西兰花副产物中萝卜硫素提取、纯化及鉴定
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摘要
以西兰花副产物为原料,研究萝卜硫素酶解浸提工艺,并通过单因素试验和响应面试验优化最佳工艺条件。在最佳条件的基础上进行放大实验,通过半制备高效液相色谱纯化和富集萝卜硫素,并应用超高效液相色谱-质谱联用技术对纯化物进行鉴定。结果表明:萝卜硫素最佳提取条件为酶解pH 5.0、酶解温度35℃、酶解时间67 min;在此条件下,萝卜硫素提取量为151.39μg/g,与预测值(156.33μg/g)无显著差异;纯化和富集后,得到28.62 mg/200 g萝卜硫素,回收率为94.52%,纯度为80.74%。根据萝卜硫素的二级质谱与碎片离子信息,进一步确定该纯化物为萝卜硫素。
The enzymatic extraction of sulforaphane from broccoli stems and leaves was optimized by one-factor-at-a-time method and response surface methodology. Scale-up experiments were carried out under optimized conditions, and the crude extract was puri?ed by preparative high performance liquid chromatography(HPLC) and identi?ed by ultra-performance liquid chromatography-mass spectrometry(UPLC-MS). The results showed that the optimal extrac tion conditions were determined as follows: pH 5.0, temperature 35 ℃ and time 67 min. Under these conditions, the maximum yield of sulforaphane of 151.39 μg/g(on a dry matter basis) was obtained, which agreed with the predicted value(156.33 μg/g). The yield of puri?ed sulforaphane was 28.62 mg from 200 g of broccoli powder, with a 94.52% recovery and 80.74% purity. The tandem mass spectrum and fragment ion information con?rmed the substance as sulforaphane.
The enzymatic extraction of sulforaphane from broccoli stems and leaves was optimized by one-factor-at-a-time method and response surface methodology. Scale-up experiments were carried out under optimized conditions, and the crude extract was puri?ed by preparative high performance liquid chromatography(HPLC) and identi?ed by ultra-performance liquid chromatography-mass spectrometry(UPLC-MS). The results showed that the optimal extrac tion conditions were determined as follows: pH 5.0, temperature 35 ℃ and time 67 min. Under these conditions, the maximum yield of sulforaphane of 151.39 μg/g(on a dry matter basis) was obtained, which agreed with the predicted value(156.33 μg/g). The yield of puri?ed sulforaphane was 28.62 mg from 200 g of broccoli powder, with a 94.52% recovery and 80.74% purity. The tandem mass spectrum and fragment ion information con?rmed the substance as sulforaphane.
引文
[1]吴广辉,毕韬韬.西兰花营养价值及深加工研究进展[J].农产品加工,2015(11):61-63.DOI:10.16693/j.cnki.1671-9646(X).2015.11.019.
[2]TSE G,ESLICK G D.Cruciferous vegetables and risk of colorectal neoplasms:a systematic review and meta-analysis[J].Nutrition&Cancer,2014,66(1):128-139.DOI:10.1080/01635581.2014.852686.
[3]DINKOVA-KOSTOVA A T,KOSTOV R V.Glucosinolates and isothiocyanates in health and disease[J].Trends in Molecular Medicine,2012,18(6):337-347.DOI:10.1016/j.molmed.2012.04.003.
[4]吴元锋.萝卜硫素制备、降解动力学及其诱导细胞凋亡研究[D].杭州:浙江大学,2014.
[5]WHITESELL J K,WONG M S.Asymmetric synthesis of chiral sulfinate esters and sulfoxides synthesis of sulforaphane[J].Cheminform,1994,25(26):597-601.DOI:10.1021/jo00082a016.
[6]THAI K M,PHAM N T A,DO V T N,et al.A new and effective approach to the synthesis of sulforaphane[J].Letters in Organic Chemistry,2016,13(1):7-10.DOI:10.2174/1570178611666151015214407.
[7]吴元锋,徐维亮,申雨珂,等.萝卜硫素制备及纯化工艺研究进展[J].食品工业科技,2016,37(19):381-386.DOI:10.13386/j.issn1002-0306.2016.19.066.
[8]谢述琼,何珺,舒华.西兰花种子中萝卜硫素酶解浸提工艺研究[J].广州化工,2016,44(8):73-75.DOI:10.3969/j.issn.1001-9677.2016.08.027.
