Sains Malaysiana 52(3)(2023): 821-835

http://doi.org/10.17576/jsm-2023-5203-11

Molecular Characterization of Ochratoxin a Producing Indigenous Aspergillus Strains from Poultry Feed in Pakistan

(Pencirian Molekul Okratoksin Penghasil Strain Aspergillus Asli daripada Makanan Poltri di Pakistan)

GULL NAZ¹, AFTAB AHMAD ANJUM^1*, TEHREEM ALI¹, MUHAMMAD NAWAZ¹, SANAULLAH IQBAL², MATEEN ABBAS³ & RABIA MANZOOR¹

¹Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

²Department of Food Science and Human Nutrition, University of Veterinary and Animal Sciences, Lahore, Pakistan

 ³Quality Operations Laboratory, Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

Diserahkan: 12 Julai 2022/Diterima: 17 Januari 2023

Abstract

Ochratoxin A (OTA) is nephrocarcinogenic and immunosuppressive toxin and OTA producing molds contaminate the food crops. Isolation and identification of ochratoxin producing fungi was carried out from poultry feed samples (n=120) followed by preliminary confirmation through macroscopic and microscopic characteristics. Purified fungal isolates identified as Aspergillus 1842(91.68%) followed by Penicillium 91 (4.53%), Mucor 52 (2.58), Alternaria 7 (0.35%), Cladosporium 6 (0.29%), Fusarium 4 (0.199%) and un-identified (07). OTA production was confirmed through thin layer chromatography (TLC) followed by high performance liquid chromatography (HPLC). Only 41 isolates (2.22%) out of 1842 Aspergillus isolates were able to produce toxin. At genetic level, characterization was performed through polymerase chain reaction (PCR) using species specific gene primers. From 41 isolates 27, 9 and 5 were characterized as Aspergillus terreus, Aspergillus parasiticus, and Aspergillus ochraceus respectively. Physical and chemical factors were optimized for OTA production. Under the effect of 37 °C temperature and 7.5 pH of Sabouraud dextrose broth (SDB) medium, higher toxin (969.45±.03 µg/mL) production was observed from ASPO-6 isolate. ASPO-4 isolate produce higher toxin amount in SDB medium with supplementation of maize 5%, wheat 1% and rice 3%. OTA stability was determined by adjusting standard concentration of 100 µg/mL in organic solvents (chloroform, acetonitrile and methanol) and organic solids. Least percentage log reduction in OTA concentration and stability of OTA was observed in opaque vials with chloroform and sucrose and transparent vials with sucrose after 6 months. OTA can be used as indigenous standard for identification of OTA from field samples.

Keywords: Aspergillus; Ochratoxin A; polymerase chain reaction; stability; thin layer chromatography

Abstrak

Okratoksin A (OTA) ialah toksin nefrokarsinogenik dan imunosupresif dan kulapuk penghasilan OTA mencemari tanaman makanan. Pengasingan dan pengenalpastian kulat penghasilan okratoksin telah dijalankan daripada sampel makanan poltri (n=120) diikuti dengan pengesahan awal melalui ciri makroskopik dan mikroskopik. Pengasingan kulat tulen dikenal pasti sebagai Aspergillus 1842(91.68%) diikuti oleh Penicillium 91(4.53%), Mucor 52(2.58), Alternaria 7(0.35%), Cladosporium 6(0.29%), Fusarium 4(0.199%) dan tidak dikenal pasti (07). Pengeluaran OTA telah disahkan melalui kromatografi lapisan nipis (TLC) diikuti oleh kromatografi cecair prestasi tinggi (HPLC). Hanya 41 pencilan (2.22%) daripada 1842 pencilan Aspergillus dapat menghasilkan toksin. Pada peringkat genetik, pencirian dilakukan melalui tindak balas rantai polimerase (PCR) menggunakan primer gen khusus spesies. Daripada 41 pencilan 27, 9 dan 5 masing-masing dicirikan sebagai Aspergillus terreus, Aspergillus parasiticus dan Aspergillus ochraceus. Faktor fizikal dan kimia telah dioptimumkan untuk pengeluaran OTA. Di bawah kesan suhu 37 °C dan 7.5 pH medium Sabouraud dextrose broth (SDB), pengeluaran toksin yang lebih tinggi (969.45±.03 µg/mL) diperhatikan daripada pencilan ASPO-6. Pengasingan ASPO-4 menghasilkan jumlah toksin yang lebih tinggi dalam medium SDB dengan tambahan jagung 5%, gandum 1% dan beras 3%. Kestabilan OTA ditentukan dengan melaraskan kepekatan piawai 100 µg/mL dalam pelarut organik (kloroform, asetonitril dan metanol) dan pepejal organik. Peratusan log pengurangan paling sedikit dalam kepekatan OTA dan kestabilan OTA diperhatikan dalam botol legap dengan kloroform dan sukrosa dan botol lutsinar dengan sukrosa selepas 6 bulan. OTA boleh digunakan sebagai piawaian asli untuk mengenal pasti OTA daripada sampel lapangan.

Kata kunci: Aspergillus; kestabilan; kromatografi lapisan nipis; Okratoksin A; tindak balas rantai polimerase

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^*Pengarang untuk surat-menyurat; email: aftab.anjum@uvas.edu.pk