普通小麦类胡萝卜素组分的超高效液相色谱分离方法
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摘要
类胡萝卜素是影响小麦面粉及面制品颜色和营养品质的重要因素。本研究以中麦175(软质麦)和中优206(硬质麦)的面粉为试验材料,旨在优化小麦类胡萝卜素提取方法 ,建立超高效液相色谱(ultra performance liquid chromatography,UPLC)测定其组分的技术体系。研究表明,用含0.1%BHT的正己烷∶丙酮(80∶20,v/v)混合液作为提取液,结合恒温振荡法(300转min–1,35℃,振荡1 h)的提取效果最好。以YMC C30胡萝卜素分析专用色谱柱(高100 mm,直径4.6 mm,粒径3μm)洗脱,乙腈∶甲醇∶水∶三乙胺(81∶14∶5∶0.05,v/v/v/v)为流动相A,甲醇∶乙酸乙酯∶三乙胺(68∶32∶0.05,v/v/v)为流动相B,流速0.4 m L min~(–1),柱温35℃,分离时间25 min;以二极管阵列检测器(photodiode array,PDA)在450 nm波长下,能有效检测叶黄素、玉米黄质和β-胡萝卜素3种组分。该技术体系可作为普通小麦类胡萝卜素组分及营养品质研究的有效方法。
Carotenoids is an important criterion in the assessment of color and nutritional qualities of end-use products in common wheat. In this study, grain powders of Zhongmai 175(soft wheat) and Zhongyou 206(hard wheat) were used as samples to develop an optimal procedure for extraction and separation of carotenoids compositions extracted from flours using ultra performance liquid chromatography(UPLC). The most effective extraction of carotenoids extraction was obtained using the solvent system of N-hexane; acetone of 80:20(v/v, 0.1% BHT w/v) under the oscillation condition of 300 r min~(–1), 35oC, and 1 h. The separation was conducted using YMC C30 Carotenoid column(100.0 mm × 4.6 mm, 3 μm) with photodiode array(PDA) detector, and the column was thermostated at 35°C. Under a gradient system consisting of acetonitrile:methanol:water:triethylamine(81:14:5:0.05, v/v/v/v)(A) and methanol: ethylacetate:triethylamine(68:32:0.05, v/v/v)(B) at a constant flow rate of 0.4 m L min~(–1), the carotenoids of common wheat flour samples can be well separated in less than 25 min. Carotenoids were detected at 450 nm. All the present results provided useful information for carotenoids compositions and quality improvement.
Carotenoids is an important criterion in the assessment of color and nutritional qualities of end-use products in common wheat. In this study, grain powders of Zhongmai 175(soft wheat) and Zhongyou 206(hard wheat) were used as samples to develop an optimal procedure for extraction and separation of carotenoids compositions extracted from flours using ultra performance liquid chromatography(UPLC). The most effective extraction of carotenoids extraction was obtained using the solvent system of N-hexane; acetone of 80:20(v/v, 0.1% BHT w/v) under the oscillation condition of 300 r min~(–1), 35oC, and 1 h. The separation was conducted using YMC C30 Carotenoid column(100.0 mm × 4.6 mm, 3 μm) with photodiode array(PDA) detector, and the column was thermostated at 35°C. Under a gradient system consisting of acetonitrile:methanol:water:triethylamine(81:14:5:0.05, v/v/v/v)(A) and methanol: ethylacetate:triethylamine(68:32:0.05, v/v/v)(B) at a constant flow rate of 0.4 m L min~(–1), the carotenoids of common wheat flour samples can be well separated in less than 25 min. Carotenoids were detected at 450 nm. All the present results provided useful information for carotenoids compositions and quality improvement.
