PCR-based screening, purification and characterization of Pediocin from indigenous fermented foods

PCR is used to identify pediocin gene, directly from fermented food samples using primers papA sense (5'-CTG CGT TGA TAG GCC AGG TTT CA-3') and papA antisense (5-`GCT TCT GTA GTA AAA ACT GTA GCC-3`) to amplify a region of the papA gene or pediocin gene. About 3 micro liter of boiled water extract from the fermented food was used as template. From 72 kinds of fermented food samples collected from various public markets in central and Southern Luzon [Philippines], 6 were found positive for the pediocin gene. More than twenty isolates showed some form of antagonistic activity using the direct assay method. Upon further testing by spot-on-lawn assay using boiled and pH neutralized supernatant, 7 isolates were found to give bacteriocin activity. This type of bacteriocins can be better utilized in food processing and preservation where thermal sterilization is being applied. The bacteriocin producers were isolated from the fermented foods positive for the pediocin gene by agar plate streaking and employing direct assays method against actively growing indicator (L. plantarum ATCC 14917) and further confirmation and quantification by the spot-on-lawn assay. Morphological observations showed that 2 isolates were Gram positive rods, 2 were Gram negative coccoids and 3 were Gram positive cocci that formed tetrads. The tetrad forming Gram-positive cocci (Pediococcus-like) that gave comparatively higher bacteriocin activity was selected for bacteriocin-purification. The bacteriocin of the most active isolate was purified by adsorption-desorption method and reverse phase high performance liquid chromatography (RP-HPLC). A reasonable yield of 35% was obtained and the bacteriocin was purified to homogeneity. The purified bacteriocin was treated with pronase, papain and amylase for 30 min at 37 deg C and the resulting activity was determined. Semi-purified bacteriocins were also subjected to various pH and temperature treatments and the residual activity estimated. The antimicrobial spectrum of inhibitory activity on bacterial strains of representative species was also assayed by spot-on-lawn method. The bacteriocin was found proteinaceous in nature because activity was lost upon treatment of pronase. However, papain did not inhibit the bacteriocin. The reason is not known but it is surmised that there is no binding site for papain on the amino acid sequence of the bacteriocin. Amylase gave no inhibitory effect on bacteriocin activity which suggests the absence of any carbohyrate moiety. The bacteriocin is tolerant to high temperatures (100 deg C for 1 hr and 121 deg C for 15 minutes). The pH range is from pH 2-8. The bacteriocin have a narrow spectrum of antagonistic activity, i.e. only against L. plantarum, L. mesenteroides, P. acidilactici, and L. monocytogenes among the bacterial species tested. Effect of the bacteriocin on the growth inhibition of L. monocytogenes during the fermentation of L.casei in fresh cow's milk from the Dairy Training and Research Institute, U.P. Los Banos was studied in comparison with P. acidilactici S3, a known pediocin producer. Milk was diluted with sterile water at 1:1 ratio and inoculated with 1% L. casei culture broth, 0.2% L. monocytogenes and 2% culture supernatants of the bacteriocin producer. Serially diluted fermentation broths were plated-out after 24 h on Listeria enrichment agar and MRS agar plates, to monitor the growth of L. monocytogenes and L. casei, respectively. Considerable degree of inhibition of L. monocytogenes resulted when the culture extract of the selected pediocin-producing isolate was added at a rate of 1% v/v during the fermentation of milk by L. casei. On the other hand, L. casei was not inhibited by the newly found bacteriocins as compared to the inhibitory activity of the bacteriocin of P. acidilactici S3, also an established pediocin producer. This result has some relevance to the control of the psychrophilic bacteria, L. monocytogenes, in fermented dairy products where beneficial lactic acid bacteria such as L. casei is being used