翟胜男^,, 刘爱峰, 李法计, 刘成, 郭军, 韩冉, 訾妍, 汪晓璐, 吕莹莹, 刘建军^,山东省农业科学院作物研究所,济南 250100

Improvement and Application of the Method for Determining Yellow Pigment Content in Wheat Grain

ZHAI ShengNan^,, LIU AiFeng, LI FaJi, LIU Cheng, GUO Jun, HAN Ran, ZI Yan, WANG XiaoLu, Lü YingYing, LIU JianJun^,Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100

通讯作者: 刘建军,E-mail： ljjsaas@163.com

责任编辑: 李莉
收稿日期:2020-04-27接受日期:2020-07-6网络出版日期:2021-01-16

基金资助:

国家自然科学基金.31701420 山东省农业良种工程.2019LGC001 山东省农业科学院农业科技创新工程.CXGC2018E01 山东省重大科技创新工程项目.2018YFJH0602

Received:2020-04-27Accepted:2020-07-6Online:2021-01-16
作者简介 About authors
翟胜男,E-mail： zsn19870322@163.com。












摘要
【目的】小麦籽粒黄色素含量是影响面制品颜色和营养品质的重要因素,为快速、准确、简便地检测小麦籽粒黄色素含量,对传统小麦籽粒黄色素测定方法AACC 14-50进行改良,为面制品颜色性状和营养品质遗传改良提供技术支撑。【方法】将酶标仪应用于小麦籽粒黄色素含量测定,对传统AACC 14-50法进行改良,分析改良方法的准确性和稳定性,探讨不同提取时间和保存时间对面粉黄色素含量的影响,利用新改良方法测定283份国内外小麦品种籽粒黄色素含量,进一步验证改良方法的有效性,发掘面制品颜色性状和营养品质优异的种质资源。【结果】改良方法与传统AACC 14-50法所测黄色素含量呈极显著正相关,相关系数达0.994（P<0.001）,方差分析显示,2种方法所测黄色素含量在品种间差异均极显著（P<0.01）。应用改良方法对144份小麦品种籽粒黄色素含量进行重复测定,3次重复两两间的相关系数分别为0.997、0.998和0.998。藁优5218、冀师02-2和郑麦366面粉黄色素含量在提取时间为30 min时达到最大值,分别为0.93、1.24和1.53 μg·g^-1,且提取时间为30—120 min时,黄色素含量保持在稳定水平（0.94—0.97、1.30—1.33和1.59—1.62 μg·g^-1）。在室温保存0—20 d的藁优5218、冀师02-2和郑麦366的面粉,其黄色素含量没有显著变化（0.93—0.97、1.29—1.33和1.60—1.64 μg·g^-1;P>0.05）。283份国内外小麦品种籽粒黄色素含量变异范围广,不同环境间相关系数为0.73—0.98。其中,黄淮麦区小麦品种黄色素含量平均值为1.15 μg·g^-1,变异范围为0.51—2.42 μg·g^-1;北部冬麦区平均值为1.57 μg·g^-1,变异范围为0.90—2.52 μg·g^-1;长江中下游麦区平均值为1.07 μg·g^-1,变异范围为0.56—2.54 μg·g^-1;国外品种平均值为1.61 μg·g^-1,变异范围为0.94—2.48 μg·g^-1。筛选到24份黄色素含量较低和26份黄色素含量较高的品种,可作为亲本材料用于面制品颜色性状和营养品质遗传改良。与传统方法相比,改良方法显著降低了工作量,缩短检测时间,效率提高12—15倍,且样本、试剂用量仅为原来的1/16,大大节约了成本。【结论】建立了一个准确稳定、经济高效、操作简便的小麦籽粒黄色素含量检测方法,可代替传统AACC 14-50法用于大规模样本黄色素含量测定。
关键词： 小麦;面制品颜色;营养品质;酶标仪;遗传改良

