Tris 1 CTAB D Elma THZ - 82 Table 1 DFT - 50 UV3 Level 3K30 Sigma ALPHA1-4 Sigma AP ph PE ~ 0. 6 mm HiTrap TM DEAE FF [11

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1 ~ 1452 Journal of Nuclear Agricultural Sciences 本文研究超声波辅助酶解法提取罗非鱼眼玻璃体中透明质酸, 通过单因素实验正交实验分析了超声波功率超声处理时间及蛋白酶种类酶解时间酶解 ph 酶量酶解温度对透明质酸提取的影响, 确定了最佳提取工艺条件 : 超声波功率 200W 超声处理时间 15min 酶作用时间 3 h 酶解温度 40 酶解 ph 值 9. 0 酶用量 6000U g - 1, 利用最优条件透明质酸的平均得率为 % 采用季铵盐络合法离子交换层析法对透明质酸粗品进行纯化得到高纯度的透明质酸, 其纯度达到 %, 利用粘度法测量透明质酸相对分子量为 100 KDa 紫外图谱中无蛋白及核酸的吸收峰, 红外光谱图显示其特征吸收峰和标准品相同本研究为罗非鱼眼的高值化利用提供了技术支撑罗非鱼眼 ; 超声波酶解 ; 透明质酸 ; 工艺条件 DOI /j. issn hyaluronic acid HA 50% HA 1 20 KHZ HA HA HA % 250U mg U mg U mg U mg U mg TS01 CARS chensjun@ hotmail. com laihaoli@ 163. com

2 Tris 1 CTAB D Elma THZ - 82 Table 1 DFT - 50 UV3 Level 3K30 Sigma ALPHA1-4 Sigma AP ph PE ~ 0. 6 mm HiTrap TM DEAE FF [11] 透明质酸含量的测定 Bitter-Muir GE Healthcare Bio-sciences KTA purifier TM 100 g g Bitter-Muir Unicorn 5 GE Healthcare Bio-sciences = / 5000 Da Mym Mym 100% Biological Company Limited [12-14] 透明质酸的相对分子量测定 NaCl 1. 2 [ 透明质酸提取方法的优化 10] g 0. 2 mol L - 1 NaCl 400 ml η r η sp C i η sp /C i ph U g - 1 Y 1 1 η 3 15min 40 4 h 95 10min 6 000r min min 100 ml 100g L - 1 CTAB h 1. 2mol L U g h C D A Temperature / min W 正交试验 X 酶种类对 HA 提取的影响 1 HA A B ph 1 Factors and levels B ph C time /h D Enzyme dosage /U g η r = t i 1 t 0 η sp = η r η = Mr 3 [15] 4h 3 95% 透明质酸的紫外光谱分析 24h 0. 5mg ml ~ 400nm 单因素试验透明质酸的红外光谱分析 [16] 4 ~ 8mg 500 ~ 4 000cm cm

3 HA HA HA HA Fig. 2 2 HA Effect of different protease dosage on the yield of HA Fig. 1 1 HA a b c d e P Effect of protease types on the yield of HA. Alphabets a b c d e mean significant difference at P 酶用量对 HA 得率的影响 2 HA 7 500U g - 1 HA HA 3 HA Fig. 3 Effect of different hydrolysis time on the yield of HA HA HA HA 200 W HA HA HA U g 酶作用时间对 HA 得率的影响 3 HA 5 HA HA 15min HA 15min HA HA HA HA HA 3 ~ 5 h 超声波功率对 HA 得率的影响 4 200W 超声处理时间对 HA 得率的影响 5 HA

4 mol L mol L Table 2 2 Orthogonal test scheme and results Test number A B C D HA Yield of HA /% Fig. 4 4 HA Effect of ultrasonic power on the yield of HA k珋 k珋 k珋 R Primary-secondary factors Optimal combination B > A > C > D A 1 B 2 C 3 D 2 5 Fig. 5 15min HA Effect of Ultrasonic treatment time on the yield of HA 6 210nm 3 a b c a b c 21 HA 20min 19 HA a c b nm HA HA a % % HA 透明质酸的相对分子量 0. 1mol L - 1 NaCl HA g L - 1 3h 40 ph U g % mL 2. 3 g mol L - 1 KDa 22

