Research On Fed Batch Fermentation For Nisin And Its Antibacterial Properties

Nisin, a kind of primary metabolites produced by Lactococcus lactis or Streptococcus lactis, can inhibit growth and reproduction of most gram-positive bacteria. Nisin with high-efficiency and safety has great prospect in business development and has been widely applied in the preservation of dairy products, meat products, beverages and other food industry.Streptococcus Thermophilus 6032 was used as fermentation strain in this study. Fed-batch fermentation technology was applied to release the inhibition of substrate for cell growth and synthesis of Nisin, thereby biomass of cells and Nisin titer unit got improved. The main research contents and experimental results were as follows:The types and addition amount of carbon, nitrogen and phosphorus source in original medium were studied by single factor experiments. Then orthogonal experiment was designed for further optimization of resulted factors. Research results showed that sucrose, yeast powder and dipotassium hydrogen phosphate were the appropriate corresponding resources. The optimal amount of fermention medium was sucrose 20g/L, yeast powder 15g/L, dipotassium hydrogen phosphate 3g/L and corn steep liquor 10%. The rest composition was similar to the primary medium. Under these conditions, Nisin titer unit and OD6oo were 4734IU/mL and 0.711.Influencing factors of fed-batch fermented Nisin were studied by single factor experiment. The optimum component of fed-batch fermentation was the whole medium, the concentration of supplement medium of sucrose and yeast powder were 40g/L and 20g/L. Addition of amino acid had no promotion in growth of cells and the syntheisis of Nisin. Other conditions for fed-batch fermentation were as follows:feeding time 14h, feeding number one, feeding volume 120mL, feeding speed 500ml/h, fermention pH 6.5, concentration of sodium hydroxide solution 5mol/L.Based on the results of Plackett-Burman method, fermentation pH, feeding time and feeding volume were selected to be the important factors of Nisin titer unit and OD600.The level of main factors were determined when the maximum response area was discovered by the design of steepest ascent. The conditions of fed-batch fermentation for Nisin was optimized by the design of Box-Behnken. The results were as follows:fermention pH6.40, feeding timel3h and feeding volume 100mL. In this way, Nisin titer unit and OD600 were 6224IU/mL and 0.914, suggesting the use of response surface method to optimize fed-batch fermentation conditions for Nisin was effective.Seed liquid was inoculated to 5L fermenter whose initial volume of fermentation medium was 3L with the amount of 5% at 34℃.1L supple-ment medium was added at the speed of 500 mL/h to original fermention medium when cell fermented to 13h. The composition of supplement medium was similar to the optimized medium except the concentration of sucrose and yeast powder were 40g/L and 20g/L. Stirring speed and diss-olved oxygen were respectively 100r/min and 40%. The Nisin titer unit and OD600 were respectively 6368 IU/mL and 0.920 when fermentation was completed. The dry weight of cell, Nisin titer unit and concentration of sucrose at different times were determined and fitted by the softwore. Resulted fermentation kinetic models of Streptococcus Thermophilus 6032 by the softwore were as follows:kinetic models of cell growth was dX/dt=0.43237X(1-X/1.15688), kinetic models of product synthesis was dP/dt= 5788.6dX/dt-49.14X, kinetic models of substrate consumption was-dS/dt=1.132dX/dt+0.0019dP/dt+0.5966X. The establishment of dynamics models provided support for the industrial use of fed-batch fermentation technology.Staphylococcus aureus and Aspergillus niger were used as indicator bacterias to research the antimicrobial properties of Nisin by single factor experiment. The experimental results indicated that the minimum inhibitory concentration of Nisin to Staphylococcus aureus and Aspergillus niger were 5mg/mL and 10mg/mL. With the increase of pH, the antibacterial activity of Nisin presented a plummeting trend. The temperature had little effect on the antibacterial activity of Nisin because of the thermal stability of Nisin. Storage time had no influence on antimicrobial properties of Nisin nearly. Sodium citrate, sodium dihydrogen phosphate and disodium hydrogen phosphate had a positive role in the protection of Nisin inhibitory effect. Addition of K+, Ca2+ and Fe3+ were also beneficial to the maintenance of antibacterial activity.