Study Of Bacteriocins Produced By Four Lactobacilli Strains

Organic acids, hydrogen peroxide, diacetyl and other metabolic end products produced byLactis acid bacteria (LAB) act as bio-preservatives by altering the intrinsic properties of the food tosuch an extent as to actually inhibit spoilage microorganisms, and the role of these metabolic endproducts has long been appreciated. The contribution of LAB-derived bacteriocins may frequentlyhave been overlooked. The widespread ability of LAB to produce bacterocins implies an importantbiological role maintained over thousands of years and the precise nature of this role has been thesubject of intensive research in recent times. Bacteriocin production could be considered asadvantageous to the producer as, in sufficient amounts, these peptides can kill or inhibit bacteriacompeting for the same ecological niche or the same nutrient pool. Although bacteriocins areproduced by many Gram-positive and Gram-negative species, those produced by the LAB are ofparticular interest to the food industry. To date, the only commercially produced bacteriocins arenisin and pediocin PA-1. While nisin has been found to be extremely effective as an additive toprevent spoilage and increase shelf-life in a number of foods. But its effectiveness has been morevariable with respect to other applications. So the new bacteriocins should be attractive to explore.Four bacteriocin-producing strains were screened from 67 strains of lactobacilli from differentsources. They were Lactobacillus Brevis KLDS1.0355, Lactobacillus kefiranofaciens KLDS1.0373,Lactobacillus coryniformis subsp. Coryniformis KLDS1.0391 and Lactobacillus buchneriKLDS1.0364. All of four strains were isolated from traditional dairy products of Inner Mongolia.The cell-free supernatants (CFSs) of four strains can inhibit indicator strains strongly excludinginhibitive effect of hydrogen peroxide and and organic acid, but the inhibitive activity decreasedsharply after treatment with proteinase K. The results confirmed that these inhibitory materials wereproteins, could be classed as bacteriocins.To optimize bacteriocin production, experments were done on the respect of incubationcondition and the media components. The optimun incubation time and tempreture used to producethe largest activity of bacteriocins were 30℃and 24h for KLDS1.0355, KLDS1.0373 andKLDS1.0364, 30℃and 28h for KLDS1.0391. The optimum media nitrogen source and carbonsouce were: trypone 1%, peptone 0.5%, meat extract 0.5%, yeast extract 0.5%, glucose 4%.Productions of bacteriocin were up to 270.64IU/ml, 333.91 IU/ml, 574.72IU/ml and 295.49IU/ml forfour strains when they were incubated at optimum conditions. The bacteriocins were precipitated by ammonium sulphate, followed by SP-Sepharose fast flowcation exchange chromatography, and the bacteriocins were washed with 0.5mol/L NaCl (in acetatebuffer, pH5.0) at flow rate of 1ml/min. The specific activitives were 74.20 IU/mg, 44.75 IU/mg,102.77 IU/mg and 54.53IU/mg of KLDS1.0355, KLDS1.0373, KLDS1.0391 and KLDS1.0364respectively. The molecular wights of KLDS1.0355, KLDS1.0373 and KLDS1.0364 bacteriocinswere 15.9 kD, 20.1kD and 21.6kD separately.Biological characterization of bacteriocins had been researched. Complete inactivation ofantimicrobial activity was observed after treatment of bacteriocin-containing cell-free supernatantsof four strains with pepsin, trypsin, proteinase K, papain,α-chymotrypsin. The results showed thatbacteriocin were proteinaceous. Partially inactivation of antimicrobial activity was observed aftertreatment of KLDS1.0355 bacteriocin-containing CFSs with chymotrypsin. There were no anychanges for antimicrobial activity after treatment of bacteriocin-containing CFSs withα-amylase,indicated that carbohydrate moieties were not required for anti-microbial activity. The bacteriocinsremained stable after 2h of incubation at pH between 2.0 and 10.0. No decrease in activity wasrecorded after treatment at 80℃for 30min and increased in activity at 121℃for 30min. Themechanism of action mode for four bacteriocins is bactericidal, as shown by a obvious decrease by97.76%, 97.67%, 97.90% and 97.67% separately in the viable cell numbers of Staphylococcusaureus ATCC25923. No increase in the activity of bacteriocins produced by KLDS1.0391,KLDS1.0364 were recorded after treatment the cells with NaC1 at pH2.0, suggesting that thebacteriocins did not adhere to the surface of the producer bacteria, whereas bacteriocins produced byKLDS1.0355, KLDS1.0373 partial adsorption to producer cells were recorded.Bactericins produced by KLDS1.0355, KLDS1.0373, KLDS1.0391 and KLDS1.0364 had alower specific activity than nisin, because the purity of these bacteriocins was too low. The furtherresearch should be focous on the purification of these bacteriocins. Compared with nisin, bactericinsproduced by four strains had the broader spectrum, including sevaral strains of Gram-positivebacteria Staphylococcus aureus, Listeria monocytogenes and some Gram-negative bacteria,especially Escherichia coli, Salmonella and Pseudomonas. They will have the broader applicationprospect in the food industry.