Halophilic bacteria from salty fermented foods and its bacteriocin encoding gene expression to use as biocontrol agent against Staphylococcus aureus in salt added foods

Abstract

This research was aimed to isolate, select and apply bacteriocin producing halophilic bacteria from salty fermented foods, Plara (Thai traditional salty fermented fish) and soya sauce as bio-control agent in foods. The isolates having inhibitory activity against different strains of Staphylococcus aureus were selected, identified and characterized prior to application as protective starter in food models. Bacterial communities of Plara and soya sauce samples were studied by two methods, a cultural dependent and cultural independent method (Reverse Transcriptase PCR DGGE (Rev-T PCR-DGGE)), and subsequently sequenced by 16s rDNA analysis. Halanaerobium spp. in Plara and Staphylococcus gallinarum in soya sauce were the main population detected by the Rev-T PCR-DGGE, while Bacillus spp. with the predominance of B. subtilis (41%) was the bacterial isolates detected by cultural plating method. Among 124 isolates from soya sauce and Plara, 37 (29.8%) isolates (35 Bacillus strains and 2 other groups of bacteria) exhibited inhibitory effect against the three different indicator Staphylococcus aureus strains. The inhibitory action was tested by deferred antagonism, spot-on-the-lawn method. The Bacillus isolates displayed different inhibitory pattern on the indicator strains, significantly different at p<0.05. The Bacillus isolates with positive inhibitory action was further investigated for the gene encoding bacteriocin production (subtilin-spa/ subtilosin-sbo). Furthermore, bacteriocin gene expression was studied, and found that two isolates of Bacillus (B. subtilis and B. licheniformis) over expressed bacteriocin gene in 5% NaCl and co-culture with cocktail S. aureus. The gene expression Bacillus subtilis isolate was selected for developing to protective culture, after subjected to safety evaluation. Minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of the selected strain were found at 20AU/ml and 80AU/ml, respectively. When application as protective cultures in two food models (cottage cheese and bamboo shoot pickle), the culture significantly reduced 2-3log cycle of S. aureus contamination. This study demonstrated a potential of Bacillus spp. in controlling the growth of S. aureus, therefore it could be a potential in further development as protective culture in food industries.