Giuseppina Avantaggiato

The widespread occurrence of emerging mycotoxins (EMs) produced by Fusarium, Aspergillus, and Penicillium species has raised increasing concerns regarding food and feed safety. Mitigation strategies currently applied to control regulated mycotoxins in feed may also be effective in reducing contamination by EMs. This study comparatively evaluated the in vitro adsorption efficacy of two leonardites, eight natural smectites, and two modified clays (organoclays) against EMs produced by Fusarium, Aspergillus, and Penicillium spp. All materials were tested at two inclusion levels (0.1 and 0.5% w/v) under two pH conditions (pH 3 and 7), simulating the gastrointestinal environment of monogastric animals. Adsorption performance was strongly influenced by mycotoxin chemistry, adsorbent type, inclusion rate, and medium pH. Organoclays exhibited the highest and most consistent efficacy, achieving near-complete adsorption of beauvericin (BEA) and enniatins (ENNs) (>98–100%) at 0.1% (w/v), as well as high removal of mycophenolic acid (MYC. A.) and citrinin (CIT) (>90%) across both pH conditions. Natural smectites showed high but more selective adsorption, removing >90% of BEA and ENNs at low inclusion rates, while displaying limited efficacy toward fusaric acid (FA) and patulin (PAT). Leonardites demonstrated intermediate and material-dependent performance; leonardite L1 adsorbed approximately 90% of BEA at 0.1% (w/v), whereas ENN adsorption ranged from ~36% to 80% at the same inclusion rate and exceeded 90% only at higher dosages. None of the tested materials effectively adsorbed patulin (PAT) at pH 7; however, at pH 3, four smectites exhibited partial adsorption, and one trioctahedral smectite achieved more than 90% PAT adsorption under acidic conditions. Overall, organoclays displayed the broadest adsorption spectrum across structurally diverse mycotoxins, while smectites exhibited high selectivity driven by surface charge density and interlayer interactions. Leonardite-based materials showed moderate but highly variable adsorption performance, likely influenced by heterogeneity in humic functional groups and physicochemical properties. These findings highlight the need for tailored adsorbent selection or combined mitigation strategies to achieve effective mycotoxin control in the animal feed industry.

Ochratoxin A (OTA), Zearalenone (ZEA), and Fumonisins (FB) are common contaminants of poultry feed associated with oxidative damage and potentially dangerous residues in products from exposed animals. We investigated the molecular effects in broilers of a short-term (10 days) dietary exposure to OTA (0.26 mg/kg), ZEA (2.9 mg/kg), or FB (60 mg/kg) on cytochrome P450 enzymes (CYP), drug transporters (DT) and the antioxidant defence system. OTA markedly decreased serum antioxidant capacity, while all mycotoxins depressed reduced glutathione content and increased lipid peroxidation in the liver, indicating a hepatic pro-oxidant effect. All the tested mycotoxins also reduced both the activities and the gene expression of selected antioxidant enzymes in the liver and duodenum as a result of the modulation of the Nrf2/Keap1 pathway. Moreover, mycotoxins differentially altered the hepatic and intestinal gene expression of CYP enzymes (i.e., CYP2A6, CYP2C45, CYP3A4, and CYP1A isoforms). Finally, the transcription of selected DT (i.e., ABCB1, ABCC2 and ABCG2) was generally enhanced in both the liver and duodenum. In conclusion, short-term exposure to OTA, ZEA, or FB at dietary concentrations higher than those recommended in the EU, but occurring in third countries, not only disrupt the antioxidant defence but also affect the expression of CYP and DT, which might potentially alter the kinetics of drugs and toxicants. Our results provide new insights into mycotoxin adverse effects in the light to assess the effectiveness of new mitigation strategies that contribute to food and feed safety.

Aflatoxin B1 (AFB1) can impair the growth of chickens and reduce the quality of eggs and meat, resulting in significant economic losses. The inclusion of mycotoxin detoxifying agents (MyDA) with binding properties in the diet is an efficient tool to reduce their absorption rate in the gastrointestinal tract. Our aim was to investigate the ability of two innovative MyDA (SeOX, a feed additive featuring a tri-octahedral smectite mixed with lignocellulose, and CHS, a di-octahedral smectite functionalized with an organic non-toxic modifier) in both reducing the bio-accessibility and mitigating the adverse effects of AFB1 in broilers exposed for 10 days to concentrations approaching the European Union maximum limits in feed (0.02 mg/kg). The amount of AFB1 in the excreta of birds, collected over four consecutive days (starting on day 7), was significantly lower (p < 0.001) in the group exposed to AFB1 alone compared to the groups treated with either SeOX or CHS. The calculated bio-accessibility was decreased by nearly 30% with both MyDA. This positive effect was reflected by a significant reduction (p < 0.001) in the oxidative stress (measured as serum antioxidant capacity and hepatic lipid peroxidation) induced by AFB1. Although antioxidant enzyme activities and glutathione levels were unaffected by any treatment, AFB1 significantly induced (p < 0.001) the upregulation of CYP2A6 and the downregulation of Nrf2; the latter was reverted by each MyDA. Overall, these results demonstrate that the selected MyDA are effective in limiting the AFB1 absorption rate, thereby mitigating or even reverting the oxidative stress induced by AFB1 in broilers.

