Streptomyces has been reported as an essential producer of bioactive substances, including antibiotics and other types of antimicrobials. This study investigated antibacterial-producing Streptomyces isolated from the gut of estuarine fish Chanos chanos, emphasizing screening for the producer of peptide-containing antibacterial compounds. Eighteen isolates were found during preliminary screening, in which four isolates showed the best antibacterial activities. Based on the morphological, physiological, and biochemical characterization, as well as 16S rRNA partial sequencing, all of the four isolates belonged to Streptomyces. Three isolates were suspected as novel isolate candidates based on homology presentations and phylogenetic tree analysis. Disk-diffusion assay of the metabolite-crude-extract from the isolates showed broad-spectrum inhibitory activities against Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa InaCC B52 with minimum inhibitory concentration and minimum bactericidal concentration ranging from 2.5–10 mg/mL and 5–10 mg/mL, respectively. The highest antibacterial activity with low MIC and MBC values was shown by isolate AIA-10. Qualitative HPLC profiling revealed that the metabolic-crude-extracts showed many peaks with intensive area at 210 and 214 nm, especially from SCA-11 and AIA-10, indicating the presence of peptide groups in the structure of the constituent compound. The results also suggested that crude extracts SCA-11 and AIA-10 had higher hydrophobicity properties than the other extracts. Further characterization of the active compound was needed to find out which compounds were responsible for the antibacterial activity. The results of this study indicated that some Streptomyces isolated from new environmental niches, i.e., gut of estuarine fish Chanos chanos, produce promising peptide-containing bioactive compounds.

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3   kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

Consumption of fresh and minimally processed food is closely related to foodborne diseases. To minimize the adverse effects, bacteriocin-like inhibitory substance (BLIS) as a natural preservative can be used. One of the bacteriocins with promising activity was produced by Streptomyces sp. Using gel filtration chromatography, the bacteriocin purification process succeeded in obtaining semi-purified fractions with broad-spectrum inhibitory activity to foodborne pathogen bacteria. These fractions are also stable up to 100 °C and pH 2.0–7.0. High-Resolution Liquid Chromatography Electrospray Ionization-Tandem Mass Spectrometry analysis followed by orthogonal projection to latent structure showed that each fraction had eight peaks with the highest positive correlation to BLIS-specific activity. Peptide identification based on MS spectrum found 597 predictive peptides, of which 42 predictive peptides with antimicrobial peptide characteristics and the highest iAMPpred antimicrobial peptide probability (>0.5) were selected. The selected predictive peptides have molecular mass of 247.13-615.37 Da and consist of at least 20% hydrophobic amino acids with a hydrophobicity value of 14.72 Kcal mol-1. The results of this study indicate the effectiveness of BLIS purification by gel filtration chromatography and the promising potential of semi-purified BLIS as a natural preservative. Besides, the active peptides in semi-purified BLIS can also be identified quickly so that the isolation process to obtain purified-BLIS can be carried out more efficiently.

Abstract. Kurnianto MA, Kusumaningrum HD, Lioe HN, Chasanah E. 2021. Antibacterial and antioxidant potential of ethyl acetate extract from Streptomyces AIA12 and AIA17 isolated from gut of Chanos chanos. Biodiversitas 22: 3196-3206. Streptomyces has been recognized as a promising and productive source of antibacterial and antioxidant compounds. The phenotypic and genomic characterizations indicated AIA12 and AIA17 are closely related to the S. globisporus ARGB01 and S. misionensis S1-SC15, respectively. Incubation on yeast-malt extract broth for nine days (AIA12) and 11 days (AIA17) proved to be the best growth medium and optimum antibacterial production time for these two strains. Crude-extract of extracellular secondary metabolites, obtained by ethyl acetate extraction, demonstrated broad-spectrum inhibitory activity against Pseudomonas aeruginosa InaCC B52, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 14028, and Listeria monocytogenes with minimum inhibitory and minimum bactericidal concentrations from 2.5 to 0.31 and 5.0 to 0.31 mg mL-1, respectively. Evaluation of antioxidant showed AIA17 crude-extract had moderate DPPH scavenging and antioxidant activities of 65.122% ± 0.56 and 28.178 ± 0.24 mg ascorbic acid equivalent antioxidant capacity (AEAC g-1), respectively. The identification of compounds through profiling with RP-HPLC showed optimum absorbance at 210 and 214 nm, which showed the presence of peptide groups in the constituent compounds' molecular structure. These findings indicate that Chanos chanos-derived Streptomyces produces valuable bioactive compounds with various promising biological activities.