引文
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[16]Gupta P K,Mir R R,Mohan A,Kumar J.Wheat genomics:present status and future prospects.Int J Plant Genomics,2008,DOI:10.1155/2008/896451
[17]Jin H,Zhang Y,Li G Y,Mu P Y,Fan Z R,Xia X C,He Z H.Effects of allelic variation of HMW-GS and LMW-GS on mixograph properties and Chinese noodle and steamed bread qualities in a set of Aroona near-isogenic wheat lines.J Cereal Sci,2013,57:146-152
[18]Mares D J,Campbell A.Mapping components of flour and noodle colour in Australian wheat.Aust J Agric Res,2001,52:1297-1310
[19]何中虎,晏月明,庄巧生,张艳,夏先春,张勇,王德森,夏兰芹,胡英考,蔡民华,陈新民,阎俊,周阳.中国小麦品种品质评价体系建立与分子改良技术研究.中国农业科学,2006,39:1091-1101He Z H,Yan Y M,Zhuang Q S,Zhang Y,Xia X C,Zhang Y,Wang D S,Xia L Q,Hu Y K,Cai M H,Chen X M,Yan J,Zhou Y.Establishment of quality evaluation system and utilization of molecular methods for the improvement of Chinese wheat quality.Sci Agric Sin,2006,39:1091-1101(in Chinese with English abstract)
[20]Symons S J,Dexter J E.Computer analysis of fluorescence for the measurement of flour refinement as determined by flour ash content,flour grade colour,and tristimulus colour measurements.Cereal Chem,1991,68:454-460
[21]He X Y,Zhang Y L,He Z H,Wu Y P,Xiao Y G,Ma C X,Xia XC.Characterization of phytoene synthase 1 gene(Psy1)located on common wheat chromosome 7A and development of a functional marker.Theor Appl Genet,2008,116:213-221
[22]Dong C H,Ma Z Y,Xia X C,Zhang L P,He Z H.Allelic variation at the Ta Zds-A1 locus on wheat chromosome 2A and development of a functional marker in common wheat.J Integr Agric2012,11:1067-1074
[23]Abdel-Aal E S M,Young J C,Wood P J,Rabalski I,Hucl P,Fregeau-Reid J.Einkorn:A potential candidate for developing high lutein wheat.Cereal Chem,2002,79:455-457
[24]Adom K K,Sorrells M E,Liu R H.Phytochemical profiles and antioxidant activity of wheat varieties.J Agric Food Chem,2003,51:7825-7834
[25]Hung P V,Hatcher D W.Ultra-performance liquid chromatography(UPLC)quantification of carotenoids in durum wheat:Influence of genotype and environment in relation to the colour of yellow alkaline noodles(YAN).Food Chem,2011,125:1510-1516
[26]周毅,付志远,李青,徐淑兔,Chander S,杨小红,李建生,严建兵.高油和普通玉米自交系类胡萝卜素和生育酚含量的比较.作物学报,2009,35:2073-2084Zhou Y,Fu Z Y,Li Q,Xu S T,Chander S,Yang X H,Li J S,Yan J B.Comparative analysis of carotenoid and tocopherol compositions in high-oil and normal maize(Zea mays L.)inbreds.Acta Agron Sin,2009,35:2073-2084(in Chinese with English abstract)
[27]DigesùA,Platani C,Cattivelli L,Mangini G,Blanco A.Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats.J Cereal Sci,2009,50:210-218
[28]Hidalgo A,Brandolini A,Pompei C,Piscozzi R.Carotenoids and tocols of einkorn wheat(Triticum monococcum ssp.monococcum L.).J Cereal Sci,2006,44:182-193
[29]Ndolo V U,Beta T.Distribution of carotenoids in endosperm,germ,and aleurone fractions of cereal grain kernels.Food Chem,2013,139:663-671
[30]Burkhardt S,B?hm V.Development of a new method for the complete extraction of carotenoids from cereals with special reference to durum wheat(Triticum durum Desf.).J Agric Food Chem,2007,55:8295-8301
[31]Oliver J,Palou A,Pons A.Semi-quantification of carotenoids by high-performance liquid chromatography:saponification-induced losses in fatty foods.J Chromatogr A,1998,829:393-399
[32]Konopka I,Czaplicki S,Rotkiewicz D.Differences in content and composition of free lipids and carotenoids in flour of spring and winter wheat cultivated in Poland.Food Chem,2006,95:290-300
