As part of its commitment to advancing scientific research and promoting innovative studies that address societal challenges, the Department of Biology at the College of Education for Pure Sciences / Ibn Al-Haytham, University of Baghdad, held the defense of a PhD dissertation entitled “Evaluation of the Efficiency of Selected Plant-Based Nanopowders in Reducing Ochratoxin A in Rice Grains and Investigating Its Histopathological Effects on Rat Kidneys,” submitted by Salam Nasseef Jasim Mohammed under the supervision of Professor Dr. Sumaya Naima Hiwar.
The research aimed to isolate and identify fungi contaminating rice grains and to evaluate the efficiency of selected plant-based nanopowders in reducing Ochratoxin A (OTA), one of the most hazardous mycotoxins affecting food safety and public health. The study also investigated the histopathological effects of the toxin on rat kidneys, providing scientific evidence that may contribute to developing innovative strategies for minimizing mycotoxin contamination and improving food quality and safety.
The dissertation was evaluated by an academic committee chaired by Professor Dr. Thamer Abdulshaheed Mohsen, with the membership of Professor Dr. Fadia Fallah Hassan, Assistant Professor Dr. Wasan Abdulwahab Faeq, Assistant Professor Dr. Atyaf Saeed Hameed, Assistant Professor Dr. Mohammed Yaseen Khudhair, and Professor Dr. Sumaya Naima Hiwar, who also served as the dissertation supervisor.
This research reflects the College’s commitment to promoting excellence in biotechnology, nanotechnology, and food safety research while encouraging innovative scientific solutions to health and environmental challenges, thereby reinforcing the University’s role in serving society and supporting sustainable development.
The dissertation contributes to the achievement of United Nations Sustainable Development Goal (SDG) 3 – Good Health and Well-being by supporting research aimed at reducing health risks associated with mycotoxins. It also advances SDG 2 – Zero Hunger through improving food safety and quality, SDG 9 – Industry, Innovation and Infrastructure by applying nanotechnology to develop innovative scientific solutions, and SDG 4 – Quality Education through strengthening scientific research and preparing highly qualified researchers capable of contributing to sustainable development.
This study was aimed to evaluate the effectiveness of certain plant-based nano-powders in reducing ochratoxin A contamination in rice grains. In addition, the study assessed the in vivo efficacy of these powders to determine their ability to mitigate toxic effects, protect target organs, and improve biochemical parameters in the human body. Rice samples were collected from twenty varieties commonly sold in Baghdad markets. Fungi isolated were isolated from rice samples (with and without surface sterilization) and then identified morphologically and microscopically. While the percentages of fungal contamination, appearance rates, and frequencies were calculated. Subsequently, the fungal isolates were tested for their ability to produce Ochratoxin A using thin-layer chromatography (TLC). The three major isolates with the highest toxin production were identified using molecular technique; in addition to detecting the presence of toxins in samples of local rice.
Plant nano-powders of clove, cinnamon, and turmeric were prepared using mechanical grinding, and characterized using atomic force microscopy (AFM). The efficacy of these plant nano-powders at concentrations of (0.2%, 0.4%, 0.6%, 0.8%, 2%, 4%, 6%, and 8%) was investigated in inhibiting the growth of the most toxin-producing fungal isolate on Potato Dextrose Agar (PDA) and liquid media. Additionally, the efficacy of plant nano-powders in inhibiting and reducing ochratoxin A production in liquid medium (in vitro) and on solid rice grains (in storage) was assessed using high-performance liquid chromatography (HPLC). Furthermore, the effect of these plant nano-powders at a concentration of 2% in female mice, for a period of one month, was evaluated by studying the toxicity of ochratoxin A, safety of these powders, their ability to bind to the toxin, and their protective and therapeutic role based on measuring the weights of the mice, biochemical parameters, and histological changes of the kidney.
