HPLC法测定谷子籽粒叶酸含量及种质资源评价
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摘要
谷子籽粒中富含叶酸,作为一种功能性食品深受北方人民的喜爱,而作物籽粒,如小米叶酸测定方法及含量分析存在较大差异,为小米品质鉴定及高叶酸谷子种质筛选造成了一定困难。本研究通过"三酶法"提取小米中的叶酸,建立了一种基于高效液相色谱技术(HPLC)高效化、规模化且稳定适用于测定谷子籽粒叶酸含量的方法,对2016年和2017年种植的45个谷子种质进行叶酸含量测定和分类评价。结果表明:叶酸标准品梯度曲线呈线性方程相关,相关系数为0.99;小米样品叶酸测定过程中精密度、稳定性和重复性的相对标准偏差(RSD)分别为0.81%、1.19%和1.65%,各项指标符合精密测定标准,准确度高,重复性好;45个谷子种质籽粒叶酸含量分布范围为0.26～2.56μg/g(2016年度)和0.53～2.86μg/g(2017年度),含量均值分别为1.19和1.84μg/g,标准差0.47和0.51μg/g,变异系数39.5%和27.7%;对上述种质进行正态分布曲线分析,发现叶酸含量整体呈现偏度分布;从中筛选出9个高叶酸(含量≥1.91μg/g)和3个低叶酸(含量≤1.15μg/g)的谷子种质。上述结论为小米叶酸含量提供了测定标准和评价体系,也为高叶酸谷子种质创新与利用奠定了基础。
Foxtail millet is a kind of functional food rich in folic acid, and it is popular in the northern China. However, there are significant differences in folic acid contents in crop grains, such as the determination method and content analysis of folate in Foxtail millet, which make it difficult to identify the quality of millet and select the germplasm with high folate content. In this study, ‘triple-enzymes method' was used to isolate folic acid from foxtail millet grains and construct a large-scale detection method by HPLC for folic acid content. Meantime, the folic acid contents were assessed among 45 accessions of foxtail millet in 2016 and 2017. The results showed that: based on HPLC, a folic acid content determination method for foxtail millet was successfully established. Folic acid standard curve showed the linear correlation between folic acid standard and peak area value, the correlation coefficient was 0.99. The relative standard deviation(RSD) was used to evaluate the precision, stability and repeatability in the detection process of folic acid content, and they were 0.81%, 1.19% and 1.65%, respectively. The range of grain folic acid content of 45 millet varieties were 0.26 ～ 2.56 μg/g in 2016 and 0.53 ～ 2.86 μg/g in 2017, with average values of 1.19 and 1.84 μg/g, standard deviation of 0.47 and 0.51 μg/g, and variation coefficient of 39.5% and 27.7%, respectively. The gaussian distribution had a little skewness. Finally, 9 high folic acid(folate content≥1.91 μg/g) and 3 low folic acid(folate content≤1.15 μg/g) accessions were screened from 45 millet accessions, respectively. This study not only provided the standard determination and evaluation system of folate in foxtail millet, and also laied a foundation for the innovation and utilization of high folate germplasm of foxtail millet.
Foxtail millet is a kind of functional food rich in folic acid, and it is popular in the northern China. However, there are significant differences in folic acid contents in crop grains, such as the determination method and content analysis of folate in Foxtail millet, which make it difficult to identify the quality of millet and select the germplasm with high folate content. In this study, ‘triple-enzymes method' was used to isolate folic acid from foxtail millet grains and construct a large-scale detection method by HPLC for folic acid content. Meantime, the folic acid contents were assessed among 45 accessions of foxtail millet in 2016 and 2017. The results showed that: based on HPLC, a folic acid content determination method for foxtail millet was successfully established. Folic acid standard curve showed the linear correlation between folic acid standard and peak area value, the correlation coefficient was 0.99. The relative standard deviation(RSD) was used to evaluate the precision, stability and repeatability in the detection process of folic acid content, and they were 0.81%, 1.19% and 1.65%, respectively. The range of grain folic acid content of 45 millet varieties were 0.26 ～ 2.56 μg/g in 2016 and 0.53 ～ 2.86 μg/g in 2017, with average values of 1.19 and 1.84 μg/g, standard deviation of 0.47 and 0.51 μg/g, and variation coefficient of 39.5% and 27.7%, respectively. The gaussian distribution had a little skewness. Finally, 9 high folic acid(folate content≥1.91 μg/g) and 3 low folic acid(folate content≤1.15 μg/g) accessions were screened from 45 millet accessions, respectively. This study not only provided the standard determination and evaluation system of folate in foxtail millet, and also laied a foundation for the innovation and utilization of high folate germplasm of foxtail millet.
