| The squid resources are very rich as a kind of common oceanic cephalopods creatures. China is one of the major countries of squid fishing and processing. In the process of squid processing, the by-products such as squid cartilage are often discarded as scraps without fully being utilized which result in a waste of resources. In this paper, the squid cartilages were chosen as research object. The best extraction technology conditions were confirmed by the single factor optimization experiment of alkali extraction and enzyme hydrolysis processes. Chondroitin sulfate was obtained after purification and its hypolipidemic activity was studied in the paper. All these could provide reference for the future comprehensive utilization of squid cartilage. This thesis major research contents are as follows:1. Solid-liquid ratio, NaOH concentration, extraction temperature and extraction time which affect the effect of chondroitin sulfate leaching conditions were optimized by single factor and response surface tests. Through the quadratic regression model, the optimum extraction process conditions were determined as follows:ratio of material to solvent was 1:8(W/V), leaching temperature was 50?, leaching time was 5.3 h, alkali concentration was 3%. Under these conditions, the yield of chondroitin sulfate was 62.89%, in keeping with the predicted value.2. On the basis of relevant references, papain enzyme was chosen for the experiment through the preliminary experiments. The several key factors influencing the papain enzyme hydrolysis was investigated by single factor test, and the further optimization was carried out by orthogonal combination design experiment on the basis of single factor tests. It is concluded that the main factors influencing the squid cartilage enzymolysis effect were, in order, pH, digestion temperature, digestion time. The optimum enzyme hydrolysis conditions were as follows:the adding quantity 2%, pH6.5, enzymolysis temperature 60?, enzymolysis time 5 h. In this condition, the protein content was 4.97%, and the chondroitin sulfate content was 70.11%.3. The purification of chondroitin sulfate was by the means of ethanol precipitation and DEAE-52 column chromatography. The chondroitin sulfate and glucuronic acid contents were determined as indicators to evaluate the efficacy of ethanol precipitation, and then the optimal alcohol precipitation conditions were gained as follows,4%NaCl concentration, pH6.5,70% ethanol concentration,20% solids concentration. Under the condition, chondroitin sulfate content in the purified product was 91.28%, glucuronic acid content was 41.03%. In the method of DEAE-52 column chromatography, the elution peak was tracking tested through sulfuric acid carbazole method. The elution results showed that the separation of components was relatively single and the separation effect was well. Collecting elution peak, the purified chondroitin sulfate was obtained after dialysis, freeze drying, and the results showed that the chondroitin sulfate content in the purified product was 93.16%, glucuronic acid content was 42.73%. The analysis of ultraviolet spectrum and infrared spectrum scanning showed that the structure of purified chondroitin sulfate excluded (or contained trace) nucleic acid and protein, and it also showed that the acetyl amino, free uronic acid and sulfate groups existed in the molecular which was basically identical to the standard chondroitin sulfate.4. Hypercholesterolemia mice model was used to study the hypolipidemic action of chondroitin sulfate from squid cartilage, and the effects of antioxidation on hypolipidemic function were explored by detecting part of oxidation indexs. It was found that chondroitin sulfate can control the weight gain in mice to some extent, and reduce the liver index, atherosclerosis index and coronary heart disease index, and it also can increase the kidney index. Chondroitin sulfate could regulate the mice serum TC, TG, LDL-C level effectively, in the meantime, the serum HDL-C content was elevated, and the volume of liver TC and TG was reduced. At the same time chondroitin sulfate could improve the activity of lipoprotein lipase and hepatic lipase, and enhance the activity of antioxidant enzymes such as the SOD and GSH-Px. The results showed that the chondroitin sulfate from squid cartilage has the function of hypolipidemic, and the fatty liver disease, atherosclerosis, coronary heart disease and other diseases might be prevented or cured after taking it. |
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