ileum on d35.</p>
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<p>Effect of combined doses of mycotoxins on intestinal lesion score. Three birds from each pen were scored for intestinal lesions on d35, using a 0–3 scale: 0 being normal, 1 indicating a mild mucus covering the small intestine, 2 indicating a necrotic small intestinal mucosa, and 3 indicating sloughed cells and blood in the small intestinal mucosa and contents. Grey bar: lesion score of 0; red bar: lesion score of 1; lesion scores were analyzed with Chi-squared test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of combined doses of mycotoxins on jejunal tight junction protein mRNA expression. All the mean values represent fold changes of up- and downregulated gene expression compared to the control group. Means (+SEM) with no common superscript differ significantly (<span class="html-italic">p</span> &lt; 0.05) (<span class="html-italic">n</span> = 6). Relative gene expression levels are shown for the jejunum (<b>A</b>): Occludin (<b>B</b>): Z−occluden (<b>C</b>): Claudin−1 (<b>D</b>): Claudin−2 (<b>E</b>): Claudin−4.</p>
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<p>Effect of combined doses of mycotoxins on splenic macrophage nitric oxide assay. On d14, d21, d28, and d35, the splenocyte MNCs (1 × 10<sup>5</sup> cells) were isolated and stimulated in vitro with 10 µg/mL of LPS for 48 h, and nitric oxide concentration was measured in the culture supernatant using the Griess assay. Means (+SEM) with no common superscript differ significantly (<span class="html-italic">p</span> &lt; 0.05) (<span class="html-italic">n</span> = 6).</p>
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<p>Effect of combined doses of mycotoxins on total bile IgA. Bile samples were analyzed for total IgA at d14, d21, d28, and d35 and expressed as optical density values. Means (+SEM) with no common superscript differ significantly (<span class="html-italic">p</span> &lt; 0.05) (<span class="html-italic">n</span> = 6).</p>
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<p>Effect of combined doses of mycotoxins on liver cytochrome mRNA expression. All the mean values represent fold changes of up- and downregulated gene expression compared to the control group. Means (+SEM) with no common superscript differ significantly (<span class="html-italic">p</span> &lt; 0.05) (<span class="html-italic">n</span> = 6). Relative expression levels are shown for liver (<b>A</b>) CYP-1A1, (<b>B</b>) CYP-1A2, and (<b>C</b>) CYP-1A4.</p>
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<p>Effect of combined doses of mycotoxins on liver cytokines mRNA expression. All the mean values represent fold changes compared to the control group. Means (+SEM) with no common superscript differ significantly (<span class="html-italic">p</span> &lt; 0.05) (<span class="html-italic">n</span> = 6). Relative expression levels are shown for liver (<b>A</b>) IL−1 and (<b>B</b>) IL−10.</p>
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11 pages, 507 KiB  
Article
Nutrient Intake and Dietary Adequacy Among Rural Tanzanian Infants Enrolled in the Mycotoxin Mitigation Trial
by Rosemary A. Kayanda, Neema Kassim, Francis M. Ngure, Rebecca J. Stoltzfus and Erica Phillips
Nutrients 2025, 17(1), 131; https://doi.org/10.3390/nu17010131 - 31 Dec 2024
Viewed by 384
Abstract
Background: The Mycotoxin Mitigation Trial (MMT) was a community-based cluster-randomized trial designed to assess the effect of dietary aflatoxin (AF) on linear growth. Similar dietary intake between arms was an important component of the trial’s program theory and essential for the trial’s internal [...] Read more.
