Studies On Culturing A Fastidious Microbe Christensenella Minuta

The Christensenellaceae?short for C.?is an intestinal bacteria family and fastidious in culturing.Studies have shown that C.family is the important participant in host metabolism.Individuals with high abundance of C.family tend to have relationship to slimmer stature.Therefore,the C family shows great potential for the treatment of obesity.C.family have not yet been cultured in high density,which seriously limits the research and application of C family.The representative strain Christensenella minuta?C.minuta?in the C.family was selected as the research material,and a SYBR Green I fluorescence quantitative PCR method was established for rapid and sensitive detection of C.minuta concentration in samples.Then,through bioinformatics analysis,the limiting factors of culture of C.minuta were discussed and the culture conditions of C.minuta were optimized by response surface methodology.Finally,the interaction between C.minuta and other probiotics in vivo was simulated simply.The results of the above research are as follows:?1?Using the genomic information of C.minuta,several pairs of specific primers were designed and validated.By constructing recombinant plasmids and using fluorescence quantitative PCR technology,the specific quantification of C.minuta in samples was realized.A SYBR Green I fluorescence quantitative PCR method for rapid detection of C.minuta abundance was established.?2?The medium suitable for C.minuta growth was predicted by Komodo,the metabolic pathway of C.minuta was analyzed by KEGG.The results showed that the modified GAM medium was C.minuta optimal medium,based on C.minuta metabolic defect,adding L-serine,L-glycine,L-cystine,L-cysteine,5-uridine monophosphate disodium salt,cytidine-5-disodium phosphate,Pyridoxine hydrochloride and cobalamin are used to optimize the composition of the medium.?3?The single factor conditions affecting C.minuta culture were investigated by temperature,pH,NaCl concentration,commensal species and prebiotic species.The results showed that the optimum culture temperature of C.minuta was 35°C,the optimum pH was7?8,the optimum NaCl concentration was 40 g/L,and the Lactobacillus bulgaricus had the most multiplication of C.minuta,reaching 3.08;inulin has the most significant effect on the growth of C.minuta.?4?PB?Plackett-Burman?experimental results show that L-cysteine,VB6 and NaCl are the three factors that have the greatest influence on the enrichment effect of C.minuta.The contour and 3D graphs of the analysis of the three factors interacting with each other show that Multiples can take the maximum value within the range of model values.When L-cysteine=65.466 mg/L,VB6=23.997 mg/L,and NaCl=42.537 g/L,Multiples_max=3.667.After testing,under the above conditions,the actual Multiples=3.601,the P-value of the response surface experimental model is less than 0.0001,indicating that the experiment is successful.