Sains Malaysiana 51(10)(2022): 3401-3414

http://doi.org/10.17576/jsm-2022-5110-23

Isolation of Lactic Acid Bacteria from Cocoa Bean Fermentation as Potential Antibacterial Agent against ESKAPE Pathogens

(Pemencilan Asid Laktik Bakteria daripada Penapaian Biji Koko sebagai Agen Antibakteria yang Berpotensi terhadap Patogen ESKAPE)

NORAZIAH MOHAMAD ZIN^1,*, AUNI NADZIRAH ABD RASHID¹, NUR ASYIKIN ZULKHAIRI¹, NUR AQILAH RIDZMAN¹, KHAIRUL BARIAH SULAIMAN²,  NUR FAIZAH ABU BAKAR¹ & ASIF SUKRI<sup>1

1</sup>Programme of Biomedical Science, Centre of Diagnostic, Therapeutic & Investigative Studies (CODTIS), Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia
²Cocoa Research & Development Centre Hilir Perak, Malaysian Cocoa Board, P. O. Box 30, Sg. Dulang Road, 36307 Sg Sumun, Perak Darul Ridzuan, Malaysia

Received: 26 April 2022/Accepted: 2 August 2022

Abstract

This study aimed to evaluate the antibacterial activity of microorganisms isolated before and during cocoa fermentation against ESKAPE pathogens. Microorganisms from cocoa fermentation process were isolated on the selective media and were tested against ESKAPE pathogens. Total titratable acidity, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cell-free supernatant from lactic acid bacteria (LAB) were determined. The presence of bacteriocin and antibacterial activity were determined. LAB was identified through molecular and biochemical tests. Ninety-five and 134 isolates were obtained from the fermentation of Sungai Balung 25 (BR25) clone and mixed clones, respectively.  Screening of antibacterial activity showed that 26 isolates of LAB from the cocoa fermentation process had antibacterial activity against ESKAPE pathogens (zone of inhibition ≥11 mm). Cell-free supernatant from some LAB demonstrated potent antibacterial activity against some ESKAPE pathogens through MIC and MBC and evaluation. Furthermore, one isolate (mix48M01) exhibited antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp., methicillin-resistant Staphylococcus aureus, and Enterococcus raffinousus. An increase in the diameter of inhibition zone was observed as the lactic acid production increased. Gram-positive bacteria isolates were more resistant to LAB than Gram-negative bacteria. Bacteriocin with good antibacterial activity was also detected from the LAB examined. Molecular analysis showed that 13 LAB shared >99% sequence similarity to Lactobacillus fermentum CECT 562 while one isolate shared sequence similarity <98%, indicating that it might be a novel Lactobacillus species. Isolated LAB from this study exhibited high antibacterial activity against ESKAPE pathogens and could be investigated further as potential probiotics and antibacterial agents in the future.

Keywords: Antibacterial; cocoa fermentation; ESKAPE pathogens; lactic acid bacteria; Lactobacillus

Abstrak

Kajian ini bertujuan menilai aktiviti antibakteria mikroorganisma dipencilkan daripada sebelum dan selepas penapaian koko. Mikoorganisma daripada penapaian koko dipencilkan di atas media selektif dan diuji ke atas patogen ESKAPE. Keasidan boleh titrat, kepekatan perencat minimum (MIC) dan kepekatan bakterisid minimum (MBC) daripada supernatan tanpa sel bakteria laktik asid (LAB) ditentukan. Kehadiran bakteriosin dan aktiviti antibakteria ditentukan. LAB dikenal pasti menggunakan kaedah biokimia dan molekul. Sembilan puluh lima dan 134 pencilan masing-masing dipencilkan daripada klon Sungai Balung (BR25) dan klon campuran. Saringan aktiviti antibakteria menunjukkan 26 pencilan mempunyai aktiviti terhadap patogen ESKAPE (zon perencatan ≥11 mm). Supernatan tanpa sel menunjukkan aktiviti antibakteria melalui penilaian MIC dan MBC. Satu pencilan (mix48M01) menunjukkan aktiviti terhadapKlebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp., rintangan metisilin Staphylococcus aureus dan Enterococcus raffinousus. Peningkatan diameter zon perencatan dapat diperhatikan apabila kepekatan asid laktik bertambah. Bakteria Gram positif adalah lebih rintang terhadap LAB berbanding dengan Gram negatif. Bakteriosin dengan aktiviti antibakteria yang baik turut dikesan. Analisis molekul menunjukkan 13 pencilan mempunyai penjujukan DNA sama >99% dengan Lactobacillus fermentum CECT 562, manakala satu pencilan mempunyai penjujukan <98%, mencadangkan spesis Lactobacillus yang baharu. Pencilan LAB yang dipencilkan dalam kajian ini menunjukkan aktiviti antibakteria yang baik terhadap patogen ESKAPE dan boleh dikaji dengan lebih mendalam lagi pada masa hadapan.

Kata kunci: Antibakteria; asid laktik bakteria; Lactobacillus; patogen ESKAPE; penapaian koko

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^*Corresponding author; email: noraziah.zin@ukm.edu.my