响应面试验优化木瓜蛋白酶法脱马齿苋多糖蛋白工艺
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摘要
为确定木瓜蛋白酶法脱马齿苋多糖蛋白的最佳工艺,以酶液-样液体积比、酶解时间、酶解温度及pH值为影响因素,以马齿苋多糖损失率与蛋白去除率为指标,通过响应面试验优化木瓜蛋白酶法脱马齿苋多糖蛋白的工艺条件。结果表明,木瓜蛋白酶法脱马齿苋多糖蛋白的最佳工艺为酶液-样液体积比0.2∶1、酶解温度60℃、酶解时间3 h、pH 6,在此条件下蛋白去除率为78.22%,多糖损失率为10.39%。马齿苋粗多糖溶液经分离纯化、高效液相色谱分析,结果表明马齿苋多糖由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖单糖组成,其组成比例为5.3∶5.9∶1.3∶27.6∶1∶18.8∶14.6。
The purpose of this study was the optimization of the papain-catalyzed deproteinization conditions of polysaccharide from Portulaca oleracea L.(POP) by response surface methodology(RSM). Polysaccharide loss and the rate of protein removal were investigated with respect to four variables: enzyme to sample ratio, hydrolysis time, hydrolysis temperature and pH. The optimum operating conditions were obtained as follows: enzyme to sample ratio 0.2:1, hydrolysis time 60 ℃, hydrolysis temperature 3 h and pH 6. Under the optimized conditions, the deproteinization efficiency was as high as 78.22% with only 10.39% loss of the polysaccharide. Monosaccharide composition analysis by high performance liquid chromatography(HPLC) showed that the purified polysaccharide was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 5.3:5.9:1.3:27.6:1:18.8:14.6.
The purpose of this study was the optimization of the papain-catalyzed deproteinization conditions of polysaccharide from Portulaca oleracea L.(POP) by response surface methodology(RSM). Polysaccharide loss and the rate of protein removal were investigated with respect to four variables: enzyme to sample ratio, hydrolysis time, hydrolysis temperature and pH. The optimum operating conditions were obtained as follows: enzyme to sample ratio 0.2:1, hydrolysis time 60 ℃, hydrolysis temperature 3 h and pH 6. Under the optimized conditions, the deproteinization efficiency was as high as 78.22% with only 10.39% loss of the polysaccharide. Monosaccharide composition analysis by high performance liquid chromatography(HPLC) showed that the purified polysaccharide was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 5.3:5.9:1.3:27.6:1:18.8:14.6.
引文
[1]WAN Y W,LI W D,LIN J,et al.Ethanol extract of Portulaca oleracea L.protects against hypoxia-induced neuro damage through modulating endogenous erythropoietin expression[J].Journal of Nutritional Biochemistry,2012,23(4):385-391.DOI:10.1016/j.jnutbio.2010.12.015.
[2]WANG P,SUN H,LIU D,et al.Protective effect of a phenolic extract containing indoline amides from Portulaca oleracea against cognitive impairment in senescent mice induced by large dose of D-galactose/NaNO2[J].Journal of Ethnopharmacol,2017,203:252-259.DOI:10.1016/j.jep.2017.03.050.
[3]OH K B,CHANG I M,HWANG K J,et al.Detection of antifungal activity in Portulaca oleracea by a single-cell bioassay system[J].Phytotherapy Research,2000,14(5):329-332.DOI:10.1002/1099-1573(200008)14:5<329::AID-PTR581>3.0.CO;2-5.
[4]DEHGHAN F,SOORI R,GHOLAMI K,et al.Purslane(Portulaca oleracea)seed consumption and aerobic training improves biomarkers associated with atherosclerosis in women with type 2 diabetes(T2D)[J].Sciences Report,2016,6:37819.DOI:10.1038/srep37819.
[5]BAI Y,ZANG X,MA J,et al.Anti-diabetic effect of Portulaca oleracea L.polysaccharideandits mechanism in diabetic rats[J].International Journal of Molecular Sciences,2016,17(8):1201.DOI:10.3390/ijms17081201.
[6]GUO G,YUE L,FAN S,et al.Antioxidant and antiproliferative activities of purslane seed oil[J].Journal of Hypertension:Open Access,2016,5(2):218.DOI:10.4172/2167-1095.1000218.
