Research On Mutagenesis And Selective Breeding Of The Probiotics Strain FGM And The Characteristics Of Mutant Strain For Fermenting Astragalus Membranaceus

Astragalus polysaccharides has attracted much interest because of its multiple important pharmacological actions. Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. In order to further enhance the fermentation performance of the strain FGM and reveal the potential mechanism of astragalus polysaccharide fermentation, FGM was subjected to mutagenesis using ultraviolet(UV) irradiation, N-methyl-N-nitro-N-nitrosoguanidine(NTG) and a combined treatment of UV and NTG. The polysaccharides in fermented product were obtained by water extraction and alcohol precipitation and the content of polysaccharides was determined with the method of phenol-sulfuric acid. Amonst all, two strains with high polysaccharide yield were screened, further studies were carried out on the aspects of stability, biochemical performance, security, fermentation related genes and their expression levels,genome sequencing and comparative genomics analysis respectively, the detailed research results are as follows:(1) With the effort of UV, NTG and UV+NTG mutagenesis on the mutagenic breeding of FGM, the study found that the UV irradiation 10~120 s would increase the polysaccharide content in the fermentation products trains,while there were no regular change under NTG treated alone.U90 was isolated from the UV treatment(90 s), the maximum yield of FAPS in case of U90 was 235.51 mg/g, increased by 58.13 % as compared to that of strain FGM; UN10-1 was isolated, which was under the combination treatment with UV(10 s) and NTG(1.0 g/L, 10min). When UN10-1 was used for astragalus membranaceus fermentation, the production of FAPS was 293.39 mg/g, 94.99 % higher than that of strain FGM.(2) U90 and UN10-1 were cultured for 10 consecutive generations and each generation was used for astragalus membranaceus fermentation. The polysaccharide content of 10 generations in fermentation was determined to evaluate the strains’ genetic stability. It has been found that the 1~ 6 generations of U90, fermentation polysaccharide content basically stable at 233.6 ± 1.66 mg/g,of which the 4th generation is the highest of 235.85 ± 0.78 mg/g. The polysaccharide yield of in different generations of strain UN10-1 was relatively stable at 292.06 ± 0.89 mg/g which may owing to its ability of using three kinds of carbon source: ribose, lactose and arabinose.(3) UN10-1 and its 48 h fermentation broth with Astragalus membranaceus were fed to mice respectively, each 0.5 m L, continuous 3 d, 7 d observation, giving the equal amount of saline at the same time as a negative control. After 7 d observation and recording the daily behavior and body weight in mice, of whole blood was collected and determined the physiological indexes, observing the appearance shape and pathological changes of main organs(heart, liver, spleen, lung, kidney, thymus and small intestine), hematoxylin- eosin(H.E.) staining microscopy observation. The results showed that there were no side effects of strain UN10-1.(4).Researches were performed on the fermentation related genes(galE,dexB,aga2,ldh and recA) of UN10-1, and successfully amplified aga2 and rec A genes with expectations, and analysing expression levels of aga2 gene in astragalus membranaceus fermentation. The results showed that aga2 gene expression levels increased gradually within 24 h of fermentation, and achieved the highest at 24 h, which was 9.54 times to that of the initial fermentation,then falling down during 24 h~60 h and 60 h~72 h a slight increasing.(5).The results of whole genome sequencing research for FGM and UN10-1 and comparative genomics indicating that there are 125 and 124 genes respectively in sugar metabolism pathway under the KEGG database. SNP showed that there were 26 nonsynonymous mutations in genes and a nonsense mutation.To sum up, UN10-1 was got under UV and NTG by probiotic FGM, which has a better performance of extracting polysaccharide from Astragalus membranaceus fermentation, with non-toxic side effects on mice; UN10-1 compaired with the original strain FGM, has a higher expression level on sugar metabolic enzyme gene aga2 in the process of fermentation; Comparative genomics analysis results showed that there were gene mutations on UN10-1.