海地瓜多糖和多肽提取纯化工艺及抗氧化活性
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摘要
以低值海地瓜(Acaudina molpadioides)体壁干品为实验原料,优化酶解超滤工艺条件,联合制备出不同分子质量段的海地瓜多糖和多肽,优化确定柱层析法分离纯化海地瓜多糖的工艺参数,对比探究不同分子质量段海地瓜多糖和多肽的体外抗氧化活性。结果表明:先用胰蛋白酶在料液比(m/V)1∶40、加酶量2.4%(质量分数)、p H值7.5、45℃下酶解8 h,然后超滤分离得到分子质量小于10 ku的海地瓜多肽,提取率达到(29.602±1.012)%;再用中性蛋白酶和木瓜蛋白酶复合使用对超滤截留液和一次酶解沉淀进行二次酶解提取多糖,先加入质量分数7%的中性蛋白酶,45℃下酶解4 h;再加入质量分数8%的木瓜蛋白酶,60℃下酶解4 h,p H值均为7.0,粗多糖提取率达到(14.511±0.162)%。确定最佳超滤条件为:操作压力0.20 MPa,料液质量分数6%,操作温度35℃。得到海地瓜多肽P_1(5~10 ku,48.47%)、P_2(1~5 ku,18.46%)和P_3(<1 ku,33.07%);得到海地瓜粗多糖G_1(<10 ku,63.09%)、G_2(10~100 ku,7.24%)、G_3(100~200 ku,4.67%)和G_4(>200 ku,25.00%)。采用Q-Sepharose-Fast-Flow阴离子交换柱层析法对G_4进行纯化,在0,0.5,1.5 mol/L洗脱盐浓度下,得到3个纯化多糖组分G_(4-1)、G_(4-2)和G_(4-3)。体外抗氧化活性检测结果显示,不同分子质量段海地瓜多肽对·OH的清除能力强弱顺序为:P_3>P_2>P_1,对DPPH·和O_2~-·的清除能力强弱顺序均为:P_2>P_3>P_1。不同海地瓜粗多糖对·OH、DPPH·和O_2~-·的清除能力强弱顺序依次为:G_4>G_1>G_3>G_2,G_4>G_3>G_1>G_2,G_1>G_4>G_2>G_3;纯化多糖对·OH的清除能力强弱顺序为:G_(4-1)>G_(4-2)>G_(4-3),对DPPH·和O_2~-·的清除能力强弱顺序均为:G_(4-2)>G_(4-1)>G_(4-3)。
In this paper,dried body walls of low-value Acaudina molpadioides were selected as research materials and enzymatic hydrolysis technologies for preparing polysaccharides and polypeptides were optimized.Polysaccharides and polypeptides with different molecular weight(MW)range were isolated by ultrafiltration,and then polysaccharides was purified by using column chromatography.The antioxidant activities of polysaccharides and polypeptides were measured in vitro.The results showed that the extraction rate of polypeptides was(29.602±1.012)%under the opimized extraction conditions as follows:ratio of material to raw material1∶40,2.4%trypsin,hydrolysis temperature 45℃,p H value of 7.5,hydrolysis time 8 h,and then polypeptides of MW blow 10 ku were obtained by ultrafiltration.The polysaccharide was extracted from the ultrafiltrate and precipitation of previous hydrolysate by the combination of neutral protease and papain.The samples were hydrolyzed with 7%neutral protease for 4 h at 45℃and p H 7.0 following with 8%papain for another 4 h at60℃.The extraction rate of crude polysaccharides was up to(14.511±0.162)%.The ultrafiltration conditions were confirmed with 0.20 MPa,material liquid concentration 6%at 35℃.Three polypeptides were obtained,48.47%between 5~10 ku named P_1,18.46%between 1~5 ku named P_2and 33.07%below 1 ku named P_3.Four crude polysaccharides were obtained,63.09%blow 10 ku named G_1,7.24%between 10~100 ku named G_2,4.67%between 100~200 ku named G_3and 25.00%above 200 ku named G_4.Three pure polysaccharides G_(4-1),G_(4-2)and G_(4-3)were obtained by using Q-Sepharose-F-F ion-exchange column chromatography.The elution concentrations were 0,0.5 and 1.5 mol/L.The results of antioxidant activity assay indicated that the·OH radical scavenging activity of polypeptides was P_3>P_2>P_1and the DPPH·and O_2~-·were P_2>P_3>P_1.The·OH,DPPH·,and O_2~-·scavenging activity of crude polysaccharides with different MW were G_4>G_1>G_3>G_2,G_4>G_3>G_1>G_2,and G_1>G_4>G_2>G_3,respectively.The DPPH·,O_2~-·and·OH scavenging activity of pure polysaccharides were G_(4-1)>G_(4-2)>G_(4-3)and G_(4-2)>G_(4-1)>G_(4-3),respectively.
