Comparative Study On The Breeding Of High Yield Polysaccharides From L.bulgaricus By Pulsed Light And Diethyel Sulfate

L.bulgaricus belongs to the lactic acid bacteria,which is a food grade bacterium.It is the most important thermophilic starter cultures of yoghurt production.The exopolysaccharides of L.bulgaricus,which has unique physical and biological characteristics,plays a vital role in the food processing industry.However,large scale application of polysaccharide is still limited due to their low yield of production.Pulsed light is a new technique for microbiological mutagenesis,but this research starts late.Pulsed light has the characteristics of environmentally friendly and simple operation compared with traditional chemical mutagens?complex operation or poisonous?.In this study,the optimal mutagenesis conditions of pulsed light were determined by orthogonal experiment.Additionally,the research has investigated the yields,the primary structures and antioxidant activities of polysaccharides produced by pulsed light-mutagenesis mutants compared with the traditional chemical mutagens one.Finally,the mutagenesis mechanism of pulsed light is discussed.The purpose of the research is to explore whether pulsed light could be a novel safety and environmentally friendly method in substitution of traditional chemical mutagens?poisonous and complex operation?for improvement the polysaccharide production capability of Lactobacillus bulgaricus.The contents and results are as follows:?1?Determination of optimal mutagenesis conditions of two mutagenesis methods and screening of high yield mutants.Taking the mutant polysaccharide yield as index,integrated single factor and orthogonal experiment,the optimal mutagenesis parameters of pulsed light are as follows:pulse voltage,1800V;pulse number,10 times;and pulse distance,7cm;the optimal mutagenesis parameters of diethyl sulfate are as follows:volume fraction,0.4%;processing time,20min;processing temperature,37?.Mutant strains L27?496.98±12.26mg/L?and D23?500.56±12.12 mg/L?are successfully obtained by screening.?2?Determination of the performance of mutant strains.The resistance to acid and bile salt of mutant strains?L27 and D23?are significantly higher than that of the wild strain?p<0.05?,and L27 increased more.The logarithmic growth period of the mutant strains is longer than that of the wild one.Mutants L27 and D23 show greater ability to produce adhesion and acetaldehyde than those of the wild one.Additionally,the acidogenic ability of mutant strain L27 is more suitable for fermented yoghurt than the wild strain and mutant D23.?3?Study on the properties of mutant polysaccharides.All polysaccharide samples produced by wild strain,mutant L27 and D23 have polysaccharide compounds and non-starth polysaccharide.There is no significant difference in the content of neutral sugar and protein content from all the polysaccharide samples?p>0.05?.There are no significant differences in chemical bonds among three EPS samples by the results of infrared spectroscopy.From scanning electron microscopy results,the surface microstructure of polysaccharide is not changed by pulse light mutagenesis.Additionally,the porous structure of polysaccharide produced by mutant L27 and the wild one has more appropriate microstructure for food processing.Compared with the wild strain and mutant D23,L27 polysaccharides have better scavenging ability of DPPH,O₂^-,.OH and Fe²⁺,showing higher antioxidant activity.The results show that the two mutagenic methods would affect the physicochemical properties and bioactivities of strain polysaccharides,and mutagenic effect of pulsed light is better than ethyl sulfate.?4?Preliminary analysis of the mechanism of mutagenesis.RAPD analysis showes that different primers have obvious changes about the DNA amplification bands of L27 and D23.This may be due to the production of CPDs by pulsed light mutagensis,resulting in damage to the mutant strain L27 genomic DNA and alkylation of diethyl sulfate resulted in the conversion of the D23 base of the mutant,frame shifting and deletion,leading to differences in the amplified bands.SDS-PAGE results find that the cell protein bands from mutants L27and D23 are different in the aspects of both presence or absence of the bands and light or dark of the bands,which indicates mutagenesis treatment affected the expression of genetic material.Additionally,the bands are different,indicating that the mutagenic mechanism of two treatments was different.