| Saccharomyces cerevisiae,as a very common fermentation engineering strain,has confirmed its safety.It can produce more than 100 kinds of natural products.It is well used in industries such as industry,chemistry and medical treatment.It has played an important role in the development of biological science and technology and human production and life.At present,the glutathione producing strain studied is mainly S.cerevisiae.Intense pulsed light efficiency mutation breeding technology is a new mutation method.It started relatively late,but it has the advantages of high intensity,instantaneity,simple operation and good stability.In this paper,the best resistant mutation breeding parameters of high glutathione production in S.cerevisiae treated with intense pulsed light were determined by response surface design method,and compared with UV mutation breeding technology.By investigating the characteristics of the starting strain and mutant strain of glutathione,and the antioxidant and stability of glutathione production by high mutant strain,the mutation breeding potential of pulsed high light technology was clarified.At the same time,this study also preliminarily discussed the mutation mechanism of intense pulsed light,and made it clear that pulsed strong light,as a new environmental friendly mutation breeding method,can promote the yield of glutathione in S.cerevisiae.The main experimental contents and conclusions include:(1)Simplify the determination method of glutathione,so that the correlation coefficients of the three methods are above 0.99,among which DTNB has the highest sensitivity;Combined with ultrasonic hot water extraction of GSH,the extraction rate was the highest.Therefore,DTNB method and ultrasonic hot water extraction were selected for the test.(2)The best mutation conditions of pulsed strong light mutation were selected by response interview.Under these conditions,the mutant with high glutathione yield and good stability was selected and compared with UV mutation method.Taking the glutathione yield of the mutant as the main index,the experiment was analyzed with the help of single factor and response surface method.It was concluded that the best conditions for pulse mutation breeding were 1420 v,17 times and 7.1cm respectively.Finally,after screening,the high-yield mutant L5 with stable genetic performance was successfully obtained,and its yield was 1.45 times that of the original strain,while the yield of UV mutant U18 was only 1.36 times that of the original strain,and the heredity was unstable.(3)Optimize the fermentation culture conditions of mutant strains.After significant factor optimization,the glucose mass fraction was 3.325%,yeast immersion powder 0.970% and ammonium sulfate 0.739%,increasing the actual yield by 41.1%.(4)Compared with the original strain of Saccharomyces cerevisiae,the growth and reproduction,environmental tolerance and fermentation ability of Saccharomyces cerevisiae mutant were determined.The results showed that compared with the original strain,the growth logarithmic period of S.cerevisiae mutant L5 was prolonged;In terms of tolerance,the temperature and ethanol tolerance of the mutant strain were better than that of the original strain,and could be better adapted to the high sugar environment of 500 g / L and the high salt environment of 100 g / L;In terms of acidity tolerance,its growth was slightly lower than that of the original strain of S.cerevisiae;According to the relevant indexes of fermentation capacity,the fermentation capacity,alcohol content,reducing sugar consumption and total acid content of the mutant strain were always better than those of the starting strain.Therefore,pulsed strong light did not adversely affect the fermentation performance of S.cerevisiae.(5)Antioxidation and stability of glutathione: within the range of p H and temperature,glutathione produced by S.cerevisiae mutant L5 and starting strain has certain scavenging ability in ferrous ion chelation,DPPH,hydroxyl radical and superoxide anion;Low temperature and low p H environment are suitable for the preservation of glutathione,and short-term addition of sodium sulfite is more conducive to the preservation of glutathione.It shows that pulse mutation can not have adverse effects on the antioxidant and stability of glutathione produced by the mutant.(6)Preliminary study on the principle of mutagenic breeding.RAPD-PCR showed that the band structure of mutant L5 changed and formed differences after primer amplification.This may be due to the production of pyrimidine dimer,which inhibits base pairing,resulting in the destruction of L5 genomic DNA of the mutant.SDS-PAGE electrophoresis also confirmed that the bacterial protein band structure of mutant L5 was 75 k Da and 100 kda more than that of the original strain,indicating that the treatment method of intense pulsed light mutation affected the normal expression of genetic material in the strain.The above conclusions show that compared with ultraviolet light,intense pulsed light technology is simple and safe to operate.It can be used as a mutation method to mutate and select S.cerevisiae high-yield glutathione strain.At the same time,its performance and effect in strain environmental tolerance and fermentation are also better than the original strain. |
