The Establishment Of Determination For The Antibacterial Activity Of Bacteriocin-Like Substance NFL And The Optimization Of The Chemically Defined Medium Composition

Bacteriocin-like substance is the inhibitory substance with the properties of the protein or polypeptide excreted by certain bacteria. Bacteriocin-like substance has a broader application prospect than bacteriocin because it is special for its broader inhibitory spectrum targeted toward Gram-positive bacteria and Gram-negative bacteria or fungi, and it has high stability over a wide pH range.At present, agar diffusion assay is the most widely used inhibitory activity detection method, but the method is time-consuming and poor stability. In this paper, a rapid turbidimetric method for determination of bacteriocin-like substance NFL inhibitory activity using a Myroides spp. strain as the indicator was established. The optimized method was as following:The indicator strain was grown to the mid-exponential growth phase, and subcultured at 2%(v/v) inoculum volume and incubated at 30℃ by agitation (150 rpm on laboratory shaker), and the OD600 and inhibition rate were determined after incubation for 5 h. Results showed that inhibition rate in the range of 14.69%-84.80% was linearly related to the concentration of bacteriocin-like substance NFL (R2=0.9911). Validation experiments of the linear regression equation showed that the average recovery rate was 100.10% and the relative standard deviation (RSD)<1.47%. So, this method was much better than the agar diffusion assay for its higher sensitivity, higher precision and shortened measurement time. Applying this method to determination the titer of nisin using Lactococcus lactis NZ9000(pNZ8148) strain as indicator, experimental results demonstrated that it was also feasible. Therefore this method established here could be applicable in the determination of bacteriocin-like substance activity in fermentation liquid and bacteriocin pure products.Organic nitrogen components of microbial culture medium is very complex, and the quality is not stable because of the different batches and different producing areas, which has a great effect on the microbial fermentation. Therefore, the aim of this study was to optimize the chemically defined medium components for the production of bacteriocin-like substance produced by Lactococcus lactis NFL. First, the best types and concentrations of carbon sources, inorganic nitrogens, amino acids, vitamins and nitrogen bases were selected by adopting the method of single factor experiment. Then the components of the medium was further optimized with an orthogonal experiment of 7 factors and 3 levels. Finally it was concluded that the optimum medium composition (g/L) was:fructose 7, ammonium sulfate 9, cystine 0.09, vitamins B7 0.025, thymine 0.004, MgSO4 0.25, MnSO4 0.03. Verification results showed that the inhibitory rate of strain NFL fermentation supernatant was 54.23%, which was increased by 11.28% than that before optimization (with the MRS broth).