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Research Journal of Microbiology
Year: 2008  |  Volume: 3  |  Issue: 1  |  Page No.: 1 - 8

Optimization of Brucella abortus Fermenter Cultural Conditions and LPS Extraction Method for Antigen Production

Reza Shapouri, Ashraf Mohabati Mobarez, Hojat Ahmadi, Bahman Tabaraie, Reza Hosseini Doust, Dariush Norozian, Ahmad Zavaran Hosseini and Davar Siadat    

Abstract: We modified Brucella fermentation medium from FAO/WHO for enhancing of Brucella abortus S99 biomass. The modified media composed of 15 g L-1 peptic digest animal tissue, 15 g L-1 pancreatic digest of casein, 10 g L-1 yeast extract and 0.10 g L-1 sodium bisulphate. Glucose was added during the incubation by fed-batch method (1-30 g L-1). Agitation speed and air flow rates were controlled at 300-500 rpm and 4-8 L min-1, respectively. Cell density was 9-10% and viable count was 3-3.3x1011 mL-1. The modified conditions enhance the biomass production to more 2 times than the FAO/WHO method. Three methods were accomplished for LPS production: Extraction by butanol with enzymatic digestion, hot phenol extraction with enzymatic digestion, modified hot phenol with trichloroacetic acid (TCA) procedure. Yield of LPS extraction was 0.2, 0.8 and 1.3%, respectively. Method III results in a greater yield of LPS which is 6 and 1.5 times the yields of methods I and II, respectively. Protein contamination of LPS was <2, <2 and <2.9% and nucleic acid contamination of LPS was <1, <1 and <1.4%, respectively. The ketodeoxyoctonate content of LPS (in each of the three methods) was in agreement with ketodeoxyoctonate values obtained previously for highly purified LPS of B. abortus. According to present study, hot phenol with trichloroacetic acid (TCA) procedure is the most suitable procedure for large-scale LPS production from Brucellae, which can be employed for the production of Brucella biomass for vaccine and antigen preparations.

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