Isolation, Screening, Identification And Fermentation Of Bacteriocin-Like Substance Producing Lactic Acid Bacteria

Bacteriocin-like substance is a kind of active material having inhibitory activity and anti-fungal ability. It is produced by bacterials. Bacteriocin-like substance is being widely used in food processing because it is safe and not toxic. Many bacterias can produce bateriocin-like substance. The bacteriocin-like substance could be different among bacterias isolated from different places and materials.In order to get high productivity and good potential in practical application, pickled vegetables were used as the resource for isolating and screening bacteriocin-like subtance-producing bacterias. One strain having high productivity of bacteriocin-like subtance was isolated and identified. Properties and potential application in food preservation of the bacteriocin-like substance were studied. Conditions for production of the acteriocin-like substance were also optimized. The main results were listed as the followings,(1) 299 Lactic Acid Bacteria(LAB) having inhibitory ability were isolated and screened out from the pickled vegetables. A new screening method was also expolored in the isolation and screening experiments. In the new screening method, 10ml Ep tubes and CaCO3 test were used to shorten and simplify the operation.(2) One strain having the highest inhibitory ability was choosed and identified by referring to the morphologic characteristics, physiological-biochemical propeties and 16S rDNA sequence. The strain was identified as Lactobacillus brevis and named SD-22.(3) Without effects of organic acids and hydrogen peroxide, the cell-free liquid culture of Lactobacillus brevis SD-22 showed high inhibitory activity against gram-positive bacterias, including Staphylococcus aureus, Bacillus subtitis and Bacillus cereus. Gram-negative bacterias, such as Escherichia coli and Salmonella typhimrnium, could also be well inhibited by SD-22. Therefore, the inhibitory substance produced by SD-22 was assumed bacteriocin-like substance and named Lactococcin SD-22. Lactococcin SD-22 showed stable to heat, active at low pH, not sensitive to proteinasea and broad antibacterial spectrum.(4) Suitable conditions for fermentation of LactococcinSD-22 by Lactobacillus brevis SD-22 was optimized at 30℃and initial pH6.5 with inoculum amount of 1% (v/v), seed age of 12h and anaerobic cultivation for 48h. Nutrients for production of LactococcinSD-22 was optimized by Central composite design composition at soya peptone 15g/L, yeast extract 15g/L, beef extract 5g/L, glucose 20g/L, K2HPO4 2g/L, NaAC 5g/L, MgSO4·7H2O 0.58g/L, tri-ammonium citrate 2g/L, MnSO4·4H2O 0.25g/L, and Tween-80 2mL/L. Under the above optimal conditions, the cell-free liquid culture of SD-22 produced inhibitory zone of 20.1mm in diameter.(5) Lactococcin SD-22 showed great potential application in preservation of fresh pork. It could extend the shelf-life of fresh pork by 2days at 4℃and 12 h at room temperature. The cell-free culture showed effective inhibition to the growth of Salmonella in fresh poak.