Mycotoxin detoxification by microorganisms offers a specific, economical, and environmentally sustainable alternative to physical/chemical methods. Three strains of B. subtilis, isolated from poultry farm environments and recognized by EFSA as safe in animal nutrition for all animal species, consumers, and the environment, were screened for their ability to remove mycotoxins. All of them demonstrated mycotoxin-dependent removal efficacy, being very effective against ZEA and its analogues (α- and β-ZOL, α- and β-ZAL, and ZAL) achieving up to 100% removal within 24 h under aerobic, anaerobic, and restrictive growth conditions with toxins as the sole carbon source. ZEA removal remained effective across a wide range of pH values (5–8), temperatures (20–40 °C), and at high toxin concentrations (up to 10 µg/mL). Additionally, up to 87% ZEA removal was achieved after 48 h of incubation (30 °C) of the strains in a contaminated liquid food model containing 1 µg/mL of the toxin. Mechanistic studies suggest that ZEA detoxification involves metabolic processes rather than physical adsorption or entrapment into bacterial cells. Enzymatic activities within the bacterial cells or associated with their cell walls likely play a role in the metabolization of the toxin. Interestingly, it has been observed that growth conditions and culture media can influence the metabolization and/or conjugation of the toxin, which can result in the production of various metabolites. Further investigation is needed to identify these metabolites and assess their safety.

The most frequent adverse effects of AFB1 in chicken are low performance, the depression of the immune system, and a reduced quality of both eggs and meat, leading to economic losses. Since oxidative stress plays a major role in AFB1 toxicity, natural products are increasingly being used as an alternative to mineral binders to tackle AFB1 toxicosis in farm animals. In this study, an in vivo trial was performed by exposing broilers for 10 days to AFB1 at dietary concentrations approaching the maximum limits set by the EU (0.02 mg/kg feed) in the presence or absence of turmeric powder (TP) (included in the feed at 400 mg/kg). The aims were to evaluate (i) the effects of AFB1 on lipid peroxidation, antioxidant parameters, histology, and the expression of drug transporters and biotransformation enzymes in the liver; (ii) the hepatic accumulation of AFB1 and its main metabolites (assessed using an in-house-validated HPLC-FLD method); (iii) the possible modulation of the above parameters elicited by TP. Broilers exposed to AFB1 alone displayed a significant increase in lipid peroxidation in the liver, which was completely reverted by the concomitant administration of TP. Although no changes in glutathione levels and antioxidant enzyme activities were detected in any treatment group, AFB1 significantly upregulated and downregulated the mRNA expression of CYP2A6 and Nrf2, respectively. TP counteracted such negative effects and increased the hepatic gene expression of selected antioxidant enzymes (i.e., CAT and SOD2) and drug transporters (i.e., ABCG2), which were further enhanced in combination with AFB1. Moreover, both AFB1 and TP increased the mRNA levels of ABCC2 and ABCG2 in the duodenum. The latter changes might be implicated in the decrease in hepatic AFB1 to undetectable levels (<LOD) in the TP supplemented group. Overall, our findings further support the use of TP as an effective feeding strategy to prevent AFB1-related adverse effects in broilers.

The Alternaria mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are mycotoxins, which can contaminate cereal-based raw materials. Today, wheat is one of the most important crops in temperate zones, and it is in increasing demand in the Western Balkans countries that are urbanizing and industrializing. This research aimed to investigate the occurrence and determine the concentration of Alternaria mycotoxins AOH, AME, and TEN in wheat samples from the Republic of Serbia and the Republic of Albania, harvested in the year 2020 in the period between 15 June and 15 July. A total of 80 wheat grain samples, 40 from each country, were analyzed by an QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. From the obtained results, it can be seen that the mean concentration of AOH was 3.3 µg/kg and AME was 2.2 µg/kg in wheat samples from Serbia, while TEN from both Serbia and Albania was under the limit of quantification (Alternaria mycotoxins have concentrations above the LOQ, which could be potentially considered a health hazard to both humans and animals.

