Research On Nisin Resistant Bacterial Strain Selection And Its Mechanism

Nisin, a lanthionine-containing peptide produced by certain strains ofLactococcus lactis, is widely used in the food industry as a safe and naturalpreservative because of its antimicrobial activity against a broad range ofgram-positive bacteria. However, the mutant study on some gram-positive bacteriashowed that some strains become resistance to nisin after inducing by graduatelyincreasement of nisin concentration These results indicated the efficacy of nisin asa food preservative could be compromised by the emergence of nisin-resistantmutants. But no nisin resistant bacterial strains from nature were reported before.The present work attempted to isolate and screen nisin resistant bacterial strainsfrom market samples in Hangzhou, China, and to explore the nisin resistantmechanism.The main results was obtained as followings:1. Isolation, screen and identification of nisin resistant bacterial strains102 bacterial strains were isolated from the samples collected at ten different markets inHangzhou, Zhejiang province, China, and screened with different nisin concentration. It wasfound that 50IU nisin inhibited 47 strains (49.0%); 100IU nisin inhibited 61 strains(59.8%);200IU nisin inhibited 84 strains (82.4%); 500IU nisin inhibited 97 strains (95.1%); theremaining five strains(NIS06,NIS13,NIS17,NIS41,N1S98)were high nisin resistant bacteria.MIC test showed that the MIC of nisin to NIS41, NIS13, NIS17, NIS98 and NIS06 was2560IU; 1280IU, 1280IU, 1280IU and 640IU, respectively. Analysis of physiological-chemicalcharacteristics and 16S rDNA gene sequences showed that the five strains were all belong toStaphylococcus, NIS41 was identified as Staphylococcus epidermidis; NIS06, NIS13 andNIS98 were all identified as Staphylococcus sciuri, N1S17 was identified as Staphylococcusxylosus.2. Observation on the mechanism of nisin resistant strains.S. epidermidis NIS 41,S. sciuri NIS06,S. sciuri NIS13,S. sciuri NIS98,S. xylosus NIS17, remained their resistance or tolerance to nisin even after 30transfers in a nisin-free medium, indicatingt the 5 nisin-resistant strains with highgenetic stability. No plasmid was extraced from the 5 bacterial strains, and no PCRproducts were detected in PCR using genomic DNA of the strains as template andpartial NisI/NisFEG sequence as primers. Nisin activity were significantly reducedafter treated with supernatant of the 5 nisin-resistant strains, indicating that theproduce of Nisin inactive substance (NIS) would be the main mechanism for theirresistance to Nisin.3. Nisin inactive substance (NIS) from Staphylococcus epidermidis NIS 41.NIS was detected in stationary growth phase of Staphylococcus epidermidisNIS 41. The NIS was not precipitated with 80% saturation of ammonium sulfateadded to cell-free supernatants. The activity of the NIS was stable duringtemperature exposure to 37 and 60℃for 20 min, but lost obviously up to 80℃.The NIS was shown to retain the activity at 4℃within 8 days, but lost completelyin 15th day. The NIS was shown to retain the activity within the pH range of 2-9,and the optimum pH for the activity was about 8.0. The NIS was not extracted byDichloromethane, chloroform, ethyl acetate, n-hexane, and anhydrous ether fromwater, also it was not precipited by Ethanol, acetone and methanol. Cell-freesupematant of S. epidermidis NIS 41 was distilled at 30℃vacuum condition, andthe NIS was found in the volatile liquid. The NIS led to suppression of nisinactivity but did no effect on the bacteriocin produced by Bacillus licheniformisZJU12. Tricine-SDS-PAGE showed that nisin was degradated by the NIS. The NISwas purified by freeze-thaw treatment and HPLC, and the molecular masswas174.9 based on ESI-MS analysis.4. Nisin purification and its effect on inhibition of tested bacterial strains.Purified Nisin was obtained from commercial Nisin by ion-exchangechromatography (CM-32) and dialysis with 1Kda cut. Then Tricine-SDS-PAGEwere applied to judge the purification. The results from MIC test for M. flavusNCIB 8166, S.epidermidis NIS 41, Escherichia.coli BL21 and Lactococcus lactisLY3 showed no obvious difference on antibacterial activity between the purified and the unpurified nisin. The purified and unpurified nisin both showed a MIC of50IU for M. flavus NCIB 8166 and a MIC of 3200IU for S. epidermidis NIS 41;both 25600IU purified nisin and unpurifed nisin failed to show antibacterialactivity against E.coli BL21 and L. lactis LY3. It is suggested that purification hadno effect on antibacterial activity of nisin. By agar plate count method, inhibitioncurve of purified and unpurifed nisin against the 4 strains remained stable in thepresence of NaCl at concentration of 0mM, 50mM and 100mM NaCl, indicatingNaCl below 100mM concentration hadno effect on antibacterial activity of nisin.