Extraction And Purification Of Intracellular Polysaccharide From Chlorella Pacifica And Its Effect On The Immune Activity Of Charybdis Japonica

This thesis mainly studies the separation and extraction of polysaccharides from Chlorella pacifica.The method of ultrasonic-assisted hot water extraction is adopted,and the extraction conditions are optimized.Chlorella pacifica polysaccharides were separated and purified by chromatographic column method,and anti-oxidation tests were carried out to study the effects of pure polysaccharides of Chlorella pacifica seaweed on the activity of partial immune enzymes of Japanese chinensis.The experimental results are as follows:(1)After the Chlorella pacifica is activated and expanded and cultured,the Chlorella pacifica in the stable phase is collected,and the collected Chlorella pacifica mud is vacuum freeze-dried to obtain Chlorella pacifica powder.0.1% activated carbon was used for decolorization,and 5% trichloroacetic acid(TCA)was used for protein removal.(2)Taking the polysaccharide extraction rate of Chlorella pacifica as the main index,single-factor experiments were carried out on the ultrasonic breaking time,extraction temperature and extraction time of Chlorella pacifica.It was found that the extraction rate of polysaccharides was the highest when the ultrasonic disruption time was 40 min,reaching 3.52%,indicating that ultrasonic waves were beneficial to the dissolution of polysaccharides from Chlorella pacifica,and that the ultrasonic disruption time was too long to destroy the internal structure of Chlorella pacifica polysaccharides,resulting in a decrease in the extraction rate of polysaccharides.When the temperature reached 90°C,the highest polysaccharide extraction rate was 4.23%,and then there was a downward trend.The possible reason was that the polysaccharide chain was broken in an excessively high temperature environment,the polysaccharide was denatured,and the extraction rate decreased slightly;the extraction time was2 h,the extraction rate was up to 4.1%,the extraction time was too short so that the Chlorella pacifica polysaccharide was not completely dissolved,and the extraction time was too long,the Chlorella pacifica polysaccharide might be degraded,and the extraction rate of polysaccharide was not high.(3)After the response surface optimization analysis,the degree of influence on the extraction of Chlorella pacifica polysaccharides from is as follows: extraction temperature greater than ultrasonication time greater than extraction time.Through actual adjustment,the optimal extraction conditions for the extraction of Chlorella pacifica polysaccharide by ultrasonic-assisted hot-water leaching method are as follows: ultrasonic breaking time 41 min,extraction temperature 85 ℃,leaching time 2 h,and the final pellets are obtained.The extraction rate of algal polysaccharide was 5.40%,which was close to the predicted extraction rate of5.46%,indicating that the response surface methodology was reliable to optimize the extraction conditions of Chlorella pacifica algal polysaccharide.(4)The crude polysaccharide of Chlorella pacifica was separated and initialized by DEAE-52 cellulose chromatography column,and finally the acidic polysaccharide component CPP-2 was eluted.Chlorella pacifica polysaccharide CPP-2 was scanned by ultraviolet spectrum at wavelengths of 200-500 nm,and it was found that the polysaccharide did not contain nucleic acid and protein components,and the purity was high.(5)When the concentration was 1 mg/m L,the ability of Chlorella pacifica sea polysaccharide CPP-2 to scavenge DPPH free radicals was 87.32%,accounting for 89.42% of the VC scavenging rate at the same concentration,which fully demonstrated that it had a significant effect on DPPH free radicals.Very good scavenging effect;CPP-2 has a stable scavenging rate of 60.57% for hydroxyl radicals,accounting for 73.19% of the VC scavenging rate at the same concentration,and the scavenging effect of hydroxyl radicals is better;when the concentration is the same as 1 mg/m L,The scavenging rate of CPP-2 to superoxide anion radicals is only 40.98% of that of VC,and the reducing power of the scavenging effect is about34.17% of that of VC.ionic capacity and reducing power.(6)For the test group,the superoxide dismutase activity in the serum of Charybdis japonica reached the highest 24 hours after injection,which was 21.34% higher than that of the control group.Significant difference;catalase activity reached the maximum at 72 h after injection,which was 40.19% higher than that of the control group,and there were significant differences between the experimental group and the control group at 24,48,and 72 h;48 h after injection The enzyme activity of lysozyme reached the highest,which was 77.47% higher than that of the control group,and there were significant differences between the experimental group and the control group at 5,24,and 48 h;the enzyme activity of acid phosphatase reached the highest value at 48 h after injection,It was 2.27 times that of the control group,and there were significant differences between the test group and the control group at 5,24,and 48 hours;the enzyme activity of alkaline phosphatase reached the highest value at 48 hours after injection,and the enzyme activity was higher than that of the control group.34.09%,there were significant differences between the experimental group and the control group at 24 and 48 h.It indicated that the polysaccharide CPP-2 of Chlorella pacifica seaweed had a strong effect on inducing immune response to Charybdis japonica.