[33]Abdel-Aal El-SM,Young J C,Rabalski I,Hucl P,Fregeau-Reid J.Identification and quantification of seed carotenoids in selected wheat species.J Agric Food Chem,2007,55:787-794
[34]Mashaba C S,Barros E.Screening South African potato,tomato,and wheat cultivars for five carotenoids.Saf J Sci,2011,107:34-39
[35]Lookhart G L,Bean S R,Bietz J A.Reversed-phase high performance liquid chromatography in grain applications.Cereal Food World,2002,48:9-16
[36]Kean E G,Hamaker B R,Ferruzzi M G.Carotenoid bioaccessibility from whole grain and degermed maize meal products.J Agric Food Chem,2008,56:9918-9926
[2]Ribaya-Mercado J D,Blumberg J B.Lutein and zeaxanthin and their potential roles in disease prevention.J Am Coll Nut,2004,23:567S-587S
[3]Nishino H,Murakoshi M,Tokuda H,Yoshiko S.Cancer prevention by carotenoids.Arch Biochem Biophys,2009,483:165-168
[4]Harjes C E,Rocheford T R,Bai L,Brutnell T P,Kandianis C B,Sowinski S G,Stapleton A E,Vallabhaneni R,Williams M,Wurtzel E T,Yan J B,Buckler E S.Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification.Science,2008,319:330-333
[5]Cazzonelli C I,Pogson B J.Source to sink:regulation of carotenoid biosynthesis in plants.Trends Plant Sci,2010,15:266-274
[6]Howitt C A,Pogson B J.Carotenoid accumulation and function in seeds and non-green tissues.Plant Cell Environ,2006,29:435-445
[7]Matthews P D,Luo R B,Wurtzel E T.Maize phytoene desaturase and zeta-carotene desaturase catalyse a poly-Z desaturation pathway:implications for genetic engineering of carotenoid content among cereal crops.J Exp Bot,2003,54:2215-2230
[8]Singh M,Lewis P E,Hardeman K,Bai L,Rose J K C,Mazourek M,Chomet P,Brutnell T P.Activator mutagenesis of the pink scutellum1/viviparous7 locus of maize.Plant Cell,2003,15:874-884
[9]Gallagher C E,Matthews P D,Li F Q,Wurtzel E T.Gene duplication in the carotenoid biosynthetic pathway preceded evolution of the grasses.Plant Physiol,2004,135:1776-1783
[10]Li F,Murillo C,Wurtzel E T.Maize Y9 encodes a product essential for 15-cis-ζ-carotene isomerization.Plant Physiol,2007,144:1181-1189
[11]Wong J C,Lambert R J,Wurtzel E T,Rocheford T R.QTL and candidate genes phytoene synthase and zeta-carotene desaturase associated with the accumulation of carotenoids in maize.Theor Appl Genet,2004,108:349-359
[12]Chander S,Guo Y,Yang X,Zhang J,Lu X,Yan J,Song T,Rocheford T,Li J.Using molecular markers to identify two major loci controlling carotenoid contents in maize grain.Theor Appl Genet,2008,116:223-233
[13]Ye X D,Al-Babili S,Kloti A,Zhang J,Lucca P,Beyer P,Potrykus I.Engineering the provitamin A(beta-carotene)biosynthetic pathway into(carotenoid-free)rice endosperm.Science,2000,287:303-305
[14]Beyer P,Al-Babili S,Ye X D,Lucca P,Schaub P,Welsch R,Potrykus I.Golden rice:Introducing the beta-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency.J Nutr,2002,132:506S-510S
[15]Paine J A,Shipton C A,Chaggar S,Howells R M,Kennedy M J,Vernon G,Wright S Y,Hinchliffe E,Adams J L,Silverstone A L,Drake R.Improving the nutritional value of Golden Rice through increased pro-vitamin A content.Nat Biotechnol,2005,23:482-487
[16]Gupta P K,Mir R R,Mohan A,Kumar J.Wheat genomics:present status and future prospects.Int J Plant Genomics,2008,DOI:10.1155/2008/896451
[17]Jin H,Zhang Y,Li G Y,Mu P Y,Fan Z R,Xia X C,He Z H.Effects of allelic variation of HMW-GS and LMW-GS on mixograph properties and Chinese noodle and steamed bread qualities in a set of Aroona near-isogenic wheat lines.J Cereal Sci,2013,57:146-152
[18]Mares D J,Campbell A.Mapping components of flour and noodle colour in Australian wheat.Aust J Agric Res,2001,52:1297-1310
