利用沼泽红假单胞菌生产类胡萝卜素的研究
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摘要
微生物发酵法生产类胡萝卜素与体积大、质量重、生长周期长的水果、蔬菜、植物相比具有很多优点,如能够节约生产成本、提高生产效率等,但是其关键环节是找到最佳培养基和培养条件,以及有效的提取工艺。本文研究了沼泽红假单胞菌产类胡萝卜素的最佳培养基和培养条件;并进一步研究了从沼泽红假单胞菌中提取类胡萝卜素的工艺条件,对细胞收集、前处理、色素初浸提、二次萃取,皂化进行了系统的研究;最后利用紫外-可见光谱、薄层层析、高效液相色谱和质谱对类胡萝卜素提取液进行了成分分析。结果表明,沼泽红假单胞菌在组成为KH2PO4 0.6 g,K2HPO4 0.9 g,酵母膏1.5 g,蛋白胨2.0 g,NaCl 0.5 g,自来水1000 mL的培养基中,在接种量为3.0 %左右,温度为28℃,光照强度为1500 lx, pH值为6.5~8.5条件下培养5天后,类胡萝卜素产量可达4.84 mg/L菌液。提取类胡萝卜素的最佳工艺条件为:碱性氯化钙沉淀收集细菌细胞;乙醇浸泡湿菌泥30 min;49℃下丙酮初浸提4 h (搅拌并加抗氧化剂),丙酮用量为5 mL/g菌泥,回收丙酮;室温下乙醚二次萃取;分离得到的乙醚萃取液在30℃下用15 %的KOH甲醇溶液皂化1.5 h(搅拌并加抗坏血酸钠);洗涤,浓缩纯化。在此工艺条件下从沼泽红假单胞菌中提取类胡萝卜素,含量可高达1.872 mg/g(以湿重计)。在展开体系为正己烷/乙酸乙酯/丙酮/甲醇:60:10:5:5薄层层析中可以清楚看到有5条色带已经被完全分离开。HPLC分析的最佳洗脱条件是:15 min, 75% B; 20 min, 0 % B,其中A为甲醇/水(9:1),B为乙酸乙酯。在此洗脱条件下,类胡萝卜素提取液在15 min内洗脱完毕,有24个峰出现,其中6个峰面积较大,占所有峰面积的80%左右。在相同洗脱条件下,叶黄素、β-胡萝卜素、虾青素、番茄红素四种标准物质的出峰时间分别是:5.692 min,15.015 min,5.176 min,15.108 min;虾青素和番茄红两种标准物质在类胡萝卜素提取液被发现,含量分别为0.15502 g/ L和1.0019 g/ L。利用高效液相色谱-质谱联合分析出了类胡萝卜素提取液的可能成分(约21个峰)。
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.
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.
引文
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