引文
[1]Bekaert S,Storozhenko S,Mehrshahl P,et al.Folate biofortification in food plants[J].Trends Plant Science,2008,13(1):28-35
[2]Hanson A D,Gage D A,Hill Y S.Plant one-carbon metabolism and its engineering[J].Trends in Plant Science,2000,5(5):206-213
[3]Ravanel S,Block M A,Rippert P,et al.Methionine metabolism in plants:Chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol[J].Journal of Biological Chemistry,2004,279(21):22548-22557
[4]马爱勤,汪之顼.叶酸营养与孕期保健[J].环境卫生学杂志,2009(6):390-392
[5]刘朝,宁艳玲,贾占玲,等.叶酸对抑郁症的辅助治疗[J].中华行为医学与脑科学杂志,2004,13(2):169-170
[6]Ramos M I,Allen L H,Mungas D M,et al.Low folate status is associated with impaired cognitive function and dementia in the Sacramento Area Latino Study on Aging[J].American Journal of Clinical Nutrition,2005,82(6):1346-1352
[7]Blancquaert D,Storozhenko S,Loizeau K,et al.Folates and folic acid:From fundamental research toward sustainable health[J].Critical Reviews in Plant Sciences,2010,29(1):14-35
[8]Iyer R,Tomar S K.Folate:A functional food constituent[J].Journal of Food Science,2009,74(9):114-122
[9]都薇,蔺娜,王彩琴,等.高效液相色谱法测定小麦粉中烟酸、叶酸含量的探讨[C].中国粮油标准质量年会暨中国粮油学会粮油质检研究分会第一次代表大会.2009:64-69
[10]He L,Zhang B,Han Y H.Foxtail millet:Nutritional and eating quality,and prospects for genetic improvement[J].Frontiers of Agricultural Science&Engineering,2015,2(2):124-130
[11]任文芳,杨润强,顾振新,等.植物源食品中叶酸的生物合成与调控及其提取与检测技术研究进展[J].食品工业科技,2018,39(9):335-341
[12]Bali S,Robinson B R,Sathuvalli V,et al.Single nucleotide polymorphism(SNP)markers associated with high folate content in wild potato species[J].Plos One,2018,13(2):e0193415
[13]RocíGarza A D L,Quinlivan E P,et al.Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(38):13720
[14]Nunes A C S,Kalkmann D C,Arag?o F J L.Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene[J].Transgenic Research,2009,18(5):661-667
[15]Gorelova V,Ambach L,RébeilléF,et al.Folates in plants:Research advances and progress in crop biofortification[J].Frontiers in Chemistry,2017,5:21
[16]常娜宁,姜凌,蒲训,等.作物叶酸检测方法的研究进展[J].中国农业科技导报,2010,12(2):44-49
[17]梁业红,Arnaud B,范云六.异源表达细菌二氢喋呤合成酶基因提高拟南芥叶酸含量的研究[J].作物学报,2006,32(2):164-168
[18]Iwatani Y,Arcot J,Shrestha A K.Determination of folate contents in some Australian vegetables[J].J.Food Comp Anal.,2003,16(1):37-48
[19]张毅.叶菜中叶酸含量的HPLC测定条件及不同处理方式对叶酸保留效果研究[D].杨凌:西北农林科技大学,2016
[20]郭健,李敏,孟妍,等.荧光分光光度法测定食品中叶酸的含量[J].中国公共卫生,2003,19(10):1259
[21]Zhang H,Jha A B,Warkentin T D,et al.Folate stability and method optimization for folate extraction from seeds of pulse crops using LC-SRM MS[J].Journal of Food Composition&Analysis,2018,71:44-55
[22]董建江,邵伏文,张林,等.不同耕作模式对稻田土壤理化性质及经济效益的影响[J].土壤,2015,47(3):509-514
[23]刘钊,魏天兴,朱清科,等.黄土丘陵沟壑区典型林地土壤微生物、酶活性和养分特征[J].土壤,2016,48(4):705-713
[24]孟繁磊,张玲,张原宇,等.大豆叶酸的三酶法提取及微生物检测技术研究[J].粮食与饲料工业,2016,12(12):56-59
[25]邵丽华,王莉.粮食及果蔬中叶酸含量分析[J].食品科学,2014,35(24):290-294
[26]董薇.水稻籽粒叶酸含量QTL分析及生物强化[D].北京:中国农业科学院,2011