[9]苏光耀,沈莲清,王向阳,等.西兰花籽中硫代葡萄糖苷酶解条件的研究[J].中国粮油学报,2008,23(2):178-182.
[10]林毅,张金娟,李晓露,等.西兰花种子中萝卜硫素的提取工艺研究[J].化学与生物工程,2014,31(12):48-50.DOI:10.3969/j.issn.1672-5425.2014.12.014.
[11]李扬.西兰花芽苗菜中莱菔硫烷的分离纯化及抗癌活性的测定[D].大庆:黑龙江八一农垦大学,2009.
[12]GUO L,YANG R,WANG Z,et al.Glucoraphanin,sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs[J].Journal of Functional Foods,2014,9(1):70-77.DOI:10.1016/j.jff.2014.04.015.
[13]MATUSHESKI N V,SWARUP R,JUVIK J A,et al.Epithiospecifier protein from broccoli(Brassica oleracea L.ssp.italica)inhibits formation of the anticancer agent sulforaphane[J].Journal of Agricultural&Food Chemistry,2006,54(6):2069-2076.DOI:10.1021/jf0525277.
[14]胡翠珍,李胜,马绍英,等.响应面优化西兰花中萝卜硫素复合提取工艺[J].食品工业科技,2016,37(4):271-277.DOI:10.13386/j.issn1002-0306.2016.04.046.
[15]LIANG H,YUAN Q P,DONG H R,et al.Determination of sulforaphane in broccoli and cabbage by high-performance liquid chromatography[J].Journal of Food Composition&Analysis,2006,19(5):473-476.DOI:10.1016/j.jfca.2005.11.005.
[16]ARES A M,VALVERDE S,BERNAL J L,et al.Development and validation of a LC-MS/MS method to determine sulforaphane in honey[J].Food Chemistry,2015,181:263-269.DOI:10.1016/j.foodchem.2015.02.085.
[17]MATUSHESKI N V,JUVIK J A,JEFFERY E H.Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli[J].Phytochemistry,2004,65(9):1273-1281.DOI:10.1016/j.phytochem.2004.04.013.
[18]MAHESHWARI P N,STANLEY D W,VAN DE VOORT F R,et al.The heat stability of allyl glucosinolate(sinigrin)in aqueous and model systems[J].Canadian Institute of Food Science&Technology Journal,1980,13(1):28-32.DOI:10.1016/S0315-5463(80)73298-4.
[19]PEREIRA F M,ROSA E,FAHEY J W,et al.Influence of temperature and ontogeny on the levels of glucosinolates in broccoli(Brassica oleracea var.italica)sprouts and their effect on the induction of mammalian phase 2 enzymes[J].Journal of Agricultural&Food Chemistry,2002,50(21):6239-6244.DOI:10.1021/jf020309x.
[20]朱磊.十字花科植物种子黑芥子酶酶学性质研究[D].北京:北京化工大学,2011.
[21]黄肖钦,马朱莉,李娜,等.西兰花种子酶解生成萝卜硫素的条件研究[J].上海工程技术大学学报,2010,24(3):277-280.DOI:10.3969/j.issn.1009-444X.2010.03.021.
[22]MAHN A,MARTIN C,REYES A,et al.Evolution of sulforaphane content in sulforaphane-enriched broccoli during tray drying[J].Journal of Food Engineering,2016,186:27-33.DOI:10.1016/j.jfoodeng.2016.04.007.
[23]TANONGKANKIT Y,CHIEWCHAN N,DEVAHASTIN S.Evolution of anticarcinogenic substance in dietary fibre powder from cabbage outer leaves during drying[J].Food Chemistry,2011,127(1):67-73.DOI:10.1016/j.foodchem.2010.12.088.
[24]ARES A M,BERNAL J,MARTíN M T,et al.Optimized formation,extraction,and determination of sulforaphane in broccoli by liquid chromatography with diode array detection[J].Food Analytical Methods,2014,7(3):730-740.DOI:10.1007/s12161-013-9766-6.
[25]KOKOTOU M G,REVELOU P K,PAPPAS C,et al.High resolution mass spectrometry studies of sulforaphane and indole-3-carbinol in broccoli[J].Food Chemistry,2017,237:566-573.DOI:10.1016/j.foodchem.2017.05.139.
