Research On Carotenoids Production By Rhodopseudomonas Palustris

Microorganism fermentation to yield carotenoids is much better than that uses fruits and vegetables of big volume and heavy weight as main materials, because of its fast growth speed, little nutrition demand and easy cultivation conditions, but the key factor is to find favorable culture medium, culture conditions and extraction process. In this paper, Rhodopseudomonas palustris was selected and identified out. First, it's favorable culture medium and culture conditions were investigated. And further, carotenoids extraction process was thoroughly researched. The modern technism of Uv-vis, TLC, HPLC and MS were used to analyze and determinate the components of carotenoids extraction solution in the end. As results, the optimum culture medium was as follows: potassium dihydrogen phosphate 0.6 g, dipotassium hydrogen phosphate 0.9 g, yeast-extract 1.5 g, peptone 2.0 g, sodium chloride 0.5 g, tap water1000 ml. The optimum cultivation conditions were initial inoculation 3.0 %, temperature 28℃, illumination intensity 1500 lx, pH value of 6.5 to 8.0. When Rhodopseudomonas palustris was cultivated in the conditions for 5 days with the medium, the total carotenoids was high up to 4.84 mg/l. The optimum extraction process was cell collection with alkaline calcium chloride, pretreatment with ethanol soak for 30 minutes, first extraction with acetone at 49℃for 4 h (5 ml acetone to 1 g bacteria mud, agitation and addition of antioxidant), callback acetone, second extraction with aether at room temperature not exceeding 30℃, saponification with 15 % potassium hydroxide methanol solution at 30℃for 1.5 h (agitation and addition of sodium ascorbate as antioxidant), washing with distilled water or sodium chloride solution, callback aether and condensation in the end. According to this process, the total carotenoids content was high up to 1.872 mg/g(wet weight), when extracted after five days'cultivation in the optimum medium and optimum conditions. Five different ribbons were seen to be separated in the TLC experiment when the solvent system of hexane/ethyl-acetate/acetone/methanol (60:10:5:5) was used. The best elution condition for HPLC was 15 min, 75% B; 20 min, 0 % B. Here A was methanol/water(9:1), B was ethyl acetate. Under this elution condition, the carotenoids extraction solution was completely eluted out in 15 minutes, and 24 peaks were separated,among which six peaks had about 80% areas in all. The retention time of standard matters were respectively astantin 5.176 min, xanthophyll 5.692 min,β-carotene 15.015 min, lycopene 15.108 min. Only astanxin and lycopene were found in the carotenoids extraction solution, respectively 0.15502 ug/ul and 1.0019 ug/ul. The possible components (21 peaks) were analyzed out by combinating MS and HPLC.