Abstract
The antimicrobial activity of bacteriocins and their application as food preservatives has been
previously proven, particularly for bacteriocins produced by lactic acid bacteria (LAB).
However, bacteriocins produced by LAB have application limitations since most of them act
against closely related microorganisms. This study investigated the antimicrobial activity of
bacteriocin extracts produced by Bacillus species isolated from fermented food. Further, the
physicochemical stability of the bacteriocin extracts was studied, as well as their identification
with LC-MS/MS.
About 15 Bacillus spp. were isolated from the fermented food and identified with 16S rRNA gene
sequencing to belong to B. velezensis, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. cereus,
B. siamensis and B. safensis. All the Bacillus spp. in this study produced bacteriocins. Out of the
15 bacteriocin extracts, 6 extracts showed good antibacterial activity against both gram-positive
(Bacillus cereus, Listeria innocua) and gram-negative bacteria (Escherichia coli, Salmonella
Typhimurium) when determined with the disc diffusion assay. The highest bacteriocin activity
(3840 AU/ml) occurred with S. Typhi, while the lowest (240 AU/ml) was obtained with some
bacteriocin extracts against both the gram-positive bacteria. The MIC of the bacteriocin extracts
ranged from 0.045 – 0.130 mg/ml, while SDS page showed a single band at approx. 11 kDa for
nearly all bacteriocin extracts. All the bacteriocins extracts showed tolerance at different pH
conditions (pH 3-9) as their antibacterial activity against all the selected microorganism was
retained. The bacteriocins extracts were stable at 50°C and 80°C but displayed reduced
antimicrobial activity after exposure to 96°C. At higher temperature (121°C), the antimicrobial
activity of the extracts was totally lost. The bacteriocins were sensitive to pepsin and proteinase K
enzymes but retained activity after treatment with α-amylase.
The SH/A4 bacteriocin extract inhibited the growth of all investigated microorganism when
compared to control (without extract). About 3.2 – 4.6 log CFU/ml reduction in the viable number
of investigated microorganism was achieved with SH/A4 bacteriocin extract, while the control
showed variable growth after 8 h. SEM images showed structural collapse on cells treated with
bacteriocin extracts compared to control cells which appeared intact. LC-MS/MS proteomic
analysis revealed the presence of five proteins with antibacterial activity in the SH/A4 bacteriocin
extract as well as proteins with peptidases activity.
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This study shows that bacteriocin extracts from Bacillus spp. can inhibit the growth of foodborne
gram-positive and gram-negative microorganisms. The bacteriocin extracts are also able to retain
their antimicrobial activity after exposure to physicochemical stresses used during food
processing. Therefore, the bacteriocin extracts can be applied to food to extend shelf life and also
to ensure food safety.