Fermentation And Purification Of Bacteriocin From A Pediococcus Acidilactici Strain

Bacteriocins, which share many varieties and different properties, arerecognized as potential biopreservatives. However, only Nisin is currentlylicenced as a food additive in over60countries. Therefore, to develop newbacteriocin from lactic acid bacteria with efforts and to study bacteriocin whichhave been found in depth is the current important task for the majority of scientists.In the early work, our laboratory has isolated a strain from the Chinese traditionalfermented cabbage, which could produce bacteriocin. Afterwards, it has beenidentified as Pediococcus acidilactici. To our Knowledge, very few papers havebeen published on Pediocin in China, this work aimed to study fermentation andpurification process of Pediocin from P. acidilactici PA003. Specifically, it isdivided into the following four parts1、A efficient laboratory-scale purification method was developed topurify a pediocin produced by P. acidilactici PA003, and then it has beenidentificated.Pediocin produced by P. acidilactici PA003was purified by ammoniumsulfate precipitation, gel filtration chromatography, ion-exchange chromatography,and reverse-phase high-performance liquid chromatography. This procedureallowed the recovery of3.8%of the bacteriocin present in the culture supernatantwith purity higher than99%and an approximately461fold increase in specificactivity. The molecular mass of the purified pediocin was about4624Da, whichwas identical to pediocin PA-1AcH, as determined by Matrix-assisted laserdesorption ionization-time of flight mass spectroscopy. By the way, we also foundP. acidilactici PA003can produce more than one kind of bacteriocins.2、The effective membrane system to separate bacteriocin was developedThis study developed an effective combined ultrafiltration（UF）–nanofiltration （NF） membrane system for ultrafiltering bacteriocinfermentation broth and concentrating the filtrate to improve the downstreamefficiency. With optimal PS-10membrane, UF processes was proposed untilvolume concentration factor （VCF） reached2.5, then diafiltration processes ofultrafiltration was proposed. The permeate obtained from UF was thenconcentrated by NF. When the VCF of NF reached4.5,1000mL of UF penetrantcan be concentrated to220mL. This two-stage membrane process employing UFfollowed by NF improved the loading solutions by4.5-fold, allowed the recovery of81.2%of the bacteriocin.3、The secondary kinetic models to describe the influence of temperatureand pH on microbial behaviour were developed.To investigate the effect of pH and temperature on the cell growth, glucoseconsumption, lactic acid and bacteriocin production of P. acidilactici PA003, thekinetic behaviour of P. acidilactici PA003was simulated in a5-L fermentor bymaking use of de Man’s Rogosa and Sharpe （MRS） broth. The secondary kineticmodels to describe the influence of temperature and pH on microbial behaviourwere developed. The optimal condition for specific growth rate and biomassconcentration was almost the same at pH6.5and35°C. At35°C and pH6.1, themaximal bacteriocin activity was also achieved.4、The fed-batch fermentation process of P. acidilactici PA003wasoptimizedThe cell growth, glucose consumption, lactic acid and bacteriocin productionby P. acidilactici PA003in modified MRS broth with various concentrations ofglucose and complex nitrogen source was investigated in5L fermentor using batchfermentation process. In fed-batch fermentation the rate of feeding of the reactorwith the substrates was either maintained constant or varied exponentially as afunction of time. The results showed that both cell growth and bacteriocin activitywere influenced by changes in the concentrations of glucose and CNS. The highestbacteriocin titre (2985AU·mL^-1) was obtained with fed-batch fermentation at anexponentially varying rate of feeding.