Abstract
【Objective】The yellow pigment content (YPC) in wheat grain is an important factor affecting the color and nutritional quality of flour end-use products. To quickly, accurately and simply detect YPC in wheat grain, the traditional method for YPC determination, AACC 14-50, was improved, providing a technical support for genetic improvement of the color and nutritional quality of flour end-use products.【Method】In this study, improvements have been made to the AACC 14-50 method using a Multiskan Spectrum microplate reader. The accuracy and stability of the modified method were analyzed, and the influence of extraction time and preservation time on flour YPC was dissected. The YPC of 283 domestic and foreign wheat varieties were determined by the improved method to further verify its effectiveness and explore excellent resources for genetic improvement of the color and nutritional quality of flour end-use products. 【Result】 The results showed that YPC determined by the modified method was significantly and positively correlated with those by the traditional AACC 14-50 method, with a correlation coefficient of 0.994 (P<0.001), and analysis of variance showed that YPC measured by the two methods was significant among varieties. The YPC of 144 wheat varieties were measured repeatedly using the modified method, and the correlation coefficients of YPC among three replicates were 0.997, 0.998 and 0.998, respectively. The flour YPC of Gaoyou 5218, Jishi 02-2 and Zhengmai 366 reached the maximum value (0.93, 1.24 and 1.53 μg·g^-1) at the extraction time of 30 min, and remained stable during 30-120 min (0.94-0.97 μg·g^-1, 1.30-1.33 μg·g^-1 and 1.59-1.62 μg·g^-1). No significant changes were observed in the flour YPC of Gaoyou 5218, Jishi 02-2 and Zhengmai 366 stored at room temperature for 0-20 days (0.93-0.97 μg·g^-1, 1.29-1.33 μg·g^-1 and 1.60-1.64 μg·g^-1; P>0.05). A wide range of YPC variation was identified in 283 domestic and foreign wheat varieties, with correlation coefficients ranging from 0.73 to 0.98 among different environments. Briefly, the mean YPC of varieties in the Huang-Huai wheat region was 1.15 μg·g^-1, ranging between 0.51-2.42 μg·g^-1. The average value of YPC in varieties from the Northern winter wheat region was 1.57 μg·g^-1, ranging from 0.90 to 2.52 μg·g^-1. The average value of YPC in the Middle-Lower reaches of the Yangtze River wheat region was 1.07 μg·g^-1, and the variation ranged from 0.56 to 2.54 μg·g^-1. The average YPC of foreign varieties was 1.61 μg·g^-1, ranging from 0.94 to 2.48 μg·g^-1. Twenty-four varieties with lower YPC and 26 with higher YPC were identified, which can be used as parents in wheat breeding programs to improve the color and nutritional quality of flour end-use products. Compared with the traditional AACC 14-50 method, the modified one reduces the workload, decreases the detection time, and increases the detection efficiency by about 12-15 times, and the consumption of samples and reagents is only 1/16 of those in the traditional method, which greatly reduces the cost. 【Conclusion】 The modified method for determining YPC in wheat grain established in this study is accurate, reliable, economical, efficient and simple, which can replace the traditional AACC 14-50 method for a large-scale determination of YPC.
Keywords：Triticum aestivum;color of flour end-used products;nutritional quality;multiskan spectrum microplate reader;genetic improvement


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本文引用格式
翟胜男, 刘爱峰, 李法计, 刘成, 郭军, 韩冉, 訾妍, 汪晓璐, 吕莹莹, 刘建军. 小麦籽粒黄色素含量检测方法的改良与应用[J]. 中国农业科学, 2021, 54(2): 239-247 doi:10.3864/j.issn.0578-1752.2021.02.001
ZHAI ShengNan, LIU AiFeng, LI FaJi, LIU Cheng, GUO Jun, HAN Ran, ZI Yan, WANG XiaoLu, Lü YingYing, LIU JianJun. Improvement and Application of the Method for Determining Yellow Pigment Content in Wheat Grain[J]. Scientia Acricultura Sinica, 2021, 54(2): 239-247 doi:10.3864/j.issn.0578-1752.2021.02.001


