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. 2021 Dec 14:2021:7190152.
doi: 10.1155/2021/7190152. eCollection 2021.

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

Muhammad A Kurnianto  1   2 , Harsi D Kusumaningrum  3 , Hanifah N Lioe  3 , Ekowati Chasanah  4
Affiliations

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

Muhammad A Kurnianto et al. Biomed Res Int. .

Abstract

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.

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Conflict of interest statement

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
The growth absorbance (in 600 nm) of test bacteria in Mueller Hinton broth exposed to cell-free supernatant (CFS); white square: E. coli; light-gray square: S. aureus; C (−): control negative using Mueller Hinton broth inoculated with test bacteria without being exposed to CFS; C (+): control positive using ampicillin 10 μg·mL−1.
Figure 2
Figure 2
Inhibition zone (mm) of concentrated cell-free supernatant (crude BLIS) against test bacteria determined using the agar well diffusion method; white square: E. coli; dark-gray square: S. aureus; light-gray square: L. monocytogenes; black square: S. Typhimurium; C (−): control negative using distilled water; C (+): control positive using ampicillin 10 μg·mL−1.
Figure 3
Figure 3
Inhibitory zone (mm) of crude BLIS after heat (a) and pH (b) treatment against E. coli; ∎: SCA-5; ●: SCA-8; ⬦: SCA-11; ▲: AIA-10; ○: AIA-12; ⬥: AIA-17.
Figure 4
Figure 4
Inhibition zone (mm) of BLIS ultrafiltration fraction based on molecular weight (<3 kDa, 3–10 kDa, and >10 kDa) against E. coli (a), S. aureus (b), L. monocytogenes (c), and S. Typhimurium (d) using the agar well diffusion method; light-gray square: >10 kDa; dark-gray square: 3–10 kDa; white square: <3 kDa.
Figure 5
Figure 5
LC-MS/MS chromatogram profile of BLIS UF-SCA-8 with MW < 3 kDa fraction.
See this image and copyright information in PMC

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