[19]何中虎,晏月明,庄巧生,张艳,夏先春,张勇,王德森,夏兰芹,胡英考,蔡民华,陈新民,阎俊,周阳.中国小麦品种品质评价体系建立与分子改良技术研究.中国农业科学,2006,39:1091-1101He Z H,Yan Y M,Zhuang Q S,Zhang Y,Xia X C,Zhang Y,Wang D S,Xia L Q,Hu Y K,Cai M H,Chen X M,Yan J,Zhou Y.Establishment of quality evaluation system and utilization of molecular methods for the improvement of Chinese wheat quality.Sci Agric Sin,2006,39:1091-1101(in Chinese with English abstract)
[20]Symons S J,Dexter J E.Computer analysis of fluorescence for the measurement of flour refinement as determined by flour ash content,flour grade colour,and tristimulus colour measurements.Cereal Chem,1991,68:454-460
[21]He X Y,Zhang Y L,He Z H,Wu Y P,Xiao Y G,Ma C X,Xia XC.Characterization of phytoene synthase 1 gene(Psy1)located on common wheat chromosome 7A and development of a functional marker.Theor Appl Genet,2008,116:213-221
[22]Dong C H,Ma Z Y,Xia X C,Zhang L P,He Z H.Allelic variation at the Ta Zds-A1 locus on wheat chromosome 2A and development of a functional marker in common wheat.J Integr Agric2012,11:1067-1074
[23]Abdel-Aal E S M,Young J C,Wood P J,Rabalski I,Hucl P,Fregeau-Reid J.Einkorn:A potential candidate for developing high lutein wheat.Cereal Chem,2002,79:455-457
[24]Adom K K,Sorrells M E,Liu R H.Phytochemical profiles and antioxidant activity of wheat varieties.J Agric Food Chem,2003,51:7825-7834
[25]Hung P V,Hatcher D W.Ultra-performance liquid chromatography(UPLC)quantification of carotenoids in durum wheat:Influence of genotype and environment in relation to the colour of yellow alkaline noodles(YAN).Food Chem,2011,125:1510-1516
[26]周毅,付志远,李青,徐淑兔,Chander S,杨小红,李建生,严建兵.高油和普通玉米自交系类胡萝卜素和生育酚含量的比较.作物学报,2009,35:2073-2084Zhou Y,Fu Z Y,Li Q,Xu S T,Chander S,Yang X H,Li J S,Yan J B.Comparative analysis of carotenoid and tocopherol compositions in high-oil and normal maize(Zea mays L.)inbreds.Acta Agron Sin,2009,35:2073-2084(in Chinese with English abstract)
[27]DigesùA,Platani C,Cattivelli L,Mangini G,Blanco A.Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats.J Cereal Sci,2009,50:210-218
[28]Hidalgo A,Brandolini A,Pompei C,Piscozzi R.Carotenoids and tocols of einkorn wheat(Triticum monococcum ssp.monococcum L.).J Cereal Sci,2006,44:182-193
[29]Ndolo V U,Beta T.Distribution of carotenoids in endosperm,germ,and aleurone fractions of cereal grain kernels.Food Chem,2013,139:663-671
[30]Burkhardt S,B?hm V.Development of a new method for the complete extraction of carotenoids from cereals with special reference to durum wheat(Triticum durum Desf.).J Agric Food Chem,2007,55:8295-8301
[31]Oliver J,Palou A,Pons A.Semi-quantification of carotenoids by high-performance liquid chromatography:saponification-induced losses in fatty foods.J Chromatogr A,1998,829:393-399
[32]Konopka I,Czaplicki S,Rotkiewicz D.Differences in content and composition of free lipids and carotenoids in flour of spring and winter wheat cultivated in Poland.Food Chem,2006,95:290-300
[33]Abdel-Aal El-SM,Young J C,Rabalski I,Hucl P,Fregeau-Reid J.Identification and quantification of seed carotenoids in selected wheat species.J Agric Food Chem,2007,55:787-794
[34]Mashaba C S,Barros E.Screening South African potato,tomato,and wheat cultivars for five carotenoids.Saf J Sci,2011,107:34-39
[35]Lookhart G L,Bean S R,Bietz J A.Reversed-phase high performance liquid chromatography in grain applications.Cereal Food World,2002,48:9-16
[36]Kean E G,Hamaker B R,Ferruzzi M G.Carotenoid bioaccessibility from whole grain and degermed maize meal products.J Agric Food Chem,2008,56:9918-9926