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0 引言

【研究意义】小麦籽粒黄色素含量对面粉及其制品表观色泽和营养品质具有重要影响,对其进行遗传改良已成为小麦品质改良的重要内容。快速、准确、简便地检测小麦籽粒黄色素含量,是面粉及其制品色泽和营养品质遗传改良的基础。【前人研究进展】黄色素是小麦籽粒中最主要的天然色素,是面粉及其制品颜色形成的主要原因。研究表明,小麦籽粒黄色素含量与面粉、面团黄度的相关系数高达0.8—0.9,与面包和面条颜色的相关系数分别为0.83和0.76^[1,2,3,4]。类胡萝卜素是构成黄色素的主要成分,两者相关系数达0.8^[5,6,7,8,9]。类胡萝卜素,尤其是β-胡萝卜素,是维生素A原,具有抗氧化、抗癌、降低心血管疾病、预防眼睛老年性黄斑病变、延缓衰老及提高免疫力等重要生理保健功能^{[10,11,12,13,14,15]}。中国传统主食馒头、面条、包子和水饺等蒸煮类食品对面粉的白度要求比较高,细腻洁白的面粉及其制品备受消费者青睐^{[16,17,18,19,20]}。中国主栽小麦品种的面粉平均白度较低,不少品种因不具备优良的颜色性状而不能满足加工需求,限制其示范推广^{[21,22,23,24]}。而日本和东南亚等国家喜爱的黄碱面条要求亮黄色,提高籽粒黄色素含量已成为这些国家的重要育种目标^[25]。此外,随着人们营养和保健意识的提高,亮黄色的面粉和面制品也越来越受到重视。因此,根据不同的市场和消费者需求,对面粉及其制品颜色性状和营养品质进行改良是小麦品质改良的重要内容。如何快速、准确、简便地检测小麦籽粒黄色素含量,是育种家们急需解决的问题。目前,小麦籽粒黄色素含量测定方法广泛采用美国谷物化学协会（American Association for Cereal Chemistry,AACC）标准方法AACC 14-50^[26]。【本研究切入点】传统的AACC 14-50方法以分光光度法测定黄色素含量,检测通量低,只能对样本进行逐个检测,需要不断更换比色皿,操作繁琐,工作量大,测定时间长,检测效率低,所需样本试剂量大,不利于面制品颜色和营养品质遗传改良育种中材料大规模检测。酶标仪诞生于20世纪50年代,与分光光度计工作原理基本一致,同样具有较高的检测精度,而且酶标仪操作更加简便,用样量少,批量操作,适用于大规模样本测定,已被广泛应用于三角褐指藻油脂含量、食用菌多糖含量、大豆总皂甙含量等检测^{[27,28,29,30]}。【拟解决的关键问题】本研究将酶标仪应用于小麦籽粒黄色素含量测定,对传统检测方法AACC 14-50进行改良,分析不同提取时间和保存时间对面粉黄色素含量的影响,并利用改良方法对283份国内外小麦品种进行黄色素含量检测,进一步验证改良方法的有效性,筛选籽粒黄色素含量较低和较高品种,为面制品颜色性状和营养品质遗传改良的亲本选择提供参考。

1 材料与方法

1.1 试验材料

根据前人研究,选用12个籽粒黄色素含量变异范围较大的小麦品种,即烟农15、汶农14、陕512、洛旱2号、观35、碧蚂1号、冀师02-1、新麦9408、陕优225、邯6172、鲁麦6号和豫麦18,分别采用传统AACC 14-50法^[26]与改良方法测定黄色素含量,用于验证改良方法的准确性。应用藁优5218、冀师02-2和郑麦366,分析不同提取时间和保存时间对面粉黄色素含量的影响。选用144份中国黄淮麦区小麦品种,用于分析改良方法的重演性和稳定性。选用283份具有代表性的国内外小麦品种,进一步验证改良方法的有效性。根据生育期和春冬性不同,于2012—2013年和2013—2014年进行分区种植。其中,144份中国黄淮麦区小麦品种种植于河南安阳和安徽濉溪;42份北部冬麦区品种和48份国外品种种植于北京和河北石家庄;49份长江中下游麦区品种种植于安徽濉溪和四川成都。

