High Production Of Nisin Strain's Selection And Culture Medium Optimization

Nisin is applied as a natural preservative in various food products, such as dairy, meat, can, beverage and so on. It shows an obvious antimicrobial activity against a wide range of Gram positive bacteria, so it can prevent food decay effectively. Compared with chemical preservative, nisin exhibits some advantages: effective, safe, nontoxic. Also it can be degraded by enzymes in human's digestive system, so there will be no cumulation of nisin. Nevertheless, the low yield of nisin and high cost of nisin production lead to a high price, which restricts its widespread use. Currently, people do a lot of research about nisin medium optimization and fermentation process optimization for improving the yield of nisin.In our research, Lactococcus lactis subsp. lactis W28 saved in our laboratory was mutanted by two rounds of UV irradiation. The mutants were screened by nisin resistance, glucose tolerance and both of them as selection marker, and then with a second shake flask experiments to obtain four mutants with genetic stability and high yields of nisin, as GNK-1, GNK-4, SNK-5, SNK-10. The four mutants with a tolerance to 7000IU/mL nisin and 15% glucose were determined, and the titers of the four strains were 2200IU/mL,2105IU/mL,2274IU/mL,2178IU/mL, which were 1.90, 1.81, 1.96, 1.88 times than the original strain Lactococcus lactis subsp. lactis W28.In our research, we choose the four mutants GNK-1,GNK-4,SNK-5,SNK-10 obtained above as the fusion population, and then do a round of genome shuffling to improve fusion cell's tolerance to nisin. The fusion cells were screened out by high concentration of nisin (8000IU/mL) and glucose (15%), and then nisin titers were determined by shake flask analysis. Two fusion cells F₂ and F6 with high yields of nisin were obtained, and the nisin titers could reach 2426IU/mL, 2525IU/mL,which were 2.09 and 2.17 times than the original strain..To search for a cheap raw material for nisin production, Waste Material from Fungal Culture (WMFC) hydrolysis liquid was used as the culture medium for Lactococcus lactis. First pretreat the WMFC with dilute sulfuric acid to obtain the hydrolysis liquid for further experiment, and then we investigated two factors affected cell's growth and nisin titer. The two factors were ion concentration and different alkalies callback pH value of the hydrolysis liquid. The results suggested that when using Ca(OH)₂ adjusted the pH value and with the hydrolysis liquid no dilution, the cell grew best, and the cell density could reach 2.9×10¹¹cfu/mL. The nisin titers didn't show a significant difference between these experiments.In our research, we also optimized the culture medium with WMFC hydrolysis liquid and determined the optimum additional carbon source, nitrogen source, phosphate and their appropriate amount. The optimal medium with WMFC hydrolysis liquid was got as: sucrose 3g/L, yeast extract 7g/L, corn steep liquor 3g/L, KH2PO4 1g/L. Under the optimal medium, the nisin titer reached 335IU/mL, which was increased 23% than the non-optimization medium. This research developed a new low-cost raw material for nisin production, and made effective use of WMFC.