Simultaneous removal of mycotoxins has been poorly addressed, and a limited number of studies have reported the efficacy of feed additives in sequestering a large spectrum of mycotoxins. In this study, a new mycotoxin-adsorbing agent was obtained by properly mixing a tri-octahedral smectite with a lignocellulose-based material. At a dosage of 1 mg mL⁻¹, these materials simultaneously adsorbed frequently occurring mycotoxins and did not exert a cytotoxic effect on intestinal cells. Chyme samples obtained by a simulated GI digestion did not affect the viability of Caco-2TC7 cells as measured by the MTT test. In addition, the chyme of the lignocellulose showed a high content of polyphenols (210 mg mL⁻¹ catechin equivalent) and good antioxidant activity. The properties of the individual constituents were maintained in the final composite, and were unaffected by their combination. When tested with a pool of seven mycotoxins at 1 µg mL⁻¹ each and pH 5, the composite (5 mg mL⁻¹) simultaneously sequestered AFB₁ (95%), FB₁ (99%), ZEA (93%), OTA (80%), T-2 (63%), and DON (22%). HT-2 adsorption did not occur. Mycotoxin adsorption increased exponentially as dosage increased, and occurred at physiological pH values. AFB₁, ZEA and T-2 adsorption was not affected by pH in the range 3–9, whereas OTA and FB₁ were adsorbed at pH values of 3–5. The adsorbed amount of AFB₁, ZEA and T-2 was not released when pH rose from 3 to 7. FB₁ and OTA desorption was less than 38%. Langmuir adsorption isotherms revealed high capacity and affinity for adsorption of the target mycotoxins. Results of this study are promising and show the potential of the new composite to remove mycotoxins in practical scenarios where several mycotoxins can co-occur.

The removal of mycotoxins from contaminated feed using lactic acid bacteria (LAB) has been proposed as an inexpensive, safe, and promising mycotoxin decontamination strategy. In this study, viable and heat-inactivated L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells were investigated for their ability to remove aflatoxin B₁ (AFB₁), ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON) from MRS medium and PBS buffer over a 24 h period at 37 °C. LAB decontamination activity was also assessed in a ZEA-contaminated liquid feed (LF). Residual mycotoxin concentrations were determined by UHPLC-FLD/DAD analysis. In PBS, viable L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells removed up to 57% and 30% of ZEA and DON, respectively, while AFB₁ and OTA reductions were lower than 15%. In MRS, 28% and 33% of ZEA and AFB₁ were removed, respectively; OTA and DON reductions were small (≤15%). Regardless of the medium, heat-inactivated cells produced significantly lower mycotoxin reductions than those obtained with viable cells. An adsorption mechanism was suggested to explain the reductions in AFB₁ and OTA, while biodegradation could be responsible for the removal of ZEA and DON. Both viable LAB strains reduced ZEA by 23% in contaminated LF after 48 h of incubation. These findings suggest that LAB strains of L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T may be applied in the feed industry to reduce mycotoxin contamination.

Aflatoxin B1 (AFB1), the mainly Aspergillus fungi derived mycotoxin, is well known for its carcinogenic effects on liver, and frequently occurs in food supplies, leading to fatal consequences in both farm animals and humans. Poultry, one of the most important segments of agro-industry, has been demonstrated to be extremely sensitive to AFB1 intake, which results in chickens’ low performance, decreased quality of both eggs and meat and a negative economic feedback. Oxidative stress caused by AFB1 plays a crucial role in chickens’ kidney damage by generating lipid peroxidation accompanied by a concomitant increase in the antioxidant enzymes involved in ROS metabolism (NADPH oxidase isoform 4 (NOX4) and its regulatory subunit p47-phox). The aim of the present work was to investigate the benefits of dietary supplementation, in chickens affected by AFB1 mycotoxicosis, using a new Feed additive (FA) containing a mixture of a tri-octahedral Na-smectite with a ligno-cellulose-based material an antioxidant adjuvant. Exposure of AFB1-treated chickens to the feed additive induced a significant down-regulation of both NOX4 and p47-phox genes expression levels. This trend was confirmed by their protein expression, demonstrating the great potential of the FA to counteract oxidative stress. To conclude, these results could open new perspectives in the methods of feeding chickens, using eco-friendly dietary supplements able to reduce AFB1-induced mycotoxicosis and to ameliorate poultry performances.