[7]LI C Y,MENG Y H,YING Z M,et al.Correction to three novel alkaloids from Portulaca oleracea L.and their anti-inflammatory effects[J].Journal of Agricultural&Food Chemical,2017,65(4):993-994.DOI:10.1021/acs.jafc.6b05659.
[8]LEE A S,KIM J S,LEE Y J,et al.Anti-TNF-αactivity of Portulaca oleracea in vascular endothelial cells[J].International Journal of Molecular Sciences,2012,13(5):5628.DOI:10.3390/ijms13055628.
[9]ASKARI V R,REZAEE S A,ABNOUS K,et al.The influence of hydro-ethanolic extract of Portulaca oleracea L.on Th1/Th2 balance in isolated human lymphocytes[J].Journal of Ethnopharmacology,2016,194:1112-1121.DOI:10.1016/j.jep.2016.10.082.
[10]ZHAO R,ZHANG T,MA B,et al.Antitumor activity of Portulaca oleracea L.polysaccharide on HeLa cells through inducing TLR4/NF-κB bignaling[J]. Nutrition and Cancer, 2017, 69(1): 131-139. DOI:10.1080/01635581.2017.1248294.
[11]翁前锋,袁凯龙,张宏颖,等.胶束电动毛细管色谱安培检测中药马齿苋中多巴胺和去甲肾上腺素[J].色谱,2005,23(1):18-21.DOI:10.3321/j.issn:1000-8713.2005.01.004.
[12] YIN A W, GAO P F, DANG L M. Anti-aging effects of flavonoids from Portulaca oleracea L.[J].Natural Product Research and Development,2017,29(6):988-993.DOI:10.16333/j.1001-6880.2017.6.015.
[13]汪荔,王征,张娇,等.传统工艺与膜分离技术联合对马齿苋多糖的提取分离与抗氧化活性研究[J].中草药,2016,47(10):1676-1681.DOI:10.7501/j.issn.0253-2670.2016.10.009.
[14]郑智音,贾晓斌,舒娈,等.鲜马齿苋多糖、生物碱和多酚类组分的制备及其降血糖活性研究[J].中草药,2014,45(18):2673-2677.DOI:10.7501/j.issn.0253-2670.2014.18.019.
[15]ZHAO C,YING Z,TAO X,et al.A new lactam alkaloid from Portulaca oleracea L.and its cytotoxity[J].Natural Product Research,2017:1-6.DOI:10.1080/14786419.2017.1385022.
[16]LI Y P,YAO L H,WU G J,et al.Antioxidant activities of novel smallmolecule polysaccharide fractions purified from Portulaca oleracea L.[J].Food Science and Biotechnology,2014,23(6):2045-2052.
[17]ZHAO R,ZHANG T,ZHAO H,et al.Effects of Portulaca oleracea L.polysaccharides on phenotypic and functional maturation of murine bone marrow derived dendritic cells[J].Nutrition&Cancer,2015,67(6):987.DOI:10.1080/01635581.2015.1060352.
[18]ZHAO R,GAO X,CAI Y,et al.Antitumor activity of Portulaca oleracea L.polysaccharides against cervical carcinoma in vitro and in vivo[J].Carbohydrate Polymers,2013,96(2):376-383.DOI:10.1016/j.carbpol.2013.04.023.
[19]SHEN H,GUO T,ZENG G,et al.Purification and characterization of an antitumor polysaccharide from Portulaca oleracea L.[J].Carbohydrate Polymers,2013,93(2):395-400.DOI:10.1016/j.carbpol.2012.11.107.
[20]GONG F,LI F,ZHANG L,et al.Hypoglycemic effects of crude polysaccharide from purslane[J].International Journal of Molecular Sciences,2009,10(3):880.DOI:10.3390/ijms10030880.
[21]牛广财,李世燕,朱丹,等.马齿苋多糖POPII和POPIII的抗肿瘤及提高免疫力作用[J].食品科学,2017,38(3):201-205.DOI:10.7506/spkx1002-6630-201703033.
[22]XIAO J L,YONG H,XUE Q G,et al.Polysaccharides from Portulaca oleracea(purslane)supplementation lowers acute exercise induced oxidative stress in young rats[J].African Journal of Pharmacy and Pharmacology,2011,5(3):381-385.DOI:10.5897/AJPP11.028.
[23]梁彦,吕艳荣.马齿苋多糖的抗衰老作用[J].江苏农业科学,2014,42(4):270-272.DOI:10.3969/j.issn.1002-1302.2014.04.102.