Saluran pencernaan (terutama usus) ikan perairan estuaria merupakan salah satu ceruk lingkungan potensial Actinobacteria yang belum tereksplorasi. Penelitian ini bertujuan untuk mengisolasi dan mengidentifikasi karakteristik morfologi Actinobacteria asal ikan bandeng (Chanos chanos) dan belanak (Mugil cephalus) serta mengevaluasi aktivitas antimikroba yang dihasilkannya. Penelitian ini diawali dengan mengambil usus ikan, kemudian digesta usus secara perlahan dipisahkan untuk dieksplorasi keberadaan Actinobacteria dengan menggunakan media isolasi selektif. Isolat yang diperoleh dikarakterisasi berdasarkan ciri makroskopik dan mikroskopik, serta dilakukan penapisan antibakteri awal menggunakan metode double-layer diffusion. Isolat dengan zona penghambatan terbaik dipilih untuk dilakukan produksi dan ekstraksi senyawa antibakteri, serta uji aktivitas antibakteri dengan metode difusi cakram terhadap bakteri uji Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa dan Escherichia coli. Sebanyak 44 isolat Actinobacteria telah diisolasi dari digesta usus ikan bandeng (Chanos chanos) dan belanak (Mugil cephalus) menggunakan media strach casein dan actinomycete isolation agar. Sebagian besar isolat yang diperoleh menunjukkan karakteristik morfologi genus Streptomyces sp., seperti koloni memiliki tekstur menyerupai serbuk, bertepung dan kasar, memiliki aerial miselium berwarna putih dan substrat miselium berwarna krim susu, serta memiliki bentuk rantai spora rectus-flexibilis. Proses penapisan antibakteri isolat Actinobacteria menunjukkan 22 isolat memiliki indeks penghambatan terhadap sedikitnya satu bakteri uji, dengan aktivitas terbaik ditunjukkan oleh isolat A-SCA-11. Uji antibakteri terhadap ekstrak kasar isolat A-SCA-11 menunjukkan aktivitas antibakteri berspektrum luas yang mampu menghambat seluruh bakteri uji dengan zona hambat tertinggi pada P. aeruginosa.  AbstractThe gut of estuary fish is one of the potential novel niches of Actinobacteria that has not yet been explored. This study aimed to isolate and identify the morphological characteristics of Actinobacteria from milkfish (Chanos chanos) and blue-spot mullet fish (Mugil cephalus) and to evaluate the antibacterial activity produced. This research was started by taking the fish gut, and then the digesta were slowly separated to explore the presence of Actinobacteria using selective isolation media. The isolates obtained were characterized by macroscopic and microscopic characteristics, and antibacterial preliminary screening of isolates was performed using a double-layer diffusion method. The isolates with the best inhibition zone were selected for production and extraction of antibacterial compounds, and antibacterial activity tests using the disk-diffusion method against the test bacteria Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Escherichia coli. A total of 44 isolates of Actinobacteria have been isolated from the gut of fish using starch casein and actinomycete isolation agar. Most isolates showed morphological characteristics of the genus Streptomyces sp., such as colonies with a tough or powdery texture, antibacterial have white aerial mycelium and milk-cream substrate mycelium, and rectus-flexibilis spore chain. The antibacterial preliminary screening of Actinobacteria isolates showed 22 isolates had inhibitory index against at least one test bacterium, with the best activity indicated by A-SCA-11. Antibacterial test of A-SCA-11 crude extract showed broad-spectrum antibacterial activity that was able to inhibit all test bacteria with the highest inhibitory zone on P. aeruginosa.