Background: The Mycotoxin Mitigation Trial (MMT) was a community-based cluster-randomized trial designed to assess the effect of dietary aflatoxin (AF) on linear growth. Similar dietary intake between arms was an important component of the trial’s program theory and essential for the trial’s internal validity and interpretation. Objective: This analysis assessed and compared dietary intake by arm within a sub-sample of infants enrolled in the MMT. Methods: Twenty paired clusters (10 per trial arm) out of the 52 MMT clusters were included in this sub-sample. Up to 15 maternal/infant dyads per cluster were randomly selected for a one-time, structured, multi-pass 24 h dietary recall. Data were collected at the midpoint of the trial, when infants were 12 months of age, over 8 calendar months. We evaluated and compared infant nutrient intake and adequacy of energy, protein, lipid, iron, zinc, calcium, and vitamin A between study arms. Nutrient intake by arm was estimated using mixed-level regression models. Results: A total of 282 mothers participated (n = 140 intervention arm and 142 standard of care (SoC) arm). The mean daily intakes of energy and lipid fed to infants were 505 kcal/day (SD = 225.9) and 13 g/day (SD = 6.9), respectively, in the intervention and SoC arms, with no difference between arms. Intervention infants consumed slightly more protein than SoC infants (13.7 v. 12.3 g/day, p = 0.02). Consumption of iron, zinc, calcium, and vitamin A were low and did not differ between arms. Conclusions: At the midpoint of the MMT, energy, lipid, and micronutrient intake did not differ between arms. Protein consumption was slightly greater in the intervention arm. Guided by the trial’s program theory, this analysis advances the interpretation of the MMT trial findings. Full article
(This article belongs to the Section Nutrition and Public Health)
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<p>Flow diagram of participants.</p>
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12 pages, 486 KiB  
Article
Effect of In Vitro Ruminal pH on Zearalenone Degradation and Interaction with Other Mycotoxins in a Static Gastrointestinal Model
by Rimvydas Falkauskas, Jurgita Jovaišienė, Gintarė Vaičiulienė, Sigita Kerzienė, Ingrida Jacevičienė, Eugenijus Jacevičius, Inga Jarmalaitė, Marija Ivaškienė, Gintaras Daunoras, Rasa Želvytė and Violeta Baliukonienė
Toxins 2025, 17(1), 13; https://doi.org/10.3390/toxins17010013 - 30 Dec 2024
Viewed by 383
Abstract
The degradation of zearalenone (ZEN) in the rumen of dairy cows is influenced by rumen pH, which is a key factor affecting this process. The aim of this study was to investigate the variation of ZEN in interaction with other mycotoxins at different [...] Read more.
The degradation of zearalenone (ZEN) in the rumen of dairy cows is influenced by rumen pH, which is a key factor affecting this process. The aim of this study was to investigate the variation of ZEN in interaction with other mycotoxins at different ruminal pH environments (physiological (pH 6.5) and acidic (pH 5.5)) using an in vitro rumen model. Rumen fluid was collected from the caudoventral part of the rumen of cows using a pharyngeal–esophageal probe. To determine the changes in different mycotoxins (ZEN; AFLB1; DON; T-2) in the rumen of cows, a model rumen system was used, and mycotoxins concentrations were detected by HPLC. The study found that at pH 6.5, ZEN alone and in combination with other mycotoxins (DON; T-2; AFLB1) significantly (p < 0.05) reduced ZEN levels compared to the rumen environment at pH 5.5. It was observed that α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) concentrations were generally higher at a rumen pH of 6.5 compared to pH 5.5, averaging 47.09 µg/L and 35.23 µg/L, respectively. Additionally, the frequency of detection for both α-ZEL and β-ZEL was greater at pH 6.5 than at pH 5.5. A comparison of α-ZEL concentrations in rumen samples at pH 5.5 showed a 20% increase from the 6th to the 9th hour of the test, while β-ZEL levels remained unchanged over the same period. Full article
(This article belongs to the Section Mycotoxins)
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<p>Change in α−ZEL and β−ZEL concentrations in physiological and acidic rumen conditions of dairy cows in vitro considering the duration of exposure.</p>
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18 pages, 2349 KiB  
Article
AcOTApks Gene-Based Molecular Tools to Improve Quantitative Detection of the Mycotoxigenic Fungus Aspergillus carbonarius
by Angelo Agnusdei, Rita Milvia De Miccolis Angelini, Francesco Faretra, Stefania Pollastro and Donato Gerin
Foods 2025, 14(1), 65; https://doi.org/10.3390/foods14010065 - 29 Dec 2024
Viewed by 362
Abstract
Ochratoxin A (OTA) is a mycotoxin, a common contaminant of grapes and their derivatives, such as wine, and classified as possible human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC). Aspergillus carbonarius is the main producer of OTA in [...] Read more.