[24]吴光杰,李玉萍,陈美琴,等.马齿苋多糖蛋白含量测定及脱蛋白方法研究[J].时珍国医国药,2015,26(7):1577-1579.
[25]曲晓兰,吴从平,曹明亮.马齿苋多糖脱蛋白工艺的研究[J].泰山医学院学报,2006,27(8):717-718.DOI:10.3969/j.issn.1004-7115.2007.03.019.
[26]蒋春燕,李代,侯红漫,等.响应面法优化榆耳多糖脱蛋白工艺[J].大连工业大学学报,2013,32(6):399-403.
[27]许海燕,徐江,王慧超,等.响应面法优化马齿苋多糖醇沉工艺[J].食品研究与开发,2017,38(8):65-70.DOI:10.3969/j.issn.1005-6521.2017.08.014.
[28]吴光杰,李玉萍,皮小芳,等.响应面法优化马齿苋多糖脱色工艺[J].食品科技,2011,36(10):166-169.
[29]吴光杰,李玉萍,皮小芳,等.马齿苋中水溶性总多糖含量测定方法研究[J].广东农业科学,2010,37(11):174-176.DOI:10.3969/j.issn.1004-874X.2010.11.055.
[30]闫攀凤,樊东升.高效液相色谱法测两头尖多糖的单糖组成[J].山西中医学院学报,2017,18(1):35-37.DOI:10.3969/j.issn.1000-7369.2017.01.013.
[2]WANG P,SUN H,LIU D,et al.Protective effect of a phenolic extract containing indoline amides from Portulaca oleracea against cognitive impairment in senescent mice induced by large dose of D-galactose/NaNO2[J].Journal of Ethnopharmacol,2017,203:252-259.DOI:10.1016/j.jep.2017.03.050.
[3]OH K B,CHANG I M,HWANG K J,et al.Detection of antifungal activity in Portulaca oleracea by a single-cell bioassay system[J].Phytotherapy Research,2000,14(5):329-332.DOI:10.1002/1099-1573(200008)14:5<329::AID-PTR581>3.0.CO;2-5.
[4]DEHGHAN F,SOORI R,GHOLAMI K,et al.Purslane(Portulaca oleracea)seed consumption and aerobic training improves biomarkers associated with atherosclerosis in women with type 2 diabetes(T2D)[J].Sciences Report,2016,6:37819.DOI:10.1038/srep37819.
[5]BAI Y,ZANG X,MA J,et al.Anti-diabetic effect of Portulaca oleracea L.polysaccharideandits mechanism in diabetic rats[J].International Journal of Molecular Sciences,2016,17(8):1201.DOI:10.3390/ijms17081201.
[6]GUO G,YUE L,FAN S,et al.Antioxidant and antiproliferative activities of purslane seed oil[J].Journal of Hypertension:Open Access,2016,5(2):218.DOI:10.4172/2167-1095.1000218.
[7]LI C Y,MENG Y H,YING Z M,et al.Correction to three novel alkaloids from Portulaca oleracea L.and their anti-inflammatory effects[J].Journal of Agricultural&Food Chemical,2017,65(4):993-994.DOI:10.1021/acs.jafc.6b05659.
[8]LEE A S,KIM J S,LEE Y J,et al.Anti-TNF-αactivity of Portulaca oleracea in vascular endothelial cells[J].International Journal of Molecular Sciences,2012,13(5):5628.DOI:10.3390/ijms13055628.
[9]ASKARI V R,REZAEE S A,ABNOUS K,et al.The influence of hydro-ethanolic extract of Portulaca oleracea L.on Th1/Th2 balance in isolated human lymphocytes[J].Journal of Ethnopharmacology,2016,194:1112-1121.DOI:10.1016/j.jep.2016.10.082.
[10]ZHAO R,ZHANG T,MA B,et al.Antitumor activity of Portulaca oleracea L.polysaccharide on HeLa cells through inducing TLR4/NF-κB bignaling[J]. Nutrition and Cancer, 2017, 69(1): 131-139. DOI:10.1080/01635581.2017.1248294.
[11]翁前锋,袁凯龙,张宏颖,等.胶束电动毛细管色谱安培检测中药马齿苋中多巴胺和去甲肾上腺素[J].色谱,2005,23(1):18-21.DOI:10.3321/j.issn:1000-8713.2005.01.004.