In this paper,dried body walls of low-value Acaudina molpadioides were selected as research materials and enzymatic hydrolysis technologies for preparing polysaccharides and polypeptides were optimized.Polysaccharides and polypeptides with different molecular weight(MW)range were isolated by ultrafiltration,and then polysaccharides was purified by using column chromatography.The antioxidant activities of polysaccharides and polypeptides were measured in vitro.The results showed that the extraction rate of polypeptides was(29.602±1.012)%under the opimized extraction conditions as follows:ratio of material to raw material1∶40,2.4%trypsin,hydrolysis temperature 45℃,p H value of 7.5,hydrolysis time 8 h,and then polypeptides of MW blow 10 ku were obtained by ultrafiltration.The polysaccharide was extracted from the ultrafiltrate and precipitation of previous hydrolysate by the combination of neutral protease and papain.The samples were hydrolyzed with 7%neutral protease for 4 h at 45℃and p H 7.0 following with 8%papain for another 4 h at60℃.The extraction rate of crude polysaccharides was up to(14.511±0.162)%.The ultrafiltration conditions were confirmed with 0.20 MPa,material liquid concentration 6%at 35℃.Three polypeptides were obtained,48.47%between 5~10 ku named P_1,18.46%between 1~5 ku named P_2and 33.07%below 1 ku named P_3.Four crude polysaccharides were obtained,63.09%blow 10 ku named G_1,7.24%between 10~100 ku named G_2,4.67%between 100~200 ku named G_3and 25.00%above 200 ku named G_4.Three pure polysaccharides G_(4-1),G_(4-2)and G_(4-3)were obtained by using Q-Sepharose-F-F ion-exchange column chromatography.The elution concentrations were 0,0.5 and 1.5 mol/L.The results of antioxidant activity assay indicated that the·OH radical scavenging activity of polypeptides was P_3>P_2>P_1and the DPPH·and O_2~-·were P_2>P_3>P_1.The·OH,DPPH·,and O_2~-·scavenging activity of crude polysaccharides with different MW were G_4>G_1>G_3>G_2,G_4>G_3>G_1>G_2,and G_1>G_4>G_2>G_3,respectively.The DPPH·,O_2~-·and·OH scavenging activity of pure polysaccharides were G_(4-1)>G_(4-2)>G_(4-3)and G_(4-2)>G_(4-1)>G_(4-3),respectively.
引文
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[13]刘程惠,朱蓓薇,董秀萍,等.海参酶解产物的分离及其体外抗氧化作用的研究[J].食品与发酵工业,2007,33(9):50-53.
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[16]PAN S K,YAO D R,ZHOU M Q,et al.Hydroxyl radical scavenging activity of peptide from sea cucumber using enzyme complex isolated from the digestive tract of sea cucumber[J].African Journal of Biotechnology,2012,11(5):1214-1219.DOI:10.5897/AJB11.2578.
[17]LIU C H,WANG X J,YUAN W P,et al.Anti-fatigue and immune functions of sea cucumber oral liquid[J].Modern Food Science and Technology,2009,25(10):1115-1119.
