Study On Bacillus Subtilis Induced By Pulsed Light And Its Fermentation Performance

Bacillus subtilis is an important food-grade probiotics in the modern industrial production.Mutagenic breeding of high tolerance and good fermentation performance can not only improve the tolerance of strains to poor environment in the producing and processing environment,but also improve enzyme production level and antibacterial effect,which plays an important role in the fields of application of food enzyme preparations and food preservative.Pulsed light is used as a mutation technique,which its unique mutagenic light source is pulsed light.Pulsed light that is instantaneous and high-intensity light can affect microbial cells,change the genetic material structure,and change the process of protein synthesis,resulting in changes in microbial traits.Nowadays,there are few reports on the mutagenesis of bacillus subtilis by pulsed light technique at home and abroad.Therefore,this study adopts pulsed light technique to mutagenize bacillus subtilis,screening the strains with excellent traits out,preliminary analyzes its mechanism of mutagenesis,and explores the feasibility of pulsed light inducing bacillus subtilis.The experiment results are as follows:1.Pulse voltage,pulse number and distance are regarded as the influencing factors and the lethal rate of stains is regarded as the index.The lethal condition is optimized by the response surface method,and the best lethal conditions are obtained as follows:2450 V,65 times,5cm for pulse voltage,pulse number and distance.The best pulse voltage and the best distance are unchanged,but pulse number is changed from 8,14,21,27,34,40 to 47 times to mutagenize strain suspension and the effects of different pulse times on the strain lethal rate are investigated.It is determined that 40 times for pulse number,screening eight resistant strains B1?B8 out.2.High temperature tolerance tests,strong acid tolerance tests and bile salt of high concentration tolerance tests are conducted for the original strain BO and eight resistant strains B1-B8 screened out by pulsed light.The superior resistant mutant strains B3,B4,B7 with three kinds of high tolerance are screened out.3.The fermentation performance of the original strain and the mutant strains has been comparatively analyzed.The results show that the fermentation performance of the mutant strains is good,and the genetic stability and antibacterial activity of the mutant strains are significantly higher than those of the original strain.(1)The ability of the strain to hydrolyze starch and the activity of a-amylase production are determined by plate-transparent circle method and Yoo modified method.The results show that the mutant strains B3,B7 have stronger ability to hydrolyze starch than that of the original strain B0.The ?-amylase activities of B3 and B7 are(114.06±0.43)U/mL and(120.89±0.32)U/mL,respectively,which are 1.67 times and 1.77 times of that of BO of(68.3±0.14)U/mL and the difference is extremely remarkable(P<0.01).B3 and B7 are subcultured five times,respectively,and the genetic stability of both is good.(2)The ability of the strain to degrade protein and the activity of protease production are determined by Phillips method and the Folin-Ciocalteu method.The results show that the degradation ability of mutant strains B3,B7 is stronger than that of the original strain B0.The protease activities of B3 and B7 are(88.3±0.35)U/mL and(96.79±0.26)U/mL,respectively,which are 1.56 times and 1.71 times of that of B0 of(56.6±0.21)U/mL and the difference is extremely remarkable(P<0.01).B3 and B7 are subcultured five times,respectively,and the genetic stability of both is good.(3)The ability of the strain to decompose fat and the activity of lipase production are determined by plate-transparent circle method and olive oil emulsification method.The results show that the mutant strains B3,B7 have stronger ability to decompose fat than that of the original strain B0.The lipase activities of B3 and B7 are(16.21±0.27)U/mL and(17.18±0.36)U/mL,respectively,which are 1.34 times and 1.42 times of that of BO of(12.1 ±0.15)U/mL and the difference is remarkable(P<0.05).B3 and B7 are subcultured five times,respectively,and the genetic stability of both is good.(4)The activity of the strain to produce a-acetolactate decarboxylase is determined.The results show that the a-acetolactate decarboxylase activities of the mutant strains B3 and B7 are(0.64±0.12)U/mL and(0.71±0.17)U/mL,respectively,which are 1.42 times and 1.58 times of that of B0 of(0.45±0.23)U/mL and the difference is remarkable(P<0.05).B3 and B7 are subcultured five times,respectively,and the genetic stability of both is good.(5)The antibacterial activity of bacillus subtilis is determined by Oxford Cup method,and the antibacterial rate against staphylococcus aureus,escherichia coli and salmonella are calculated.The results show that mutant strains B3 and B7 have the best antibacterial effect on the three pathogens,all of which belong to grade one.The antibacterial effect of original strain B0 on E.coli and salmonella is slightly worse,both of which belong to grade two.The antibacterial rate of B3,B7 on the three pathogens is higher than that of B0,when the concentration of suspension was 103/mL and 104/mL,the difference is remarkable(P<0.05).The antibacterial activity of the mutant strains B3 and B7 is good.4.SDS-PAGE experiment is conducted on the mutant strains B3,B7 and the original strain B0.The results show that the bacillus subtilis can be affected by pulsed light.The stripes on the electric lane of strain protein with different molecular weights are different in the aspects of both presence or absence of the stripes and light or dark color of the stripes,which illustrates that pulsed light has caused the change of strain protein expression.