[26]MATUSHESKI N V,WALLIG M A,JUVIK J A,et al.Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea[J].Journal of Agricultural and Food Chemistry,2001,49(4):1867-1872.DOI:10.1021/jf0013860.
[27]王兆玲,冯尚彩,况鹏群.响应面法优化萝卜芽苗中还原型萝卜硫素的制备工艺研究[J].药学研究,2018,37(8):460-465.DOI:10.13684/j.cnki.spkj.2017.12.037.
[28]代梅,高林,吴继红.西兰花中硫代葡萄糖苷的研究进展[J].食品研究与开发,2017,38(6):212-217.DOI:10.3969/j.issn.1005-6521.2017.06.048.
[29]苏光耀.西兰花种子中硫苷酶解产物萝卜硫素的提取分离与结构鉴定[D].杭州:浙江工商大学,2007.
[30]刘锡建,王明强,徐一麟,等.SP850树脂对萝卜硫素吸附特性的研究[J].离子交换与吸附,2013,29(6):561-568.DOI:10.16026/j.cnki.iea.2013.06.004.
[31]LIANG H,LI C,YUAN Q,et al.Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal[J].Journal of Agricultural and Food Chemistry,2008,56(17):7746-7749.DOI:10.1021/jf801318v.
[2]TSE G,ESLICK G D.Cruciferous vegetables and risk of colorectal neoplasms:a systematic review and meta-analysis[J].Nutrition&Cancer,2014,66(1):128-139.DOI:10.1080/01635581.2014.852686.
[3]DINKOVA-KOSTOVA A T,KOSTOV R V.Glucosinolates and isothiocyanates in health and disease[J].Trends in Molecular Medicine,2012,18(6):337-347.DOI:10.1016/j.molmed.2012.04.003.
[4]吴元锋.萝卜硫素制备、降解动力学及其诱导细胞凋亡研究[D].杭州:浙江大学,2014.
[5]WHITESELL J K,WONG M S.Asymmetric synthesis of chiral sulfinate esters and sulfoxides synthesis of sulforaphane[J].Cheminform,1994,25(26):597-601.DOI:10.1021/jo00082a016.
[6]THAI K M,PHAM N T A,DO V T N,et al.A new and effective approach to the synthesis of sulforaphane[J].Letters in Organic Chemistry,2016,13(1):7-10.DOI:10.2174/1570178611666151015214407.
[7]吴元锋,徐维亮,申雨珂,等.萝卜硫素制备及纯化工艺研究进展[J].食品工业科技,2016,37(19):381-386.DOI:10.13386/j.issn1002-0306.2016.19.066.
[8]谢述琼,何珺,舒华.西兰花种子中萝卜硫素酶解浸提工艺研究[J].广州化工,2016,44(8):73-75.DOI:10.3969/j.issn.1001-9677.2016.08.027.
[9]苏光耀,沈莲清,王向阳,等.西兰花籽中硫代葡萄糖苷酶解条件的研究[J].中国粮油学报,2008,23(2):178-182.
[10]林毅,张金娟,李晓露,等.西兰花种子中萝卜硫素的提取工艺研究[J].化学与生物工程,2014,31(12):48-50.DOI:10.3969/j.issn.1672-5425.2014.12.014.
[11]李扬.西兰花芽苗菜中莱菔硫烷的分离纯化及抗癌活性的测定[D].大庆:黑龙江八一农垦大学,2009.
[12]GUO L,YANG R,WANG Z,et al.Glucoraphanin,sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs[J].Journal of Functional Foods,2014,9(1):70-77.DOI:10.1016/j.jff.2014.04.015.
[13]MATUSHESKI N V,SWARUP R,JUVIK J A,et al.Epithiospecifier protein from broccoli(Brassica oleracea L.ssp.italica)inhibits formation of the anticancer agent sulforaphane[J].Journal of Agricultural&Food Chemistry,2006,54(6):2069-2076.DOI:10.1021/jf0525277.
[14]胡翠珍,李胜,马绍英,等.响应面优化西兰花中萝卜硫素复合提取工艺[J].食品工业科技,2016,37(4):271-277.DOI:10.13386/j.issn1002-0306.2016.04.046.
