Exploration of Bacillus sp.-derived Secondary Metabolite Compounds as Anti-Foodborne Pathogen Bacteria using Metabolomic and Genomic Analysis

Tsania Taskia Nabila, Ir. Jaka Widada, M.P., P.hD; Dr. Ema Damayanti, M.Biotech

2024 | Tesis | S2 Bioteknologi

Mikroorganisme patogen dapat menyebabkan kontaminasi pangan dan menjadi perhatian utama di dalam dunia industri. Penelitian ini bertujuan untuk mengeksplorasi metabolit-metabolit dari isolat Bacillus sp. BP1 yang berpotensi melawan dua jenis foodborne pathogens (FBPs) yaitu Staphylococcus aureus (S. aureus) dan Salmonella enterica serovar Typhimurium (S. Typhimurium) menggunakan pendekatan metabolomik dan genomik. Konsep one strain many compounds (OSMAC) diaplikasikan terhadap Bacillus sp. BP1 menggunakan beragam jenis media fermentasi yang meliputi tryptic soy broth (TSB), TSB-chitosan (TSB-chi), mineral salts medium-glucose (MG) dan -fructose (MGF), carboxy methyl cellulose (CMC), dan starch nitrate broth (SNB). Ekstrak dari pellet (P) dan supernatant (S) berjumlah dua belas dan seluruhnya diskrining aktivitas antibakterinya. Ekstrak S-TSB-chi dan P-TSB menunjukkan aktivitas antibakteri tertinggi. Berdasarkan hasil pengujian time-kill dan scanning electron microscope (SEM), kedua ekstrak mampu menunjukkan potensi penghambatan terhadap FBPs dan metabolit sekunder akan mulai diproduksi pada jam ke-18 dan 24 pasca kultivasi. Berdasarkan hasil profiling metabolit menggunakan Fourier Transform Infrared Spectroscopy (FTIR) dan Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) yang dilanjutkan dengan dua jenis analisis multivariat, diketahui gugus fungsi utama dan senyawa-senyawa penting yang meliputi biosurfaktan seperti N,N-Bis(2-hydroxyethyl)dodecanamide dan cyclo(phenylalanyl-prolyl). Bilangan gelombang FTIR 1704.88735 merupakan variabel penting dan diinterpretasi sebagai gugus amida I. Analisis genome mining menunjukkan adanya beberapa jenis senyawa bioaktif potensial yang dikode oleh dua belas biosynthetic gene cluster (BGC) dan terdapat  satu BGC yang mirip 86?ngan BGC pengkode surfaktin, sehingga mengindikasikan adanya senyawa baru. Molecular docking pada tiga senyawa terpilih dengan ampicillin sebagai antibiotik standard terhadap protein-protein S. aureus dan S. Typhimurium menunjukkan hasil bahwa cyclo(phenylalanyl-prolyl) memiliki afinitas pengikatan yang tinggi dan N,N-Bis(2-hydroxyethyl) dodecanamide berinteraksi paling lemah jika dibandingkan dengan ampicillin. Surfaktin menunjukkan afinitas pengikatan paling tinggi untuk seluruh jenis protein S. aureus dan S. Typhimurium. Senyawa-senyawa terpilih yang diproduksi oleh B. velezensis BP1 ini terbukti mampu menunjukkan potensinya sebagai antibakteri S. aureus dan S. Typhimurium.

Kata kunci: foodborne pathogen, Bacillus sp. BP1, OSMAC, genomik, metabolomik

Food contamination by pathogenic microorganisms is a significant concern in the food industry. This research aimed to explore the metabolites from Bacillus sp. BP1 two foodborne pathogens (FBPs), Staphylococcus aureus (S. aureus) and Salmonella enterica serovar Typhimurium (S. Typhimurium), using metabolomic and genomic approaches. The one strain many compounds (OSMAC) framework was applied to the bacterium under various fermentation media including tryptic soy broth (TSB), TSB-chitosan (TSB-chi), mineral salts medium-glucose (MG) and -fructose (MGF), carboxy methyl cellulose (CMC), and starch nitrate broth (SNB). Twelve supernatants (S) and pellets (P)-derived extracts were tested, with S-TSB-chi and P-TSB showing the highest antibacterial activity. Time-kill assays and SEM confirmed their potency against FBPs, and the secondary metabolites were likely produced after 18 to 24 hours of cultivation. Metabolite profiling using Fourier Transform Infrared Spectroscopy (FTIR) and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) supported by two multivariate analyses identified the key functional groups and compounds including biosurfactants like N,N-Bis(2-hydroxyethyl)dodecanamide and cyclo(phenylalanyl-prolyl). FTIR wavenumber of 1704.88735 was an important variable and belongs to an amide I group. Genome mining revealed several promising bioactive compounds with twelve biosynthetic gene clusters including surfactin with 86?duced amino acids indicating predicted novel compound. Molecular docking of selected compounds against protein targets in both S. aureus and S. Typhimurium showed that cyclo(phenylalanyl-prolyl) had the higher binding affinity and N,N-Bis(2-hydroxyethyl) dodecanamide had the lower binding affinity than ampicillin as reference antibiotic. Surfactin showed the strongest binding affinity for targets in both S. aureus and S. Typhimurium. These selected compounds that produced by B. velezensis BP1 has potency as antibacterial to treat S. aureus and S. Typhimurium.

Key words: foodborne pathogens, Bacillus sp. BP1, OSMAC, genomic, metabolomic

Kata Kunci : foodborne pathogens, Bacillus sp. BP1, OSMAC, genomic, metabolomic