Aspergillus carbonarius, a common food-contaminating fungus, produces ochratoxin A (OTA) and poses a risk to human health. This study aimed to assess the inhibitory activity of tea tree essential oil and its main components, Terpene-4-ol (T4), α-terpineol (αS), and 3-carene (3C) against A. carbonarius. The study showed αS and T4 were the main antifungal components of tea tree essential oil, which primarily inhibit A. carbonarius growth through cell membrane disruption, reducing antioxidant enzyme activities (catalase, peroxidase, superoxide dismutase) and interrupting the tricarboxylic acid cycle. Furthermore, αS and T4 interacted with enzymes related to OTA biosynthesis. Molecular docking and molecular dynamics show that they bound mainly to P450 with a minimum binding energy of -7.232 kcal/mol, we infered that blocking the synthesis of OTA precursor OTβ. Our hypothesis was preliminarily verified by the detection of key substances in the OTA synthesis pathway. The results of UHPLC-QTOF-MS2 analysis demonstrated that T4 achieved a degradation rate of 43 % for OTA, while αS reached 29.6 %, resulting in final breakdown products such as OTα and phenylalanine. These results indicated that α-terpinol and Terpene-4-ol have the potential to be used as naturally safe and efficient preservatives or active packaging to prevent OTA contamination.

BACKGROUND: Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential.
RESULTS: A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL-1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions.
CONCLUSIONS: This study provides a polymerase chain reaction-like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors.

Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.

The contamination of mycotoxins poses a serious threat to global food security, hence the urgent need for simultaneous detection of multiple mycotoxins. Herein, two SERS nanoprobes were synthesized by embedded SERS tags (4-mercaptopyridine, 4MPy; 4-mercaptobenzonitrile, TBN) into the Au and Ag core-shell structure, and each was coupled with the aptamers specific to ochratoxin A (OTA) and zearalenone (ZEN). Meanwhile, a rigid enhanced substrate Indium tin oxide glass/AuNPs/Graphene oxide (ITO/AuNPs/GO) was combined with aptamer functionalized Au@AgNPs via π-π stacking interactions between the aptamer and GO to construct a surface-enhanced Raman spectroscopy (SERS) aptasensor, thereby inducing a SERS enhancement effect for the effective and swift simultaneous detection of both OTA and ZEN. The presence of OTA and ZEN caused signal probes dissociation, resulting in an inverse correlation between Raman signal intensity (1005 cm-1 and 2227 cm-1) and the concentrations of OTA and ZEN, respectively. The SERS aptasensor exhibited wide linear detection ranges of 0.001-20 ng/mL for OTA and 0.1-100 ng/mL for ZEN, with low detection limits (LOD) of 0.94 pg/mL for OTA and 59 pg/mL for ZEN. Furthermore, the developed SERS aptasensor demonstrated feasible applicability in the detection of OTA and ZEN in maize, showcasing its substantial potential for practical implementation.

Ochratoxin A (OTA) is a prevalent mycotoxin found in feed that causes significant kidney injury in animals. Further investigation was needed to devise strategies for treating OTA-induced kidney damage through the gut-kidney axis. Evidence indicates the crucial role of intestinal microbiota in kidney damage development. Inulin, a dietary fiber, protects kidneys by modulating intestinal microbiota and promoting short-chain fatty acid (SCFA) production. However, its precise mechanism in OTA-induced kidney damage remained unclear. In this study, chickens were orally administered OTA and inulin for 2 weeks to investigate inulin\'s effects on OTA-induced kidney damage and underlying mechanisms. The alteration of intestinal microbiota, SCFAs contents, and SCFA receptors was further analyzed. Results demonstrated that inulin supplementation influenced intestinal microbiota, increased SCFAs production, and mitigated OTA-induced kidney damage in chickens. The importance of microbiota in mediating inulin\'s renal protection was further confirmed by antibiotic and fecal microbiota transplantation experiments. Additionally, inulin exhibited antioxidant and anti-inflammatory properties, alleviating NLRP3 inflammasome activation and pyroptosis. In summary, inulin protected chickens from OTA-induced kidney damage, which might provide a potential strategy to mitigate the harmful effects of mycotoxins through prebiotics and safeguard renal health.

Ochratoxin A (OTA), as one of the most important and harmful mycotoxins, is classed as possible human carcinogen (group 2B). As we all know, DNA damage may cause genomic instability, cell cycle disorder, activation of DNA damage pathway, and stimulation of DNA repair system. To explore the roles of DNA damage repair protein (hMLH1) on OTA-induced G2 arrest, the DNA damage, chromosome aberration, cell cycle distribution and p53-p21 signaling pathway were evaluatd after different time OTA exposure (6, 12, 24, 48h) in immortalized human gastric epithelial cells (GES-1). Our results demonstrated that OTA exposure could trigger genomic instability, DNA damage and G2 phase arrest of GES-1 cells. At the same time, OTA treatment could increase the expression of hMLH1, and induce phosphorylation of the p53 protein, as well as p21, in response to DNA damage. Finally, inhibition of hMLH1 by siRNA effectively prevented the activation of p53-p21 signaling pathway and rescued the G2 arrest elicited by OTA. This study demonstrated that hMLH1-p53-p21 signaling pathway played an important role in DNA damage and G2 cell cycle arrest the mediated by OTA in GES-1 cells.