[15]LIANG H,YUAN Q P,DONG H R,et al.Determination of sulforaphane in broccoli and cabbage by high-performance liquid chromatography[J].Journal of Food Composition&Analysis,2006,19(5):473-476.DOI:10.1016/j.jfca.2005.11.005.
[16]ARES A M,VALVERDE S,BERNAL J L,et al.Development and validation of a LC-MS/MS method to determine sulforaphane in honey[J].Food Chemistry,2015,181:263-269.DOI:10.1016/j.foodchem.2015.02.085.
[17]MATUSHESKI N V,JUVIK J A,JEFFERY E H.Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli[J].Phytochemistry,2004,65(9):1273-1281.DOI:10.1016/j.phytochem.2004.04.013.
[18]MAHESHWARI P N,STANLEY D W,VAN DE VOORT F R,et al.The heat stability of allyl glucosinolate(sinigrin)in aqueous and model systems[J].Canadian Institute of Food Science&Technology Journal,1980,13(1):28-32.DOI:10.1016/S0315-5463(80)73298-4.
[19]PEREIRA F M,ROSA E,FAHEY J W,et al.Influence of temperature and ontogeny on the levels of glucosinolates in broccoli(Brassica oleracea var.italica)sprouts and their effect on the induction of mammalian phase 2 enzymes[J].Journal of Agricultural&Food Chemistry,2002,50(21):6239-6244.DOI:10.1021/jf020309x.
[20]朱磊.十字花科植物种子黑芥子酶酶学性质研究[D].北京:北京化工大学,2011.
[21]黄肖钦,马朱莉,李娜,等.西兰花种子酶解生成萝卜硫素的条件研究[J].上海工程技术大学学报,2010,24(3):277-280.DOI:10.3969/j.issn.1009-444X.2010.03.021.
[22]MAHN A,MARTIN C,REYES A,et al.Evolution of sulforaphane content in sulforaphane-enriched broccoli during tray drying[J].Journal of Food Engineering,2016,186:27-33.DOI:10.1016/j.jfoodeng.2016.04.007.
[23]TANONGKANKIT Y,CHIEWCHAN N,DEVAHASTIN S.Evolution of anticarcinogenic substance in dietary fibre powder from cabbage outer leaves during drying[J].Food Chemistry,2011,127(1):67-73.DOI:10.1016/j.foodchem.2010.12.088.
[24]ARES A M,BERNAL J,MARTíN M T,et al.Optimized formation,extraction,and determination of sulforaphane in broccoli by liquid chromatography with diode array detection[J].Food Analytical Methods,2014,7(3):730-740.DOI:10.1007/s12161-013-9766-6.
[25]KOKOTOU M G,REVELOU P K,PAPPAS C,et al.High resolution mass spectrometry studies of sulforaphane and indole-3-carbinol in broccoli[J].Food Chemistry,2017,237:566-573.DOI:10.1016/j.foodchem.2017.05.139.
[26]MATUSHESKI N V,WALLIG M A,JUVIK J A,et al.Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea[J].Journal of Agricultural and Food Chemistry,2001,49(4):1867-1872.DOI:10.1021/jf0013860.
[27]王兆玲,冯尚彩,况鹏群.响应面法优化萝卜芽苗中还原型萝卜硫素的制备工艺研究[J].药学研究,2018,37(8):460-465.DOI:10.13684/j.cnki.spkj.2017.12.037.
[28]代梅,高林,吴继红.西兰花中硫代葡萄糖苷的研究进展[J].食品研究与开发,2017,38(6):212-217.DOI:10.3969/j.issn.1005-6521.2017.06.048.
[29]苏光耀.西兰花种子中硫苷酶解产物萝卜硫素的提取分离与结构鉴定[D].杭州:浙江工商大学,2007.
[30]刘锡建,王明强,徐一麟,等.SP850树脂对萝卜硫素吸附特性的研究[J].离子交换与吸附,2013,29(6):561-568.DOI:10.16026/j.cnki.iea.2013.06.004.
[31]LIANG H,LI C,YUAN Q,et al.Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal[J].Journal of Agricultural and Food Chemistry,2008,56(17):7746-7749.DOI:10.1021/jf801318v.