Ochratoxin A (OTA) is a mycotoxin, a common contaminant of grapes and their derivatives, such as wine, and classified as possible human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC). Aspergillus carbonarius is the main producer of OTA in grapes. The stability of the molecule and the poor availability of detoxification systems makes the control of A. carbonarius in vineyards the main strategy used to reduce OTA contamination risk. Several molecular methods are available for A. carbonarius detection, but the correlation between the abundance of fungal population and OTA contamination needs to be improved. This study aimed at the development of innovative quantitative PCR (qPCR) and digital droplet PCR (ddPCR) tools to quantify the mycotoxigenic fractions of A. carbonarius strains on grapes, based on the key gene AcOTApks in the pathway of OTA biosynthesis. Different primers/probe sets were assessed, based on their specificity and sensitivity. This method allowed to quantify up to 100 fg∙µL−1 [cycle of quantification (Cq) = 37] and 10 fg∙µL−1 (0.38 copies∙µL−1) of genomic DNA (gDNA) from A. carbonarius mycelium in qPCR and ddPCR, respectively. The sensitivity as to artificially contaminated must samples was up to 100 conidia (Cq = 38) and 1 conidium (0.13 copies∙µL−1) with qPCR and ddPCR, respectively. Finally, the methods were validated on naturally infected must samples, and the quantification of the fungus was in both cases highly correlated (r = +0.8) with OTA concentrations in the samples. The results showed that both analytical methods can be suitable for improving the sustainable management of OTA contamination in grapes and their derivatives. Full article
(This article belongs to the Section Food Analytical Methods)
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<p>Linear regressions obtained by (<b>A</b>) Scorpion-qPCR assay on DNA from pure mycelium of <span class="html-italic">A. carbonarius</span> (AC49), ten-fold serial diluted from 10 fg·µL<sup>− 1</sup> to 10 ng·µL<sup>−1</sup>, and (<b>B</b>) Scorpion-qPCR assay on DNA from musts artificially contaminated with AC49 conidial suspension, from 10<sup>0</sup> to 10<sup>8</sup> conidia·mL<sup>−1</sup>.</p>
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<p>Linear regression obtained by (<b>A</b>) <span class="html-italic">AcOTApks</span>-based qPCR on DNA from pure mycelium of <span class="html-italic">A. carbonarius</span> (AC49), ten-fold serial diluted from10 fg·µL<sup>−1</sup> to 10 ng·µL<sup>−1</sup>, and (<b>B</b>) <span class="html-italic">AcOTApks</span>-based qPCR assay on DNA from musts artificially contaminated, with AC49 conidial suspension from 10<sup>0</sup> to 10<sup>8</sup> conidia·mL<sup>−1</sup>.</p>
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<p>Linear regression generated assessing ten-fold serial dilution (from10 fg·µL<sup>−1</sup> to 10 ng·µL<sup>−1</sup>) of DNA from pure mycelium of <span class="html-italic">A. carbonarius</span> (AC49), using (<b>A</b>) primer/probe set 2 and (<b>B</b>) primer/probe set 3. The model was elaborated by relating the logarithm of target DNA concentration to the logarithm of quantification in copies·µL<sup>−1</sup>. Plot obtained from ddPCR on DNA from pure mycelium of <span class="html-italic">A. carbonarius</span> (AC49) using (<b>C</b>) set 2 and (<b>D</b>) set 3. Blue dots represent positive events, while black dots under the threshold line (pink) represent negative events. (<b>E</b>) Linear regression model generated from ddPCR analysis of DNA from musts artificially contaminated with AC49 conidial suspension, from 10<sup>0</sup> to 10<sup>8</sup> conidia·mL<sup>−1</sup>.</p>