The detection results of the surface contamination of non-sterilized rice grains showed that from most contaminated to least contaminated as following: Al-Waha/Anbar Al-Mashkhab, Al-Khadrawi, Mahmoud, Abu Araba, Mahrani, Kobani, Joker, Judi, Al-Walima, Mashkour, Al-Kharafi, Ali Pasha, Malik Al-Punjab, Al-Barari, Ahlna, Daawat, Jihan, and Al-Awsi, as the percentages of fungal contamination reached (100, 76.3, 64.6, 63.3, 63.3, 46.4, 43.3, 43.3, 40, 40, 40, 33.3, 33, 30, 28, 20.4, 13.3%) respectively. Whereas the surface-sterilized rice grains showed that the most contaminated to the least as following: Mahmoud, Abu Araba, Al-Khadrawi, Judi, Malik Al-Punjab, Ali Pasha, Kubani, Al-Waha, Daawat, Mashkour, Al-Walima, Al-Barari, Joker, Al-Awsi, Al-Kharafi, Jihan, Mahrani, and Ahlna, as the contamination rates reached (45.3, 43.3, 40, 36.6, 33, 32.6, 30, 26.6, 26, 23.3, 20, 20, 16.6, 13, 10, 6.3, 0, 0)% respectively. The percentage of fungal genera appearance in collected samples of non-surface-sterilized grains revealed the presence of nine genera: Penicillium spp., Aspergillus spp., Cladosporium spp., Fusarium spp., Moniliella spp., Alternaria spp., Mucor spp., Curvularia spp., Trichothecium spp., with percentages of appearance of 17.5%, 15%, 7%, 5%, 2%, 0.6%, 0.6%, 0.5%, 0.2% respectively. While the percentages of frequency of the same genders were 35%, 28%, 16%, 10%, 3%, 2%, 2%, 1%, and 0.4% respectively.
On the other hand, nine fungal genera were recorded in surface-sterilized grain samples: Penicillium spp., Aspergillus spp., Cladosporium spp., Fusarium spp., Moniliella spp., Yeast, Alternaria spp., Mucor spp., and Monascus spp., with the following percentages of occurrence: 15%, 12%, 5%, 4%, 1%, 0.5%, 0.2%, 0.2%, and 0.2%, respectively. The highest percentage of fungal appearance was 25% for Aspergillus, followed by Penicillium at 22%, Cladosporium (13%), Fusarium (8%), Moniliella (2%), Yeast (1%), Alternaria (0.6%), Mucor (0.6%), and Monascus (0.6%), respectively.
Thin-layer chromatography (TLC) analysis showed that all Penicillium isolates (29 isolates), A. ohraceous (1 isolate), and A. terreus (6 isolates) were OTA toxin-producing, while A. niger (4 isolates) and A. carbonarius (5 isolates) did not show the ability to produce the toxin. Moreover, three isolates of Penicillium were distinguished by their high toxin productivity, which are PM11, PM18 and PM20, as these isolates recorded high concentrations of ochratoxin A estimated by (HPLC), reaching 561.77, 518.95, and 495.9 ppb/kg respectively. The three isolates (PM11, PM18, PM20) were identified using molecular diagnosis, as they matched 99% with Penicillium polonicum in the Global GenBank (NCBI), representing the first record of this fungus as a rice grain contaminant and producer of OTA toxin in Iraq. In addition, the results of the atomic force microscopy (AFM) examination of the nano-powders of clove, cinnamon and turmeric observed that their particle sizes reached the required nanoscale range (94.4, 115.3, 42.19 nm) for clove, cinnamon and turmeric respectively.
