Mutation Breeding Of Bacteriocin-Like Substance-Producing Lactococcus Lactis NFL And The Optimization Of Determination Method Of The Antimicrobial Activity Of Bacteriocin-Like Substance

Bacteriocins are ribosomally encoded peptides or proteins that usually exhibit antimicrobial activity against species genetically closely related to the producer strains.Bacteriocin-like substances are another type of anti-microbial peptides,which are not only can inhibit G+ bacteria,but also restrain the growth of G-bacteria and/or eucaryotes.This characteristics determines bacteriocin-like substances will have wider application prospects than bacteriocins.Earlier study showed that the bacteriocin-like compound produced by Lactococcus lactis NFL is easily distinguished by its broader bactericidal activity against common G+and G-bacteria,remarkable stability to heat and sensitivity to protease,which indicated the great prospect of this substance used as natural bio-preservative.In order to increase the yields of the bacteriocin-like substance,this study explored simple and effective mutation methods for breeding a high-yield bacteriocin-like substance-producing L.lactis strain.The wild-type strain L.lactis NFL was mutated by 5 mutagenisis methods,i.e.,heat-induced mutation,UV radiation,UV-heat stress compound mutation,heat-UV synchronous mutation and H₂O₂-induced mutation.The antibacterial activity of the supernatant against Escherchia coli CMCC44102 was tested with double layer agar diffusion method.In addition,agar diffusion method is widely used for determine the antimicrobial activities,but errors due to human factors always influence the accuracy of the results.The objective of this study was to set up a spectrophotometer turbidimetry with characteristics of accuracy,simpleness,and no special requirement for equipments to determine the potency of all kinds of antimicrobial substances.The main results were as follows:The optimal condition of heat-induced mutation was treatment with 60? water bath for 3.5 min,under this condition the mutant strain H-29 was obtained,whose bacteriocin-like substance production was increased by 27.1%.The optimal condition of UV radiation was treatment with UV lamp of 15 w,vertical distance of 30 cm and radiation time of 4 min,under this condition the mutant strain UV-1 was obtained,whose bacteriocin-like substance production was increased by 12.1%.The optimal condition of UV-heat stress compound mutation was incubation temperature of 44 ?,under this condition the mutant strain UV-HS-8 was obtained,whose bacteriocin-like substance production was increased by 20%.The optimal condition of heat-UV synchronousmutation was treatment with radiation time of 10 s at the moment of 2 min during the 50?water bath for 4 min with vertical distance of 20 cm,under this condition the mutant strain H-UV-19 was obtained,whose bacteriocin-like substance production was increased by19.4%.The optimal condition of heat-induced mutation was treatment with 80 mmol/L H₂O₂ for 15 min,under this condition the mutant strain HP-2 was obtained,whose bacteriocin-like substance production was increased by 11.4%.The results of 10 passage experiments showed that above 5 optimal mutant strains had good generic stability.Among the tested 5 mutation methods,the most effective mutagenesis is heat-induced mutation,of which positive mutation rate was increased by 13% compared to that of UV radiation.The next is UV-heat stress compound mutation and heat-UV synchronous mutation.H₂O₂-induced mutagenic effect is almost equal to UV radiation.Results revealed that the calibration curves of three antimicrobial substances,including bacteriocin nisin,bacteriocin-like substance NFL and antibiotic gentamycin sulfate,were linear in the experimental range at the sampling time point which corresponded to the maximal inhibitory rate with middle inoculum size?R2>0.99?.This method is suitable for precise determination of all kinds of antimicrobial substances in simple laboratory conditions.