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<p>(<b>A</b>) Amplitude of fluorescence in the ddPCR plot of the droplets generated from the amplification of DNA samples from isolates AC49, AC32, AC35, AC44, AC46 and AC72. (<b>B</b>) <span class="html-italic">AcOTApks</span> partial genome sequences obtained with primer Seq-F1/Seq-R1. Single nucleotide polymorphisms (SNPs) in the isolates AC32, AC35, AC44, AC46 and AC72 were highlighted in positions 5931 and 5404. (<b>C</b>) Rate of ochratoxin A (OTA) production in the <span class="html-italic">A. carbonarius</span> isolates assessed. (<b>D</b>) Clustering of <span class="html-italic">A. carbonarius</span> isolates as a function of OTA accumulation at 8 days. The K-means clustering analysis categorized the 15 isolates into four groups, identified as representing 95.1% of the variability by use of the elbow method.</p>
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16 pages, 988 KiB  
Article
Assessment of Alternaria Toxins and Pesticides in Organic and Conventional Tomato Products: Insights into Contamination Patterns and Food Safety Implications
by Tommaso Pacini, Teresa D’Amore, Stefano Sdogati, Emanuela Verdini, Rita Bibi, Angela Caporali, Elisa Cristofani, Carmen Maresca, Serenella Orsini, Alessandro Pelliccia, Eleonora Scoccia and Ivan Pecorelli
Toxins 2025, 17(1), 12; https://doi.org/10.3390/toxins17010012 - 29 Dec 2024
Viewed by 717
Abstract
Alternaria toxins (ATs) are a group of toxins produced by Alternaria fungi that frequently contaminate tomatoes and tomato products. Recently, the European Food Safety Authority evaluated ATs for their genotoxic and carcinogenic properties. Alternaria infestation is often controlled using ad hoc treatment strategies [...] Read more.
Alternaria toxins (ATs) are a group of toxins produced by Alternaria fungi that frequently contaminate tomatoes and tomato products. Recently, the European Food Safety Authority evaluated ATs for their genotoxic and carcinogenic properties. Alternaria infestation is often controlled using ad hoc treatment strategies (fungicides). In this study, two analytical methods were developed, validated and applied for the determination of five ATs and 195 pesticides in tomato products collected from the Italian market. Two distinct groups, organic (n = 20) and conventional (n = 20) Italian tomato sauces, were characterized in depth. Tenuazonic acid, alternariol and alternariol monomethyl ether were found up to 517, 27 and 7.1 µg/kg, respectively, while pesticides were detected between 0.0026 and 0.0421 mg/kg in conventional products, and, interestingly, up to 0.0130 mg/kg in organic products. No correlation emerged between the detected levels of ATs and pesticides and the type of tomato cultivation, but the probability of pesticide contamination in conventional products was eight times higher than in organics. Some considerations about exposure assessment and risk characterization for ATs were also proposed in the overall population and in more sensitive and/or exposed subgroups, underlining the need for new focused toxicological and monitoring studies to establish reliable reference values. Moreover, these data highlight that fungicide treatments may not protect tomatoes from ATs contamination, although it may remove fungi infestation. As organic product consumption is increasing, it is important to lay down dedicated regulations for maximum permitted levels to ensure the food safety of these products that are often perceived by consumers as a healthier and environmentally friendlier choice. Full article
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<p>Heatmap of ATs contamination. Color range is set for each analyte from green (&lt;LOD) to red (maximum detected value). LOD: 23 µg/kg (TeA); 0.5 µg/kg (other ATs). Maximum detected values: 517 µg/kg (TeA); 27 µg/kg (other ATs).</p>
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<p>Heatmap of pesticide contamination. Color range is set for each analyte from green (&lt;LOD) to red (maximum detected value). LOD: 0.0008 mg/kg. Maximum detected value: 0.0421 mg/kg.</p>
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