| ?-glucan is one of the most important sources of cell wall of the yeast strains(Saccharomyces cerevisiae),it is now known that ?-glucan not only exhibits a broad spectrum of biological activities including immune-modulating,anti-inflammatory,anti-cancer,and anti-aging properties,but also can be used in food production as moisturizer,dietary fibers(DF).Yeast is a cheaper source to produce ?-glucan than others,in view of the practical relevance of studies,efforts were undertaken to enhance yeast ?-glucan for more effective production processes.Therefore,it is imperative to breed yeast strains with higher ?-glucan production and optimize the fermentation process.In this study,heavy ion radiation processing was offered to induce mutagenesis of baker's yeast,screened out the high ?-glucan content mutants after the irradiation,established a high-throughput trace measurements to make screening process more precise,quickly,and high efficient.Furtherly,the fermentation conditions of ?-glucan production and extraction conditions was optimized respectively.This study provided a scientific basis for 12C6+-ion beams irradiation mutation on Saccharomyces cerevisiae and mutant breeding,and will establish the principle and experimental foundations for the industrial production of yeast ?-glucan.1.The 12C6+-ion beams irradiation mutation on Saccharomyces cerevisiae and mutant breedingAfter using 12C6+-ion beams irradiation mutation on start yeast strains(CK),the survival rate of yeast cells was obtained by statistical data.The result showed that the dose of half lethal was around 100 Gy compared to the unexposed control group.It was good to screen the positive mutants at the irradiation dose around100 Gy.Phenol-sulfuric acid method was used to determine the content of yeast?-glucan,and the high-throughput micro detection method of ?-glucan detection was established for quick screening after heavy ion beams irradiation by using 96-well microliter plate and the microplate reader,which could replace the ultraviolet spectrophoto-meter for detecting.The sample size in this detection was 200 ul each hole,and 96 samples can be detected at a time.The precision is R2 > 0.99 during theconcentration ranges of 0.001-0.25mg/ml.Five S.Cerevisiae mutant strains 100G-1?100G-9?80G-25?140G-7 and 100G-2 were screened out with high ?-glucan production after the shake flask fermentation,which are significantly higher compared with control strains.After nine consecutive generations fermentation,the high ?-glucan collection were still stable.2.Optimization of growth conditions by high ?-glucan production S.Cerevisiae mutant strainsAccording to the experiment,the carbon sources,nitrogen sources,and concentration of metal ions were optimized respectively to get the optimal fermentation conditions for the high ?-glucan production 100G-9 S.Cerevisiae mutant strain.The optimal carbon sources was sweet sorghum-culture with the ratio of4.0%,the optimal nitrogen sources was 0.5% peptone,1% beef extract and 0.5% yeast extract,and the concentration of metal ions were 0.1% K+,0.1%Mg2+respectively.Compared with the original fermentation conditions,the biomass accumulation of 100G-9 S.Cerevisiae mutant strain was increased to 8.16g/L,5.8%higher than the contrl group.This optimized experiment showed a high effective way to improve biomass production of 100G-9 S.Cerevisiae mutant strain.3.Optimization of ?-glucan extraction conditions by S.Cerevisiae mutant strainsTo obtain the optimized extraction conditions of yeast ?-glucan,the orthogonal experiment was used and the optimum extraction conditions were as follows: NaOH concentration is 2.0 mol/L,the extraction temperature is 90?,and the extraction time is 2.5h.The most important influence factor in ?-glucan extraction conditions is NaOH concentration,then comes the extraction temperature and extraction time.In theses optimized conditions,the extraction rate has increased 3.23% compared to with those under the original group,and the purity of ?-glucan is 95.21%,which is also4.61% higher than the report. |
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