微拟球藻对营养盐的响应及其多糖酶解物抗氧化活性
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摘要
为探讨在温度、光照一定时,NaNO_3和NaH_2PO_4对微拟球藻(Nannochloropsis sp.,N.sp.)生长的影响,为其规模化培养提供实验数据;同时研究微拟球藻胞内多糖(Intracellular polysaccharide from Nannochloropsis sp.,NSP)及其酶解物的抗氧化活性,以探究N.sp.在食品、医药行业中的应用价值.利用分光光度法对经不同浓度NaNO_3和NaH_2PO_4处理的微拟球藻的生物指标进行测定.运用水提醇沉法获得NSP粗品,并用紫贻贝酶(Enzyme from Mytilus edulis Linnaeus)对NSP粗品进行酶解,以羟基自由基(·OH)、1,1-二苯基-2-苦味酰基自由基(DPPH·)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐自由基(ABTS·+)、超氧阴离子自由基(O_2~-·)和2,2'-偶氮-双-(2-脒基丙烷)氯化二氢(AAPH)的清除能力为指标,评价NSP粗品及其酶解物的抗氧化活性.结果表明,微拟球藻生长的最适NaNO_3和NaH_2PO_4浓度分别为0.1125 g/L和0.0075 g/L;NSP粗品对·OH、DPPH·、ABTS·+、O_2~-·和AAPH均具有一定的清除能力.NSP酶解物对DPPH·的清除能力与NSP粗品的无显著差异,对·OH、ABTS·+和O_2~-·的清除能力均强于NSP粗品,其中对·OH和ABTS·+的半清除浓度(IC_(50))值分别为(0.9206±0.0147)mg·m L~(-1)和(0.0751±0.0093)mg·m L~(-1),NSP酶解物对O_2~-·的清除率是酶解前效果的约2倍.NSP酶解物的ORAC值为(940.1±7.5)μmol TE/g,是酶解前的4.7倍左右.
This study explored the effects of inorganic nitrogen and inorganic phosphorus on growth of Nannochloropsis sp. under a constant temperature and light-dark cycles in order to provide experimental data for its largescale cultivation. The antioxidant activities of polysaccharides(NSP)and its enzymolysis products(enzymolysis NSP)in vitro from N.Sp.were further studied in order to predict the application value of N.sp.in the food and drug industry.The biological indexes of Nannochloropsis sp.treated with different concentrations of inorganic nitrogen and inorganic phosphorus concentrations were determined by spectrophotometry.The NSPs were obtained by water extraction and Enzyme from Mytilus edulis Linnaeus was used to hydrolyze NSP.The NSP and antioxidant activities of its Enzymolysis product were evaluated using scavenging capacity of hydroxyl free radica(l·OH),1,1-Diphenyl-2-picrylhydrazyl free radical(DPPH·),2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)diammonium salt radical(ABTS·+),free radical superoxide anion free radica(lO_2~-·)and 2,2'-azobis[2-methylpropionamidine]dihydrochloride(AAPH).The concentrations of inorganic nitrogen and inorganic phosphorus for optimal growth of N.sp.were0.1125 g/L and 0.0075 g/L respectively.The NSP had radicals scavenging ability on·OH,DPPH·,ABTS·+,O_2~-·and AAPH.There was no significant difference between the scavenging ability of enzymolysis NSP on DPPH·and the scavenging ability of NSP but,the scavenging ability of enzymolysis NSP on·OH,ABTS·+,and O_2~-·were stronger than that of NSP.The half-scavengingof enzymolysis NSP on·OH and ABTS·+were(0.9206±0.0147)mg·mL~(-1)respectively.The scavenging rate of NSP on O_2~-·after enzymolysis was about 2 times as good as that before enzymolysis.The ORAC value of enzymolysis NSP was(940.1±7.5)μmol TE/g which was 4.7 times as much as before enzymolysis.
This study explored the effects of inorganic nitrogen and inorganic phosphorus on growth of Nannochloropsis sp. under a constant temperature and light-dark cycles in order to provide experimental data for its largescale cultivation. The antioxidant activities of polysaccharides(NSP)and its enzymolysis products(enzymolysis NSP)in vitro from N.Sp.were further studied in order to predict the application value of N.sp.in the food and drug industry.The biological indexes of Nannochloropsis sp.treated with different concentrations of inorganic nitrogen and inorganic phosphorus concentrations were determined by spectrophotometry.The NSPs were obtained by water extraction and Enzyme from Mytilus edulis Linnaeus was used to hydrolyze NSP.The NSP and antioxidant activities of its Enzymolysis product were evaluated using scavenging capacity of hydroxyl free radica(l·OH),1,1-Diphenyl-2-picrylhydrazyl free radical(DPPH·),2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)diammonium salt radical(ABTS·+),free radical superoxide anion free radica(lO_2~-·)and 2,2'-azobis[2-methylpropionamidine]dihydrochloride(AAPH).The concentrations of inorganic nitrogen and inorganic phosphorus for optimal growth of N.sp.were0.1125 g/L and 0.0075 g/L respectively.The NSP had radicals scavenging ability on·OH,DPPH·,ABTS·+,O_2~-·and AAPH.There was no significant difference between the scavenging ability of enzymolysis NSP on DPPH·and the scavenging ability of NSP but,the scavenging ability of enzymolysis NSP on·OH,ABTS·+,and O_2~-·were stronger than that of NSP.The half-scavengingof enzymolysis NSP on·OH and ABTS·+were(0.9206±0.0147)mg·mL~(-1)respectively.The scavenging rate of NSP on O_2~-·after enzymolysis was about 2 times as good as that before enzymolysis.The ORAC value of enzymolysis NSP was(940.1±7.5)μmol TE/g which was 4.7 times as much as before enzymolysis.