5 Fig. 6 6 HA Elution profile of HA by Ion exchange chromatography Fig. 7 7 The relationship between solution concentration and reduced viscosity of HA 透明质酸的紫外光谱 nm 260 nm 210 nm cm - 1 HA cm cm - 1 C O C N N-H 透明质酸的红外光谱 9 8 Fig. 8 HA UV spectra of HA cm cm - 1 O C O cm CH 3 3

6 Fig. 9 HA FT-IR spectra of HA W 225 W 10min Km Vm HA HA HA % 20 HA % HA 1. 33% 1. 2% HA HA HA 24 7h 50 3h 45 HA U g U g % HA 4 40 ph U g W 35KHz 15min 3h % % 100kDa 1 Eun J O Kitae P Ki S K Jiseok K Jeong A Yang Ji H K Min Y L Allan S H Sei K H. Target specific and long-acting delivery of protein peptide and nucleotide therapeutics using hyaluronic acid derivatives J. Journal of Controlled Release Grigorij K Ladislav Robert S Peter G. Hyaluronic acid a natural biopolymer with a broad range of biomedical and industrial applications J. Biotechnology Letters Kakehi K Kinoshita M Yasueda S. Hyaluronic acid separation and biological implications J. Journal of Chromatography B Analytical Technologies in the Biomedical and Life Science M J J J J D Guillermo L Loida O Jose A. CremataJose A C Carlos P Gabriel C Efren L John F K. Isolation purification and characterization of hyaluronan from human umbilical cord residues J. Carbohydrate Polymers Bitter T Muir H M. A modified uronic acid carbazole reaction J. Analytical Biochemistry J Laurent T C Ryan M Pietruszkiewicz A. Fractionation of hyaluronic acid the polydispersity of hyaluronic acid from the bovine

7 1452 Journal of Nuclear Agricultural Sciences ~ 1452 vitreous body J. Biochimica ET Biophysica Acta Amagai I Tashiro Y Ogawa H. Improvement of the extraction procedure for hyaluronan from fish eyeball and the molecular 20. characterization J. Fisheries Science Jae K Periasamy S Jae H K Jong I C Hyun J P Myung W B Ju J W L. Structural and antioxidant properties of gamma irradiated J hyaluronic acid J. Food Chemistry J J Ev enia D Vladimír V Vlasta S Zdenka H Anna E. Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating J. Carbohydrate Polymers D Lanfranco C David R. Process for preparing a hyaluronic acid fraction having a low polydispersion index USA US A P J J D Extraction of Hyaluronic Acid from Eyeball of Tilapia by Ultrasonic-assisted Enzyme Method CHEN Sheng-jun 1 CHEN Hui 1 2 GAO Rui-chang 2 LI Lai-hao 1 YU Gang 1 HU Xiao 1 MA Hai-xia 1 DENG Jian-chao 1 1 Key Laboratory of Aquatic Product Processing Ministry of Agriculture National R&D Center for Aquatic Product Processing South China Sea Fisheries Research Institute CAFS Guangzhou Guangdong Food and Biological Engineering College Jiangsu University Zhenjiang Jiangsu Abstract In order to make full use of tilapia raw material the condition of ultrasound-assisted enzymatic extraction of hyaluronic acid HA from the tilapia eyes was studied. Through single factor experiment and orthogonal experiment the influence of the ultrasonic power ultrasonic time kinds of protease time of enzyme digestion ph of solution amount of enzyme and temperature of enzyme solution on the extraction process of hyaluronic acid were analyzed and the optimum extraction conditions have been confirmed as follows ultrasonic power 200 W ultrasonic time 15min composite enzyme mixture of trypsin and compound protease with the enzyme activity 1 1 enzyme action time 3 hrs hydrolysis temperature 40 ph 9. 0 enzyme dosage 6 000U g - 1 the average yield of hyaluronic acid was %. The hyaluronic acid was purified by CTAB and ion exchange chromatography and the purity was %. The relative molecular weight of hyaluronic acid was 100 KDa measured by Ubbelohde viscometer. The ultraviolet spectrum of production did not show the existence of protein and nucleic acid. Infrared spectra of HA was the same as the standard. This study provides the technical support for the high-value utilization of the tilapia eyes. Key words Eyeball of tilapia Ultrasonic Hyaluronic acid Technolgical condition

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%