[27]邵丽华,王莉,白文文,等.山西谷子资源叶酸含量分析及评价[J].中国农业科学,2014,47(7):1265-1272
[2]Hanson A D,Gage D A,Hill Y S.Plant one-carbon metabolism and its engineering[J].Trends in Plant Science,2000,5(5):206-213
[3]Ravanel S,Block M A,Rippert P,et al.Methionine metabolism in plants:Chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol[J].Journal of Biological Chemistry,2004,279(21):22548-22557
[4]马爱勤,汪之顼.叶酸营养与孕期保健[J].环境卫生学杂志,2009(6):390-392
[5]刘朝,宁艳玲,贾占玲,等.叶酸对抑郁症的辅助治疗[J].中华行为医学与脑科学杂志,2004,13(2):169-170
[6]Ramos M I,Allen L H,Mungas D M,et al.Low folate status is associated with impaired cognitive function and dementia in the Sacramento Area Latino Study on Aging[J].American Journal of Clinical Nutrition,2005,82(6):1346-1352
[7]Blancquaert D,Storozhenko S,Loizeau K,et al.Folates and folic acid:From fundamental research toward sustainable health[J].Critical Reviews in Plant Sciences,2010,29(1):14-35
[8]Iyer R,Tomar S K.Folate:A functional food constituent[J].Journal of Food Science,2009,74(9):114-122
[9]都薇,蔺娜,王彩琴,等.高效液相色谱法测定小麦粉中烟酸、叶酸含量的探讨[C].中国粮油标准质量年会暨中国粮油学会粮油质检研究分会第一次代表大会.2009:64-69
[10]He L,Zhang B,Han Y H.Foxtail millet:Nutritional and eating quality,and prospects for genetic improvement[J].Frontiers of Agricultural Science&Engineering,2015,2(2):124-130
[11]任文芳,杨润强,顾振新,等.植物源食品中叶酸的生物合成与调控及其提取与检测技术研究进展[J].食品工业科技,2018,39(9):335-341
[12]Bali S,Robinson B R,Sathuvalli V,et al.Single nucleotide polymorphism(SNP)markers associated with high folate content in wild potato species[J].Plos One,2018,13(2):e0193415
[13]RocíGarza A D L,Quinlivan E P,et al.Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(38):13720
[14]Nunes A C S,Kalkmann D C,Arag?o F J L.Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene[J].Transgenic Research,2009,18(5):661-667
[15]Gorelova V,Ambach L,RébeilléF,et al.Folates in plants:Research advances and progress in crop biofortification[J].Frontiers in Chemistry,2017,5:21
[16]常娜宁,姜凌,蒲训,等.作物叶酸检测方法的研究进展[J].中国农业科技导报,2010,12(2):44-49
[17]梁业红,Arnaud B,范云六.异源表达细菌二氢喋呤合成酶基因提高拟南芥叶酸含量的研究[J].作物学报,2006,32(2):164-168
[18]Iwatani Y,Arcot J,Shrestha A K.Determination of folate contents in some Australian vegetables[J].J.Food Comp Anal.,2003,16(1):37-48
[19]张毅.叶菜中叶酸含量的HPLC测定条件及不同处理方式对叶酸保留效果研究[D].杨凌:西北农林科技大学,2016
[20]郭健,李敏,孟妍,等.荧光分光光度法测定食品中叶酸的含量[J].中国公共卫生,2003,19(10):1259
[21]Zhang H,Jha A B,Warkentin T D,et al.Folate stability and method optimization for folate extraction from seeds of pulse crops using LC-SRM MS[J].Journal of Food Composition&Analysis,2018,71:44-55
[22]董建江,邵伏文,张林,等.不同耕作模式对稻田土壤理化性质及经济效益的影响[J].土壤,2015,47(3):509-514
[23]刘钊,魏天兴,朱清科,等.黄土丘陵沟壑区典型林地土壤微生物、酶活性和养分特征[J].土壤,2016,48(4):705-713
[24]孟繁磊,张玲,张原宇,等.大豆叶酸的三酶法提取及微生物检测技术研究[J].粮食与饲料工业,2016,12(12):56-59
[25]邵丽华,王莉.粮食及果蔬中叶酸含量分析[J].食品科学,2014,35(24):290-294
[26]董薇.水稻籽粒叶酸含量QTL分析及生物强化[D].北京:中国农业科学院,2011
[27]邵丽华,王莉,白文文,等.山西谷子资源叶酸含量分析及评价[J].中国农业科学,2014,47(7):1265-1272