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[20]LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein measurement with the Folin phenol reagent[J].Biological Chemistry,1951,193:265-275.
[21]陈山,杨晓泉,郭祀远,等.大豆肽超滤分离纯化过程的研究[J].食品与发酵工业,2003,29:49-52.
[22]厉朝龙.生物化学与分子生物学实验技术[M].杭州:浙江大学出版社,2003:16-22.
[23]李贵荣.枸杞多糖的提取及其对活性氧自由基的清除作用[J].中国现代应用药学杂志,2002,19(2):94-96.
[24]梁引库.黄精多糖的脱色和脱蛋白及体外抗氧化活性研究[J].江苏农业科学,2013,41(5):240-243.
[25]张娅,李宝才.松茸多糖超声提取物抗氧化活性研究[J].化学与生物工程,2011,28(9):75-82.
[26]HATANO T,KAGAWA H,YASUHARA T,et al.Two new flavonoids and other constituents in licorice root:their relative astringency and radical scavenging effects[J].Chemistry of Pharmacology Bulletin,1988,36(6):2090-2097.DOI:10.1248/cpb.36.2090.
[27]WU H,CHEN H,SHIAU C.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9):949-957.DOI:10.1016/S0963-9969(03)00104-2.
[28]冯骏.海参多糖的分离纯化与化学组成的研究[D].厦门:集美大学,2014.
[29]展学孔,周海妹,马小花,等.海参多糖提取新工艺[J].中国实验方剂学杂志,2011,17(15):40-42.
[30]冀利,孙曙光,孙玖玉,等.从浓缩枣汁超滤截留液中提取金丝小枣多肽和多糖[J].山东食品发酵,2013,168(1):3-10.
[31]罗晓航.PEF结合酶法提取鲍鱼脏器粗多糖及其抗氧化活性研究[D].福州:福建农林大学,2012.
[32]李霞,张峰,李永才,等.兰州百合不同部位多糖含量及抗氧化活性的比较[J].食品工业科技,2012(24):88-91.
[2]王方国,张海生,周怀阳.海地瓜的营养成分及其开发利用设想[J].中国海洋药物,1998,66(2):52-54.
[3]伏纬华,吴凤梧,戚宝凤,等.海地瓜的研究-Ⅱ:海地瓜与黄玉海参营养成分的比较[J].中国海洋药物,1994,51(3):28-30.
[4]TAKASHI H,NOBUHIRO Z,KYOKO Y,et al.Recent advances in researches on physiologically active substances in holothurians[J].Journal of Ocean University of China,2005,4(3):193-197.
[5]LIU X,SUN Z,ZHANG M,et al.Antioxidant and antihyperlipidemic activities of polysaccharides from sea cucumber Apostichopus japonicus[J].Carbohydrate Polymers,2012,90(4):1664-1670.DOI:10.1016/j.carbpol.2012.07.047.
[6]LUO L,WU M,XU L,et al.Comparison of physicochemical characteristics and anticoagulant activities of polysaccharides from three sea cucumbers[J].Marine Drugs,2013,11(2):399-417.
[7]CHOO P S.Population status,fisheries and trade of sea cucumbers in Asia[J].Fao Fisheries&Aquaculture Technical Paper,2008,5(16):81-118.
[8]PURCELL S W.Value,market preferences and trade of Bechedemer from Pacific Island sea cucumbers[J].Plos One,2014,9(4):1-8.
[9]BORDBAR S,ANWAR F,SAARI N.High-value components and bioactives from sea cucumbers for functional foods:a review[J].Marine Drugs,2011,9(10):1761-1805.DOI:10.3390/md9101761.
[10]KARIYA Y,MULLOY B,IMAI K,et al.Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis[J].Carbohydrate Research,2004,339(7):1339-1346.DOI:10.1016/j.carres.2004.02.025.
[11]WU M,XU S,ZHAO J,et al.Physicochemical characteristics and anticoagulant activities of low molecular weight fractions by free-radical depolymerization of a fucosylated chondroitin sulphate from sea cucumber Thelenata ananas[J].Food Chemistry,2010,122(3):716-723.