A novel electrochemical aptasensor was prepared for the simultaneous determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA). Composites of Au nanoparticles and polyethyleneimine-reduced graphene oxide (AuNPs/PEI-RGO) with good electrical conductivity and high specific surface area were employed as the supporting substrate, demonstrating the ability to provide more binding sites for aptamers and accelerate the electron transfer. Aptamers were immobilized on a AuNPs/PEI-RGO surface to specifically recognize AFB1 and OTA. A metal-organic framework of UiO-66-NH2 served as the signal carrier to load metal ions of Cu2+ and Pb2+, which facilitated the generation of independent current peaks and effectively improved the electrochemical signals. The prepared aptasensor exhibited sensitive current responses for AFB1 and OTA with a linear range of 0.01 to 1000 ng/mL, with detection limits of 6.2 ng/L for AFB1 and 3.7 ng/L for OTA, respectively. The aptasensor was applied to detect AFB1 and OTA in cereal samples, achieving results comparable with HPLC-MS, with recovery results from 92.5% to 104.1%. With these merits of high sensitivity and good selectivity and stability, the prepared aptasensor proved to be a powerful tool for evaluating contaminated cereals.

Phytic acid (PA) is a natural antioxidant with various biological activities, providing protective effects in multiple animals. Ochratoxin A (OTA) is a mold toxin commonly found in feed, which induces multi-organ damage, with kidney being the target organ of its toxicity. This study investigates the protective effects of PA on OTA-induced renal damage and its potential mechanisms in chicks. The results demonstrates that PA treatment restores OTA-induced renal pathological injuries, reverses the diminished activities of antioxidant enzymes, reduces the accumulation of malondialdehyde, and normalizes the expression of pro-inflammatory cytokines, which confirms that PA can alleviate OTA-induced renal damage. Further investigations reveal that OTA-induced renal injury accompanied by an increase in tissue iron content and the transcription levels of ferroptosis-related genes (TFR, ACSL4, and HO-1), and a decrease in the levels of SLC7A11 and GPX4. PA treatment reverses all these effects, indicating that PA mitigates OTA-induced renal ferroptosis. Moreover, PA supplementation improves intestinal morphology and mucosal function, corrects OTA-induced changes in the intestinal microbiota. Besides, PA microbiota transplantation alleviates renal inflammation and oxidative stress caused by OTA. In conclusion, PA plays a protective role against renal damage through the regulation of ferroptosis and the intestinal microbiota, possibly providing novel insights into the control and prevention of OTA-related nephrotoxicity.

As a highly toxic mycotoxin, ochratoxin A (OTA) is widely contaminating agricultural products and has various toxicological effects. Bioenzymes for OTA degradation have shown promising potential for detoxification. Other than the efficient amidohydrolase ADH3 previously, two novel amidohydrolases ADH1 and AMD3 were obtained in this study. During Escherichia coli expression, the expressed protein solubility was very low and will limit future industrial application. Here, high copy number integrations were screened, and the amidohydrolases were efficiently secretory expressed by Pichia pastoris GS115. The protein yields from 1.0 L of fermentation supernatant were 53.5 mg for ADH1, 89.15 mg for ADH3, and 79.5 mg for AMD3. The catalytic efficiency (Kcat/Km) of secretory proteins was 124.95 s-1 mM-1 for ADH3, 123.21 s-1 mM-1 for ADH1, and 371.99 s-1 mM-1 for AMD3. In comparison to E. coli expression, the active protein yields substantially increased 15.78-51.53 times. Meanwhile, two novel amidohydrolases (ADH1 and AMD3) showed much higher activity than ADH3 that produced by secretory expression.

1. This trial investigated the effect on embryo injected with ochratoxin A (OTA) and the growth performance, jejunal morphology and barrier of ducklings to 21 d old.2. Two hundred forty, fertilised eggs were individually weighed and randomly assigned to two groups, a control (CON) and the OTA treatment, according to average egg weight. On d 13 of embryonic development, the treatment group was injected with 8 ng OTA/g egg and the CON group was injected with NaHCO3 solution as a placebo. All newly hatched ducklings were assigned to the CON or OTA group based on the different treatments. Each treatment consisted of six replicates and each included 10 ducklings and the experiment lasted until 21 d of age.3. The results showed that embryos injected with OTA affected the 21 d body weight (BW) and average daily gain (ADG) of ducklings (p < 0.05). OTA exposure increased the relative weights of the liver, pancreas, gizzard, proventriculus and jejunum (p < 0.05); and decreased the relative length of the jejunum of ducklings (p < 0.05). Moreover, jejunal crypt depth increased (p < 0.05) and the villus height-to-crypt depth ratio (Vh/Cd) decreased in the OTA-injected group (p < 0.05). Compared with those in the CON group, the mRNA expression of Zonula Occludens-1; (ZO-1) (p = 0.0582) and Occludin; (p = 0.0687) in the OTA treatment group was downregulated.4. The findings demonstrated that a single low-dose injection of OTA increased body weight and daily gain in ducklings. Moreover, embryo exposure to OTA had negative effects with increased relative weight of organs and the jejunal crypt depth, decreased relative length of the intestine and mRNA expression of tight junctions (ZO-1, Occludin).