Optimization Of Culture Conditions And Improved Detection Methods Of Nocardia Rubra Cell Wall Skeleton

At present, tumour and many disease already becomes significant healthy threat of human. Some researches show that the extract of the Nocardia rubra Cell Wall Skeleton such as Polysaccharide etc. are effective in treating tumour and female-pathology. Bacteria cell wall skeleton preparation is capable of regulating immune function, can enhance the T cells, macrophages, NK cell activity, while a letter for the human lymphocyte mitogen activity, and can induce interferon. Through years of experimental and clinical studies have shown that it has anti-tumor effect and resistant to bacteria, virus infection. Now about the research of the N-CWS are almost focus on the inhibiting effect in tumor growth or some cure of the inflammation, but the research of the optimization of culture condition are still stay in a low lever.So this project through a series of experiment have worked out that the best culture medium for the Nocardia rubra is the glucose/yeast extract, and the best pH value is between 7-8, shake cultivation with 26℃and 320 r/min are most suitable, and when the liquid nutrient medium are 50 mL, the most suitable vaccination quantity is 1.5%(0.75 mL).Studied the ultrasonic method, self-dissolving method and the alkaline conditions of Nocardia broken. The results showed that the three kinds of cell wall broken law and alkaline ultrasonic methods period is short, good results; while autolysis France a long cycle is inefficient, ineffective. Broken whole time and output power is broken ultrasonic main factors, using short-term approach and broken many times the concentration of 1.5 g/L of cell suspension in favor of the broken cells. In the Nocardia broken in the self-dissolving method, since the dissolving time is the main factors, NaCl mass fraction of 1%, the temperature is 50℃conducive to cell autolysis, a long-time processing time,10% of NaCl and fluid pH9 environment conducive to self-dissolve. Lye (KOH solution) a concentration of 5.5% is most beneficial actinomycetes cells, broken, broken time is 30 minutes. At the same time the use of orthogonal pairs of ultrasonic broken the terms of research, obtained under certain bacteria concentration for the best set broken ultrasonic ultrasonic time 15s, interval 15s, cycles 40 times, power 500W.Traditional Nocardia cell wall skeleton (N-CWS) finished detect certain deficiencies exist in this experiment through the following three improvements to enhance the level of detection. Thin-layer chromatography instead of paper chromatography and some of the factors affecting the chromatography, finding the best chromatographic separation, so that diaminopimelic acid clearer identification experiments; For arabinose and glucose between the regularity of the standard curve, using glucose instead of arabinose, production of the standard curve for determination of cell wall sugar; Finished with the spectrophotometer optical absorption peak in methanol instead of whether the absorption peak of color recognition method to enable more reliable detection of methanol residues.