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Studies on the Extraction, degradation and Bioactive of Hyaluronic Acid from Squid Eyes

Author: LuJiaFang
Tutor: YangWenGe
School: Ningbo University
Course: Of Food Science
Keywords: squid eyes hyaluronic acid purification degradation moisture retention antioxidation
CLC: TS254.1
Type: Master's thesis
Year: 2010
Downloads: 152
Quote: 1
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Abstract


Hyaluronic acid （Hyaluronic Acid, HA） extensively distributes in the connective tissue of animals and humans. As an absorbable and degradable biomaterials, hyaluronic acid has been widely used in clinical medicine and advanced cosmetic production, showed a huge market potential and broad application prospects.Squid eyes containing abundant of HA are large quantities of processing waste in coastal areas, and can be used as resources for preparing hyaluronic acid. Using squid eyes as raw material, the hyaluronic acid was extracted with enzyme, purified by ultrafiltration and gel chromatography, then degraded in physical or chemical methods. Moreover, the moisture and antioxidant activity in vitro of hyaluronic acid with different molecular weight were evaluated. The purpose of this paper is to provide the theoretical basis for improving the added value of squid waste and developing the function of hyaluronic acid .The research contents of this paper include the following aspects:1.Extraction, purification and identification of hyaluronic acid from squid eyes.Using the extraction ratio of HA as index, three methods including hot water, enzyme and ultrasonic extraction were compared. Duncan analysis revealed the extraction ratio of HA in different methods had significant differences. Therefore, subtilisin was choosed to extracte the hyaluronic acid from squid eyes, and the process conditions were optimized by single factor and orthogonal tests. The optimal technology as follows: zymolysis time 1h, zymolysis temperature 60℃and the ratio of enzyme 4%. Under this optimal conditions, the yield of HA was up to 14.10%.Crude hyaluronic acid was decolorized by macroporous resins. The decoloration rate and loss rate of HA were 81% and 18%, respectively by using D101 macroreticular resin absorption. HA was further purified by DEAE-Sephadex A-25 ion exchange chromatography, two kinds of hyaluronic acid marked HA-1 and HA-2 were separated and their yields were 5.22% and 84.37%, respectively, two component-class shared of the total rate of 89.60%.Both of HA-1 and HA-2 had no protein and nucleic acid left and their purity were shown to be on a single elution peak by UV spectrum and gel-filtration chromatography. The average molecular weight of HA-1 and HA-2 were 6.77×105 and 1.73×106, respectively. The infraction spectrum of depurated HA was according to that of the HA Standarded.2. The optimal degradation conditions of hyaluronic acid were determined.Using the molecular weight of HA as index, the physical and chemical methods were applied to degrade the hyaluronic acid from squid eyes. The effect of degradation methods and conditions on the molecular weight of HA were studied to determine the optimal degradation conditions. The results indicated that the hyaluronic acid can be degraded effectivly by the reaction system of ascorbate and hydrogen peroxide, and the best condition as follows: the mole ratio of hydrogen peroxide and ascorbic acid was 3:1, pH4.0, reaction time 15min, temperature 50℃. In this optimal condition, the molecular weight of HA could be degraded to 5433.3. The bioactive of hyaluronic acid and its degradation products were evaluated.The moisture retention, reductive capability on Fe3+, scavenging ability on DPPH·and·OH of hyaluronic acid and its degradation products were determined. The results showed that the moisture retention of hyaluronic acid were better than glycerol. Higher the molecular weight of hyaluronic acid, better the moisture retention. The moisture retention of HA-2 is the best and its moisture rate reach to 91.66% at low concentrations. Moreover, degraded products of HA showed definite scavenging activities and obviously dose-depengdent inhibitory effects. In a word, with molecular weight decreasing, degraded HA presented the trend of increasing of scavenging ability on DPPH·and·OH, reductive capability on Fe3+. HA （ Mr <6000） showed the best antioxidative activity, whereas the components of HA-1, HA-2 without degradation had weaker antioxidative activity.

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