Research On Mutation Breeding And Optimization Of Fermentation Conditions Of Propionibacterium Shermanii Strains

It is easy to be contaminated by kinds of microorganism for food during the storage period, causing series of changes in properties and the generation of toxic and harmful substances. Therefore, it is nesserary to add some substances which can inhibit the growth of microorganism in food. Moreover, these additives must be non-toxic and harmless to human beings. The preservatives which are widely used in food industry are potassium sorbate, sodium benzoate, nisin. It was found that the metabolites of Propionibacterium can inhibite numbers of microbes in 1992. Since then a kind of metabolites of P. shermanii called MicrogardTM were approved and used in dairy products widely. Nowadays, the industrialized production of metabolites from Propionibacterium has not yet started interiorly. Long fermentation period and Low antibacterial activities were the most important factors which effect processes of the industrialized production of metabolites.In order to improve the antibacterial activities of the metabolites and lay the foundation for the industrialized production of metabolites from Propionibacterium, this article made a study on mutation breeding and fermentation condition optimization of P. shermanii H1310.The main results of this research were listed as follow:1) The studies were carried on to determaine the metabolic characteristics of P.Shermanii H1310, the results indicated that the culture period reached logarithmic growth phase for 24～48h when the culture of strain in SLB medium using screw bottles.The fermentation of strain using screw bottles could be separated into three periods.In the first period(0～72h), the strain grew rapidly and pH of the broth decreased to 4.91, the concent of reducing sugar decreased from 15g/L to 3.3g/L, the antibacterial activity increased gradually. In the second period (72～96h), the biomass changed little and pH dropped to 4.68, all of the reduing suger were consumed, the antibacterial activity reached to 18.01AU/mL. In the last period (96～168h), the biomass of broth was reduced greatly and pH was constant at 4.6, the antibacterial activity fluctuated between 15.68～19.21AU/mL. The culture period reached logarithmic growth phase for 14-38h when the culture of strain in SLB medium using 15L fermentation tank. Besides that, the growing states of stain in 15L fermentation tank and 1T fermentation tank were similar to the growing state in screw bottles. The fermentation supernatant of Propionibacterium were prepared into powder using spray drying method. The highest antibacterial activity of powders were 223AU/g.The powders had an inhibition effect on E. Coli、Saccharomyces Cerevisiae and Alicyclobacillusacidoterrestris.2) The optimal ultraviolet (UV) mutagenesis conditions were listed:the mutation distance was 30cm, the power of UV lamp was 15W, the time of irradiation was 20s. The optimal nitrosoguanidine (NTG) mutagenesis conditons were listed:the pH of TM buffer was 8.0, the concentration of NTG was 0.5mg/mL, the action time was 60min.Two screening methods (double-layers screening method and screw tube-screw bottle screening method) were established respectively and the optimal screening method was determained. In the double-layers screening method, firstly diluting the broth of P. putida to OD600nm 0.2, then taking 3mL soft ager medium which was mixed with 0.1 mL diluted broth to cover on the single colonies of P. shermanii, selecting the colonies which were surrounded by clear inhibition zone after cultivating for 12h, incubating them using screw bottles to screen again. In screw tube-srew bottle screening method, the single colony of P. shermanii was activated in SLB medium for 36h, and then cultured in lOmL fermentation medium with 5% inoculation volume using screw tube for 4 days. Using the ager diffusion method to test the antibacterial activity roughly. Then incubating the strains which showed larger inhibition zones using screw bottles for 4 days. Using the critical dilution method to measure the antibacterial activity. Strains were cultured at static cultivation deeply, and the culture temperature was 30℃. As it was more time-saving and efficient for double-layers screening method in comparison to screw tube-screw bottle screening method, double-layers screening method was chosen as the better method for screen.Through the double-layers screening method and screw tube-screw bottle screening method,7 strains were screened from 6398 strains, strain NTG110605 was obtained with genetic stability and the antimicrobial activity of the metabolites from strain NTG110605 was increased by 46.3% compared with the original strain.3) Adding carbon source in the fermentation broth, the results showed that adding glucose and lactic acid sodium made the antibacterial activity of the supernate increase by 104% significantly (P<0.05). The concrete adding mean was listed as that:adding sodium lactate once after 48h fermentation, the adding amount was 7.8g/L, adding glucose at intervals of 24h after 72h fermentation for three times, the adding amount was 4.4g/L. Adding 0.8% sodium chloride in fermentation broth, the antibacterial activity of the supernate increase by 38% compared with the control group, and the powders FM828 could replace the original nitrogen source (yeast extract LM800).This research studied in two aspects, and the results showed that not only the strain breeding but also the fermentation condition optimization experiment were effective methods to improve the antibacterial activity of metabolites from P. shermanii.