The statistical analysis using ANOVA test found a significant difference in nano-powders in inhibiting of P. polonium growth on PDA medium; clove (Clv-NP) and the mixture (Mix-NP) achieved the highest efficacy, as clove inhibition rate was at 100% at concentration of 0.4%. Meanwhile, high concentrations of cinnamon (Cnn-NP) at (6-8%) were significantly inhibited the growth, and turmeric (Tur-NP) showed a slight difference at a concentration of 2% compared to the control (P≤0.05). High-performance liquid chromatography (HPLC) analysis revealed that clove, cinnamon, turmeric, and the mixture of nano-powders were highly effective in inhibiting ochratoxin A production by P. polonium in liquid rice medium. All treatments completely inhibited the toxin production (100%) at all used concentrations (2-8%), except for the turmeric nano-powder at 2% and 4% concentrations, which showed inhibition of 87% and 92%, respectively. Furthermore, the nano-powder of clove, cinnamon and the mixture showed complete inhibition of the biomass of P. polonicum at all concentrations (2-8%) in comparison to control. Conversely, nano-powder of turmeric promoted P. polonicum growth. HPLC analysis revealed the high efficiency of the nanopowders in reducing the concentration of the toxin originally produced in the liquid rice medium. Nano-powdered of clove reduced toxin production by 100% at concentrations of 0.8% and above, while nano-turmeric powder outperformed nano-cinnamon powder, achieving 100% reduction at concentrations of 6% and 8%.
HPLC analysis of stored rice grains noticed that treatment with nano-clove powder completely inhibited toxin production at concentrations of 4% and above. Nano-cinnamon powder completely inhibited toxin production at a concentration of 8%, while nano-turmeric powder achieved 100% inhibition at both concentrations of 6% and 8%. Interestingly, the three nano-powder treatments ranked highly in their ability to reduce ochratoxin A present in rice grains during a one-month storage period. Clove nano-powder topped the list, achieving reduction rates ranging from 79%-100% as the concentration increased from 0.4%-8%. Turmeric nano-powder was the second, followed by cinnamon nano-powder, as the reduction rate reached 100% for cloves and turmeric at a concentration of 8%, and 98% for cinnamon at the same concentration.
The statistical analysis of female mice weight revealed significant differences (P≤0.05) in the toxin group (G3) during the second and fourth weeks as compared to the control group (G1). In contrast, the results for group G2 showed no significant differences between the cinnamon and turmeric groups (G2B and G2C) and the control group in the fourth week, while the clove treatment (G2-A) recorded a significant difference in comparison to control group in the same week. In the prevention group (G4), comparison with the toxin group (G3) observed a significant differences favoring the clove (G4-A) and cinnamon (G4-B) groups during the second week only. Furthermore, the results of the treatment (G5) and toxin binding (G6) groups showed significant differences compared to the toxin group (G3); this was particularly evident in the turmeric (G5C and G6C) treatments during the first week and the cinnamon (G5B and G6B) treatments during the second week.
The results of the biochemical parameters revealed a clear toxic effects of ochratoxin A, as the group treated with the toxin only (G3) recorded a significant increase (p≤0.05) in all levels of studied parameters compared to the control group (G1), including creatinine, urea, liver enzymes (ALP, AST, ALT), uric acid, total protein, total bilirubin and albumin levels, confirming the occurrence of damage to kidney and liver functions. On the other hand, treatment with studied nano-powders alone (G2) showed an improvement in some indicators in comparison to control, but with a slight increase in some values. The preventive (G4), therapeutic (G5), and combination (G6) treatment groups showed varying degrees of efficacy in mitigating these toxic effects, as the levels of all liver enzymes (ALP, AST, ALT), uric acid, total protein, total bilirubin and albumin, creatinine, and urea were reduced in these groups compared to the toxin group (G3).
The histological examinations of female mice kidneys noticed severe damage in the group treated with ochratoxin A only (G3); it was observed a severe degeneration and necrosis of the renal tubules, cellular swelling and tubular dilation, and degeneration of the renal glomerular cells. However, the groups treated with nano-powders only (clove, cinnamon, turmeric) showed largely normal kidney tissue with some minor changes. In the protective group (G4), the clove treatment (G4-A) showed moderate degeneration of the renal tubules, while the cinnamon (G4-B) and turmeric (G4-C) treatments were similar to the negative control with minor changes. In the treatment group (G5 group), all treatments found a notable improvement compared to the toxin group, exhibiting slight degeneration of the renal tubules while preserving the normal structure and interstitial tissue of the glomeruli. While in the concurrent treatment and toxin binding group (G6 group), the cinnamon (G6-B) and turmeric (G6-C) treatments were superior, showing only slight swelling in some cells, while the clove (G6-A) treatment showed granulomatous degeneration in the renal tubules.