引文
[1]刘婉君.微拟球藻多不饱和脂肪酸合成途径及去饱和酶功能的研究[D].暨南:暨南大学,2016:1.
[2]谭仁祥,石建功,郭跃伟,等.功能海洋生物分子—发现与应用[M].北京:科学出版社,2007:3-17.
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[4]Joventino I P,Alves H G R,Neves L C,et al.The microalga Spirulina platensis presents anti-inflammatory action as well as hypoglycemic and hypolipidemic properties in diabetic rats[J].Journal of Complementary and Integrative Medicine,2012,9(1):17-45.
[5]席波,宋东辉,孙晶,等.十种微藻粗多糖的抑菌作用及海水小球藻粗多糖的抗氧化活性[J].天津科技大学学报,2015(5):20-25.
[6]Chen Y X,Liu X Y,Xiao Z,et al.Antioxidant activities of polysaccharides obtained from Chlorella pyrenoidosa via different ethanol concentrations[J].International Journal of Biological Macromolecules,2016,91:505-509.
[7]周妍.两种海洋微藻多糖的免疫及抗肿瘤活性[D].烟台:烟台大学,2010:32.
[8]Mansour H A,Shoman S A,Kdodier M H.Antiviral effect of edaphic cyanophytes on rabies and herpes-1 viruses[J].Acta Biol Hung,2011,62(2):194-203.
[9]Majdoub H,Mansour M B,Chaubet F,et al.Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis[J].Biochimica et biophysica acta,2009,1790(10):1377.
[10]庞辉,汤桂芳,玉艳红.螺旋藻多糖的抗溃疡作用及机制[J].广西医学,2006,28(11):1772-1773.
[11]刘玉兰,颜鸣.螺旋藻多糖的抗衰老作用[J].中国药理学通报,1998(4):362-364.
[12]Wong C Y,Chu W L,Marchant H,et al.Comparing the response of Antarctic,tropical and temperate microalgae to ultraviolet radiation(UVR)stress[J].Journal of Applied Phycology,2007,19(6):689-699.
[13]Park J K,Kim Z H,Lee C G,et al.Characterization and immunostimulating activity of a water-soluble polysaccharide isolated from Haematococcus lacustris[J].Biotechnology&Bioprocess Engineering,2011,16(6):1090-1098.
[14]东方.微藻多糖药理活性及应用研究进展[J].齐齐哈尔医学院学报,2016,37(30):3811-3812.
[15]张琳.微拟球藻脂酰辅酶A合成酶与脂肪酸延伸酶的研究[D].青岛:中国海洋大学,2011:4.
[16]梁鑫,朱葆华,杨官品,等.一株分离微拟球藻LAMB0002的鉴定[J].中国海洋大学学报(自然科学版),2014,44(7):039-045.
[17]Zhang J,Wan L,Xia S,et al.Morphological and spectrometric analyses of lipids accumulation in a novel oleaginous microalga,Eustigmatos cf.polyphem(Eustigmatophyceae)[J].Bioprocess&Biosystems Engineering,2013,36(8):1125-1130.
[18]Pal D,Khozin-Goldberg I,Cohen Z,et al.The effect of light,salinity,and nitrogen availability on lipid production by Nannochloropsis sp.[J].Applied Microbiology&Biotechnology,2011,90(4):1429.
[19]石恩慧,李红.响应面法优化超声提取板栗总苞多酚工艺条件[J].中国食品学报,2013,13(5):69-76.
[20]GB/T 5009.3-2016食品中水分的测定方法[S].北京:中国标准出版社,2016.
[21]吴鑫,黄杰涛,张莲姬.苯酚-硫酸法测定榛花多糖含量分析条件的优化[J].延边大学农学学报,2017,39(3):44-49.
[22]刘海英,山成龙.药用植物大黄对·OH的清除作用的研究[J].光谱实验室,2008,5(4):614-617.
[23]许明录,左浩,汪高博,等.翼蓼块根的抗氧化活性和抑制癌细胞增殖的研究[J].河南科技学院学报(自然科学版),2016,44(2):53-58.
[24]倪慧艳,陈伟,宋文静,等.黄芪多糖抗氧化作用研究[J].中医学报,2017,32(9):1795-1707.
