The Study Of Stress Effects About Seawater And Ultrasound On Chlorella Vulgaris And Its Lutein

Chlorella vulgaris is a kind of spherical single-cell green algae distributed in fresh water,which is rich in carotenoids.Lutein is one of the most important carotenoids in Chlorella vulgaris,possessing the characteristic of antioxidant,anti-cancer,prevention of atherosclerosis and diabetic retinopathy.Studies have shown that high salt stress in seawater can promote microalgae to synthesize more carotenoids,but inhibit the growth of microalgae in seawater,so that microalgae can not be cultured in seawater.Ultrasound is a green technology,which has resonance effect when interacting with liquid and can produce cavitation.The introduction of appropriate ultrasound treatment can stimulate the growth of microalgae.Therefore,in this paper,the stress culture of Chlorella vulgaris was carried out by seawater and ultrasound to study its growth condition and the accumulation of lutein.What’s more,the lutein in Chlorella vulgaris was further analyzed and purified by ultra-high performance liquid chromatography and high-speed countercurrent chromatography.The main research contents and results are as follows:1.The effects of seawater and ultrasonic stress on the growth of Chlorella vulgaris were studied.Chlorella vulgaris was respectively cultured in fresh water,seawater,seawater and ultrasound for 10 days,while the growth of Chlorella vulgaris was observed by measuring the changes of cell density,OD_680nm,biomass concentration,chlorophyll a,chlorophyll b and carotenoid content.The results showed that the growth of Chlorella vulgaris was inhibited due to high salt stress,presented as the cell density,OD_680nm,biomass concentration and chlorophyll a content decreased.In the process of seawater culture,the introduction of ultrasonic treatment alleviated the inhibition of growth and increased the density of algal cells by 39.5%.In addition,under the stress of seawater and ultrasound,the contents of chlorophyll b and carotenoid of Chlorella vulgaris reached to 1.83 mg/L and 1.19 mg/L on the 10th day,which were more than50%higher than that of freshwater group.The results further demonstrated that microalgae would protect the growth of cells under stress by synthesizing more specific pigments as a defense mechanism.2.The changes of lutein content in Chlorella vulgaris cultured under seawater and ultrasonic stress were systematically studied.Firstly,an ultra-high performance liquid chromatography method for rapid qualitative and quantitative analysis of lutein in Chlorella vulgaris was established to observe the changes of lutein content in microalgae under different culture methods.The results showed that the content of lutein in Chlorella vulgaris that cultured in seawater（12.24 mg/g）was significantly higher than that in fresh water group（9.27 mg/g）.After adding ultrasound in seawater,the lutein content of Chlorella vulgaris reached to the highest value（13.93 mg/g）,which was 50.3%and 13.8%higher than that of fresh water group and seawater group,respectively.The significant change of lutein content suggested that Chlorella vulgaris mainly protected cells by synthesizing more lutein when cultured under salt stress.It was the lutein that played the most important role in the pigment defense mechanism.Finally,the Chlorella vulgaris cultured under seawater and ultrasonic stress was compared with commercial Chlorella vulgaris from three aspects.The results found that there were significant differences in the contents of lutein,chlorophyll and pheophytin,which further proved the importance of pigment in the growth process of Chlorella vulgaris as well as the potential and advantages of seawater and ultrasonic culture of microalgae.3.A high efficient separation and preparation method of lutein from Chlorella vulgaris by countercurrent chromatography was established.After crude extraction of lutein from Chlorella vulgaris cultured by seawater and ultrasonic with absolute ethanol,high-purity lutein was separated from the crude extract by high-speed countercurrent chromatography.The two-phase solvent system with low toxicity was selected:n-heptane,ethanol and water.By measuring the partition coefficient of lutein in the two-phase solvent system,n-heptane:ethanol:water（20:16:6,v:v:v）was finally selected as the two-phase solvent system to separate lutein.Taking the retention rate of stationary phase as the reference index,the more suitable chromatographic parameters were selected:the rotating speed was 800 rpm and the flow rate was 5.0 ml/min.In the experiment,lutein was separated and purified by using the upper phase of the solvent system as the stationary phase and the lower phase as the mobile phase with the detection wavelength of 405 nm.Under the above conditions,28.3 mg lutein with purity of 92.6%was successfully isolated from 300 mg Chlorella vulgaris crude extract within60 min.In addition,in the process of separating lutein by countercurrent chromatography,three other fractions were obtained.The compounds of two fractions mainly contained chlorophyll and pheophytin,which were determined by ultra-high performance liquid chromatography and ultraviolet visible full wavelength scanning.The experimental results demonstrated that the high-speed countercurrent chromatography method for the separation and preparation of lutein in Chlorella vulgaris is an efficient,economic and low toxicity preparation chromatography method,providing research ideas for the extraction and purification of high-purity carotenoids from algaes.