Studies On Extraction Process Of Polysaccharides From Chlorella Pyrenoidosa And Its Anti-aging Activity

Chlorella Pyrenoidosa is a unicellular green alga,spreading widely both in fresh and marine water.With the value of Chlorella Pyrenoidosa came to be known,Chlorella Pyrenoidosa was produced as single-cell protein by Japan,America,Former Soviet Union and so on.In the 80s,Japan used the Chlorella Pyrenoidosa to develop functional foods and beauty products.In 2012,Chlorella Pyrenoidosa has been named as new resource food in China.Recent researches exhibited that the polysaccharides from Chlorella Pyrenoidosa possess great bioactivities,including immunomodulatory,anticancer,antitumor,antivirus,antibateria and antioxidant.But in the study of Chlorella Pyrenoidosa,our country was still in the stage of development,and most of products were raw algae powder,which lack of technology,especially lack of deep-processing.Therefore,Chlorella Pyrenoidosa was studied in the paper.Based on the optimization of cell disruption conditions and polysaccharides extraction conditions,the antioxidant polysaccharides from Chlorella Pyrenoidosa were selected in vitro.Then the anti-aging activity of polysaccharides from Chlorella Pyrenoidosa was evaluated by the Drosophila melanogaster model.The paper aimed to provide some theoretical basises for further development of Chlorella Pyrenoidosa.Firstly,the cell disruption conditions of Chlorella Pyrenoidosa were optimized.Three methods,including freezing-thawing,enzyme and ultrasonic,were used and optimized by orthogonal test.The results indicated that the optimum cell disruption conditions of freezing-thawing were as follows:freezing-thawing times 4,freezing time 1h and cell density 2×10^7.Under the optimum conditions,the cell disruption degree was 61.21±1.4%.The optimum cell disruption conditions of enzyme were as follows:enzymatic pH 4.0,enzymatic amount 2%,and enzymatic time 4 h.Under the optimum conditions,the cell disruption degree was 80.15±2.1%.The optimum cell disruption conditions of ultrasonic were as follows:ultrasonic time 24 min,ultrasonic temperature 60?,ultrasonic power 300 W.Under the optimum conditions,the cell disruption degree was 64.1 8±1.5%.It indicated that among three methods,enzyme method has the highest cell disruption degree,ultrasonic followed,but the time that ultrasonic cell disruption used was the least.Based on the optimization of cell disruption method,the method of enzyme was selected.Extraction time,extraction temperature,solid to liquid ratio and enzymatic amount were investigated.The response surface methodology(RSM)was used to optimize the polysaccharides extraction conditions of Chlorella Pyrenoidosa.The results exhibited that the optimum extraction conditions were as follows:extraction time 2 h,extraction temperature 93?,the ratio of solid to liquid 1:30 and enzymatic amount 2%.Under the optimum conditions the polysaccharide yield was 5.87±0.24%.The polysaccharides of Chlorella Pyrenoidosa were preliminarily separated by the alcoholic precipitation using fractionation and step-by-step methods.Six components of CPPA60,CPPA70,CPPA85,CPFA40,CPFA60 and CPFA85 were obtained.Another six components of CPPB60,CPPB70,CPPB85,CPFB40,CPFB60 and CPFB85 were obtained after removing of free protein and small molecules.The antioxidant activities of 12 polysaccharides from Chlorella Pyrenoidosa were evaluated by the scavenging activities on hydroxyl,DPPH and superoxide radicals.The main component analysis method was used to select the strongest antioxidant polysaccharide.The results showed that CPPA70 had the strongest antioxidant activity,followed by CPFB40.CPFB40 was selected for a further anti-aging study,using MRC-5 cell model induced aging by H2O2.It exhibited that the rate of senescent cells of CPFB40 group was dropped significantly when compared with negative control(P<0.05),but there was no significant difference among three dose of CPFB40(P>0.05)Lastly,the anti-aging activity of CPFB40 was investigated by the Drosophila melanogaster model.The results showed that the maximum life-span,least life-span and average life-span of Drosophila melanogaster have been extended in CPFB40 groups.The average life-span of male Drosophila melanogaster was extended up to 8.43%,11.47%and 8.71%,respectively in three dose of CPFB40.And female Drosophila melanogaster was extended up to 4.03%,5.71%and 10.56%,respectively.The anti-aging activity relates to the gender of Drosophila melanogaster.In the experiments of the activity of many antioxidase in vivo of Drosophila melanogaster,the activity of SOD,GSH and CAT were improved in CPFB40 groups.