[12]WANG Y,SU W,ZHANG C,et al.Protective effect of sea cucumber(Acaudina molpadioides)fucoidan against ethanol-induced gastric damage[J].Food Chem,2012,133(4):1414-1419.DOI:10.1016/j.foodchem.2012.02.028.
[13]刘程惠,朱蓓薇,董秀萍,等.海参酶解产物的分离及其体外抗氧化作用的研究[J].食品与发酵工业,2007,33(9):50-53.
[14]LI Z,WANG H,LI J,et al.Basic and clinical study on the antithrombotic mechanism of glycosaminoglycan extracted from sea cucumber[J].Chinese Medical Journal,2000,113:8-13.
[15]ALTHUNIBAT O Y,HASHIM R B,TAHER M,et al.In vitro antioxidant and antiproliferative activities of three Malaysian sea cucumber species[J].European Journal of Scientific Research,2009,37(3):376-387.
[16]PAN S K,YAO D R,ZHOU M Q,et al.Hydroxyl radical scavenging activity of peptide from sea cucumber using enzyme complex isolated from the digestive tract of sea cucumber[J].African Journal of Biotechnology,2012,11(5):1214-1219.DOI:10.5897/AJB11.2578.
[17]LIU C H,WANG X J,YUAN W P,et al.Anti-fatigue and immune functions of sea cucumber oral liquid[J].Modern Food Science and Technology,2009,25(10):1115-1119.
[18]MAMELONA J,SAINT L R,PELLETIER E.Nutritional composition and antioxidant properties of protein hydrolysates prepared from echinoderm by products[J].International Journal of Food Science&Technology,2010,45(1):147-154.DOI:10.1111/j.1365-2621.2009.02114.x.
[19]DUBOIS M,GILLES K A,HAMILTON J K,et al.Colorimetric method for determination of sugars and related substances[J].Analytical Biochemistry,1956,28(3):350-356.
[20]LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein measurement with the Folin phenol reagent[J].Biological Chemistry,1951,193:265-275.
[21]陈山,杨晓泉,郭祀远,等.大豆肽超滤分离纯化过程的研究[J].食品与发酵工业,2003,29:49-52.
[22]厉朝龙.生物化学与分子生物学实验技术[M].杭州:浙江大学出版社,2003:16-22.
[23]李贵荣.枸杞多糖的提取及其对活性氧自由基的清除作用[J].中国现代应用药学杂志,2002,19(2):94-96.
[24]梁引库.黄精多糖的脱色和脱蛋白及体外抗氧化活性研究[J].江苏农业科学,2013,41(5):240-243.
[25]张娅,李宝才.松茸多糖超声提取物抗氧化活性研究[J].化学与生物工程,2011,28(9):75-82.
[26]HATANO T,KAGAWA H,YASUHARA T,et al.Two new flavonoids and other constituents in licorice root:their relative astringency and radical scavenging effects[J].Chemistry of Pharmacology Bulletin,1988,36(6):2090-2097.DOI:10.1248/cpb.36.2090.
[27]WU H,CHEN H,SHIAU C.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9):949-957.DOI:10.1016/S0963-9969(03)00104-2.
[28]冯骏.海参多糖的分离纯化与化学组成的研究[D].厦门:集美大学,2014.
[29]展学孔,周海妹,马小花,等.海参多糖提取新工艺[J].中国实验方剂学杂志,2011,17(15):40-42.
[30]冀利,孙曙光,孙玖玉,等.从浓缩枣汁超滤截留液中提取金丝小枣多肽和多糖[J].山东食品发酵,2013,168(1):3-10.
[31]罗晓航.PEF结合酶法提取鲍鱼脏器粗多糖及其抗氧化活性研究[D].福州:福建农林大学,2012.
[32]李霞,张峰,李永才,等.兰州百合不同部位多糖含量及抗氧化活性的比较[J].食品工业科技,2012(24):88-91.
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