Isolation, Characterization, Partial Purification and Plasmid Curing Analysis of Bacteriocin of Pediococcus acidilactici KX688798 Isolated from Fermented Vigna radiate

Microbial proteases have wide industrial applications and proteases of the lactic acid bacteria (LAB) have received special attention because of their importance in the food and dairy industry. Of all the LAB, the proteolytic system of the pediococci is the least studied. Therefore, this study was aimed at characterizing and purifying the protease produced by Pediococcus acidilactici grown in de Man, Rogosa and Sharpe (MRS) broth. Casein concentration of 2% (w/v) and 2.5 ml of the crude enzyme were optimal for the activity of the protease. The crude protease had temperature and pH optima of 28 oC and 4.0 respectively thus indicating that the enzyme is a mesophilic and acidic protease. Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced purification fold (1.24 - 1.59) with specific activity ranging between 25.39 - 32.54 units/mg protein. The protease showed a characteristic band on SDS-PAGE and the molecular weight was estimated between 45 and 66 kDa. The potential applications of the protease are discussed.Keywords: Protease, lactic acid bacteria, Pediococcus acidilactici, enzyme purification.

Comparison of biopreservatives obtained from a starter culture of Pediococcus acidilactici by different techniques

Lactic acid‐producing bacteria (LAB) isolated from fowl, fish intestines and mushroom were identified and characterized with reference to the production of bacteriocins and its genetic markers. Among the isolated cultures, two isolate each of Lactobacillus casei ssp casei C‐40 and M‐50, Pediococcus pentosaceus C‐6 and M‐10, and Pediococcus acidilactici C‐20 and F‐58 and one isolate of Pediococcus sp. F‐7 were potent producers of bacteriocin active against related strains of LAB. With reference to the genetic characterization of bacteriocin production, the potent cultures appeared to posses either one or two plasmids of 4–8 Kb size and a megaplasmid. Plasmid curing of bacteriocinogenic strain of Lact. casei ssp. casei C‐40 resulted in the loss of bacteriocin production and an altered pattern of sugar utilization. Antibiogram of bacteriocinogenic cultures of LAB and bacteriocin indicators was carried out to evaluate as a markers in genetic characterization. The bacteriocinogenic cultures were resistant to most of the antibiotics, particularly those belonging to the ß‐lactam and cephalosporin groups. Besides, most of these cultures were sensitive to 30 μg of chloramphenicol and 15 ng of erythromycin. The potential role of bacteriocinogenic LAB is significance in maintaining the intestinal environment, however; sensitive/resistance to antibiotics is to be viewed with concern as feeds do contain antibiotics.

Sixteen pediococcal strains, including eleven Pediococcus acidilactici and five P. pentosaceus strains were screened for inhibitory potential using a deferred overlay spot method against a limited collection of foodborne pathogens. Of those screened, P. acidilactici PC, an organism isolated from fermented sausage, was effective and subsequently screened for inhibitory potential against 46 foodborne pathogens and 28 other lactic acid bacteria. Strain PC produced an antimicrobial agent capable of inhibiting members of the genera Listeria, Clostridium, Leuconostoc and Pediococcus. Gram-negative microorganisms from seven genera, Lactococcus, Streptococcus and Lactobacillus strains were unaffected by the inhibitory substance. The inhibitory agent was sensitive to proteolytic enzymes and exhibited a bactericidal mode of action, confirming the identity as a bacteriocin. In addition, the partially purified bacteriocin was thermally stable up to 100°C for 60 min and maintained inhibitory potential over a wide range of pH values. Plasmid curing studies suggested linkage of bacteriocin production to a 5.5-MDa plasmid. Plasmid profiles were identical for P. acidilactici PC, PAC1.0 and PO2. Genetic analysis of total genomic DNA via DNA fingerprinting and ribosomal RNA (rRNA) typing provided further evidence that these strains were identical. DNA fingerprinting and rRNA typing also showed utility in discrimination between and within other species of pediococci.