Study On Cultivation And Dynamic Changes Of The Anti-oxidizing Ingredients In Rhodobacter Sphaeroides

Objective:Biological Liuwei is obtained by fermentate classical Traditional Chinese Medicine( TCM) Liuwei Dihuangtang with Rhodobacter sphaeroides. During the fermentation process, some substances in bacteria will change as well as some basic substances in the medicine which makes the Biological Liuwei shows stronger antioxidant capacity than original parties. Our research group has done a lot of studies about the changes in the basic material and pharmacological experiments of the medicing after metabolism, but lack of bacterial related research. In order to make the synergistic mechanism of Biological Liuwei on antioxidant effect clearer, we’ll analysis of the changes of antioxidants in biomass during growth and metabolism process by the use of modern analytical methods. This could provide a basis for further research of Biological Liuwei.Methods:①Activation and passaged of Rhodobacter sphaeroides: according to common bacteria activation and passage technology, build culture method for this study. ②Study of species preservation methods: combination of typical species preservation methods, established a method suitable for this study. ③Establish analysis method for antioxidant in the bacteria: Investigated methodology combined with modern chemical and instrumental technology such as UV visible spectrophotometry, HPLC, etc. ④Research of extraction technology: Optimized the extraction method of antioxidant in bacteria through response surface method and orthogonal test. ⑤Research on the changes of antioxidant: analysis of the changes of antioxidant in bacteria during the growth and metabolism process basis on the analysis method and extraction method established before. ⑥Determinate the content of antioxidant ingredients in Biological Liuwei tablet by means of UV or HPLC.Results:①Established a proper culture methods of Rhodobacter sphaeroides, laid the foundation for further study. ②Preservation method of bacteria is by using of 15% glycerol as a protective agent deposited in the refrigerator with temperature of-20 ℃ or-40 ℃. Also can use of 10% skim milk as a protective agent for freeze-dried preservation. ③Established analysis method for antioxidant by use of UV-visible spectrophotometry to determinate lycopene and HPLC to determinate Co Q10. Where the HPLC conditions: Column: DIKMA Platisil ODS C18 column(250 mm × 4.6 mm, 5 μm), mobile phase of methanol to ethanol was 20 to 80, column temperature of 35 ℃, the injection volume of 10 μL, a flow rate of 1 m L / min. ④Optimum extraction process of lycopene in Rhodobacter sphaeroides are : by using acetone as solvent extraction, saponification time of 28 min, solid-liquid ratio of 1:86(g / m L), extraction time of 11 min, extracted twice. Optimum extraction of coenzyme Q10 are: acetone as extraction solvent, 1: 50(g / m L) of the solid-liquid ratio, ultrasonic power of 200 W, ultrasonic time of 15 min, extracted two times. ⑤Studied the changes of lycopene and coenzyme Q10 during the growth and metabolism process of bacteria, the results show that the growth of bacteria in different environments in which the content of lycopene and coenzyme Q10 will be a corresponding change. ⑥ After measurement, the content of lycopene and coenzyme Q10 in the Biological Liuwei Tablets are 194.27±33.73 μg/g、21.21±0.98 μg/g.Conclusion:The method of bacterical activation and expansion combined with required is practical and reliable. Methods for measuring and extracting the lycopene and Co Q10 in Rhodobacter sphaeroides during the process of bacterical growth and metabolism are simple, accurate and reliable. Research on the changes of lycopene and Co Q10 in the bacteria under different cultural conditions provides basis for further study of Biological Liuwei and provides an important reference for the bacterical fermentation of lycopene and Co Q10 by use of Rhodobacter sphaeroides. The measurement results of lycopene and Co Q10 in Biological Liuwei shows that the increase in antioxidant activity of Biological Liuwei maybe related to the reserved of antioxidants in the bacteria.