[25]臧延青,何飞,李执坤,等.野生蒲公英花多酚的提取和体外抗氧化活性研究[J].黑龙江八一农垦大学学报,2017,29(4):62-66.
[26]续洁琨,姚新生,栗原博.抗氧化能力指数(ORAC)测定原理及应用[J].中国药理学通报,2006,22(8):1015-1021.
[2]谭仁祥,石建功,郭跃伟,等.功能海洋生物分子—发现与应用[M].北京:科学出版社,2007:3-17.
[3]陈宇婷.两种海洋微藻多糖抑制肿瘤血管生成活性的研究[D].大连:大连海洋大学,2017:1-2.
[4]Joventino I P,Alves H G R,Neves L C,et al.The microalga Spirulina platensis presents anti-inflammatory action as well as hypoglycemic and hypolipidemic properties in diabetic rats[J].Journal of Complementary and Integrative Medicine,2012,9(1):17-45.
[5]席波,宋东辉,孙晶,等.十种微藻粗多糖的抑菌作用及海水小球藻粗多糖的抗氧化活性[J].天津科技大学学报,2015(5):20-25.
[6]Chen Y X,Liu X Y,Xiao Z,et al.Antioxidant activities of polysaccharides obtained from Chlorella pyrenoidosa via different ethanol concentrations[J].International Journal of Biological Macromolecules,2016,91:505-509.
[7]周妍.两种海洋微藻多糖的免疫及抗肿瘤活性[D].烟台:烟台大学,2010:32.
[8]Mansour H A,Shoman S A,Kdodier M H.Antiviral effect of edaphic cyanophytes on rabies and herpes-1 viruses[J].Acta Biol Hung,2011,62(2):194-203.
[9]Majdoub H,Mansour M B,Chaubet F,et al.Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis[J].Biochimica et biophysica acta,2009,1790(10):1377.
[10]庞辉,汤桂芳,玉艳红.螺旋藻多糖的抗溃疡作用及机制[J].广西医学,2006,28(11):1772-1773.
[11]刘玉兰,颜鸣.螺旋藻多糖的抗衰老作用[J].中国药理学通报,1998(4):362-364.
[12]Wong C Y,Chu W L,Marchant H,et al.Comparing the response of Antarctic,tropical and temperate microalgae to ultraviolet radiation(UVR)stress[J].Journal of Applied Phycology,2007,19(6):689-699.
[13]Park J K,Kim Z H,Lee C G,et al.Characterization and immunostimulating activity of a water-soluble polysaccharide isolated from Haematococcus lacustris[J].Biotechnology&Bioprocess Engineering,2011,16(6):1090-1098.
[14]东方.微藻多糖药理活性及应用研究进展[J].齐齐哈尔医学院学报,2016,37(30):3811-3812.
[15]张琳.微拟球藻脂酰辅酶A合成酶与脂肪酸延伸酶的研究[D].青岛:中国海洋大学,2011:4.
[16]梁鑫,朱葆华,杨官品,等.一株分离微拟球藻LAMB0002的鉴定[J].中国海洋大学学报(自然科学版),2014,44(7):039-045.
[17]Zhang J,Wan L,Xia S,et al.Morphological and spectrometric analyses of lipids accumulation in a novel oleaginous microalga,Eustigmatos cf.polyphem(Eustigmatophyceae)[J].Bioprocess&Biosystems Engineering,2013,36(8):1125-1130.
[18]Pal D,Khozin-Goldberg I,Cohen Z,et al.The effect of light,salinity,and nitrogen availability on lipid production by Nannochloropsis sp.[J].Applied Microbiology&Biotechnology,2011,90(4):1429.
[19]石恩慧,李红.响应面法优化超声提取板栗总苞多酚工艺条件[J].中国食品学报,2013,13(5):69-76.
[20]GB/T 5009.3-2016食品中水分的测定方法[S].北京:中国标准出版社,2016.
[21]吴鑫,黄杰涛,张莲姬.苯酚-硫酸法测定榛花多糖含量分析条件的优化[J].延边大学农学学报,2017,39(3):44-49.
[22]刘海英,山成龙.药用植物大黄对·OH的清除作用的研究[J].光谱实验室,2008,5(4):614-617.
[23]许明录,左浩,汪高博,等.翼蓼块根的抗氧化活性和抑制癌细胞增殖的研究[J].河南科技学院学报(自然科学版),2016,44(2):53-58.
[24]倪慧艳,陈伟,宋文静,等.黄芪多糖抗氧化作用研究[J].中医学报,2017,32(9):1795-1707.
[25]臧延青,何飞,李执坤,等.野生蒲公英花多酚的提取和体外抗氧化活性研究[J].黑龙江八一农垦大学学报,2017,29(4):62-66.
[26]续洁琨,姚新生,栗原博.抗氧化能力指数(ORAC)测定原理及应用[J].中国药理学通报,2006,22(8):1015-1021.