[12] YIN A W, GAO P F, DANG L M. Anti-aging effects of flavonoids from Portulaca oleracea L.[J].Natural Product Research and Development,2017,29(6):988-993.DOI:10.16333/j.1001-6880.2017.6.015.
[13]汪荔,王征,张娇,等.传统工艺与膜分离技术联合对马齿苋多糖的提取分离与抗氧化活性研究[J].中草药,2016,47(10):1676-1681.DOI:10.7501/j.issn.0253-2670.2016.10.009.
[14]郑智音,贾晓斌,舒娈,等.鲜马齿苋多糖、生物碱和多酚类组分的制备及其降血糖活性研究[J].中草药,2014,45(18):2673-2677.DOI:10.7501/j.issn.0253-2670.2014.18.019.
[15]ZHAO C,YING Z,TAO X,et al.A new lactam alkaloid from Portulaca oleracea L.and its cytotoxity[J].Natural Product Research,2017:1-6.DOI:10.1080/14786419.2017.1385022.
[16]LI Y P,YAO L H,WU G J,et al.Antioxidant activities of novel smallmolecule polysaccharide fractions purified from Portulaca oleracea L.[J].Food Science and Biotechnology,2014,23(6):2045-2052.
[17]ZHAO R,ZHANG T,ZHAO H,et al.Effects of Portulaca oleracea L.polysaccharides on phenotypic and functional maturation of murine bone marrow derived dendritic cells[J].Nutrition&Cancer,2015,67(6):987.DOI:10.1080/01635581.2015.1060352.
[18]ZHAO R,GAO X,CAI Y,et al.Antitumor activity of Portulaca oleracea L.polysaccharides against cervical carcinoma in vitro and in vivo[J].Carbohydrate Polymers,2013,96(2):376-383.DOI:10.1016/j.carbpol.2013.04.023.
[19]SHEN H,GUO T,ZENG G,et al.Purification and characterization of an antitumor polysaccharide from Portulaca oleracea L.[J].Carbohydrate Polymers,2013,93(2):395-400.DOI:10.1016/j.carbpol.2012.11.107.
[20]GONG F,LI F,ZHANG L,et al.Hypoglycemic effects of crude polysaccharide from purslane[J].International Journal of Molecular Sciences,2009,10(3):880.DOI:10.3390/ijms10030880.
[21]牛广财,李世燕,朱丹,等.马齿苋多糖POPII和POPIII的抗肿瘤及提高免疫力作用[J].食品科学,2017,38(3):201-205.DOI:10.7506/spkx1002-6630-201703033.
[22]XIAO J L,YONG H,XUE Q G,et al.Polysaccharides from Portulaca oleracea(purslane)supplementation lowers acute exercise induced oxidative stress in young rats[J].African Journal of Pharmacy and Pharmacology,2011,5(3):381-385.DOI:10.5897/AJPP11.028.
[23]梁彦,吕艳荣.马齿苋多糖的抗衰老作用[J].江苏农业科学,2014,42(4):270-272.DOI:10.3969/j.issn.1002-1302.2014.04.102.
[24]吴光杰,李玉萍,陈美琴,等.马齿苋多糖蛋白含量测定及脱蛋白方法研究[J].时珍国医国药,2015,26(7):1577-1579.
[25]曲晓兰,吴从平,曹明亮.马齿苋多糖脱蛋白工艺的研究[J].泰山医学院学报,2006,27(8):717-718.DOI:10.3969/j.issn.1004-7115.2007.03.019.
[26]蒋春燕,李代,侯红漫,等.响应面法优化榆耳多糖脱蛋白工艺[J].大连工业大学学报,2013,32(6):399-403.
[27]许海燕,徐江,王慧超,等.响应面法优化马齿苋多糖醇沉工艺[J].食品研究与开发,2017,38(8):65-70.DOI:10.3969/j.issn.1005-6521.2017.08.014.
[28]吴光杰,李玉萍,皮小芳,等.响应面法优化马齿苋多糖脱色工艺[J].食品科技,2011,36(10):166-169.
[29]吴光杰,李玉萍,皮小芳,等.马齿苋中水溶性总多糖含量测定方法研究[J].广东农业科学,2010,37(11):174-176.DOI:10.3969/j.issn.1004-874X.2010.11.055.
[30]闫攀凤,樊东升.高效液相色谱法测两头尖多糖的单糖组成[J].山西中医学院学报,2017,18(1):35-37.DOI:10.3969/j.issn.1000-7369.2017.01.013.