所有供试材料采用随机区组设计,3次重复,4行区,行长2 m,行距25 cm,按常规方法进行田间种植管理。对2次重复的籽粒进行磨粉,用于黄色素含量测定。

1.2 制粉

应用单籽粒谷物特性测试仪SKCS 4100（Perten,Sweden）测定籽粒硬度。利用近红外分析仪Foss-Tecator 1241（Foss,H?g?nas,Sweden）测定籽粒蛋白质含量和含水量。根据硬度等级计算润麦所需加水量：软质麦（硬度指数<40）为14%、混合麦（40<硬度指数<60）为15%、硬质麦（>60）为16%。室温润麦16—18 h,应用Junior试验磨（Brabender,Germany）制粉,出粉率约为60%。面粉4℃保存,用于黄色素含量检测。

1.3 黄色素含量测定

2013—2014年于中国农业科学院作物科学研究所何中虎研究员实验室,分别利用传统AACC 14-50法^[26]与改良方法测定12个籽粒黄色素含量变异范围较大的小麦品种黄色素含量。应用改良方法测定283份国内外小麦品种籽粒黄色素含量。

传统AACC 14-50法^[26]：（1）称取8.0 g面粉放入125 mL玻璃瓶中;（2）加入40 mL水饱和正丁醇提取液（体积比5﹕1）,振荡1 min,静置30 min;（3）再次摇匀,应用Whatman no.1滤纸过滤;（4）以水饱和正丁醇为对照,利用分光光度计测定436.5 nm处反应液吸光值A,计算黄色素含量。

改良方法：（1）称取0.5 g面粉放入10 mL离心管中;（2）加入2.5 mL水饱和正丁醇提取液（体积比5﹕1）,旋涡振荡、混匀;（3）将离心管放入PE离心管盒内,并固定在沉降值测定仪（Brabender,Germany）上,上下振荡30 min,以利于黄色素的充分提取;（4）应用Eppendorf离心机（Centrifuge 5804 R,Germany）,5 000 r/min离心10 min;（5）吸取200 μL上清液加入96孔酶标板（Costar 3590,USA）,以水饱和正丁醇溶液为对照,利用酶标仪SpectraMax Plus 384（Molecular Devices,LLC,USA）测定436.5 nm处上清液吸光值A。

黄色素含量=A×30.1,单位为μg·g^-1。每个品种2次生物学重复,每个重复样品3次技术重复,均值作为样品黄色素含量的表型值。

1.4 不同提取时间和保存时间对面粉黄色素含量的影响

利用水饱和正丁醇分别对藁优5218、冀师02-2和郑麦366面粉进行不同时间（15、30、45、60、75、90、105和120 min）的提取,分析不同提取时间对面粉黄色素含量的影响。

利用改良方法,分别对室温条件下不同保存时间（0、5、10、15和20 d）的藁优5218、冀师02-2和郑麦366面粉进行黄色素含量的测定,分析不同保存时间对面粉黄色素含量的影响。

1.5 统计分析

利用SAS V9.2（http://www.sas.com）软件,进行黄色素含量基本统计量分析,利用PROC CORR程序进行相关性分析,应用PROC GLM程序进行方差分析。利用Microsoft Excel软件进行图表绘制。

2 结果

2.1 传统AACC 14-50法与改良方法所测黄色素含量比较

选用12个代表性小麦品种,分别采用传统AACC 14-50法与改良方法测定籽粒黄色素含量,每个样本重复3次,平均值作为样本黄色素含量表型值。对2种方法所测黄色素含量进行比较（图1）,传统AACC 14-50法所测黄色素含量较高,平均值为3.58 μg·g^-1,变异范围为2.51—5.22 μg·g^-1,相对标准偏差为0.09%—11.13%,变异系数为24.3%。改良方法所测黄色素含量值较低,平均值为1.21 μg·g^-1,变异范围为0.76—1.79 μg·g^-1,相对标准偏差为0.01%—6.47%,变异系数为27.6%。2种方法所测黄色素含量呈极显著正相关（r=0.994,P<0.001）。

图1

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图12种方法所测黄色素含量的相关性

Fig. 1The correlation of yellow pigment content measured by the two methods

2.2 传统AACC 14-50法与改良方法所测黄色素含量方差分析

对传统AACC 14-50法与改良方法所测12个小麦品种的黄色素含量进行方差分析,由表1可知,应用2种方法所测得的12个小麦品种籽粒黄色素含量在品种水平差异均极显著。

Table 1
表1
表12种方法测定黄色素含量的方差分析
Table 1Analyses of variance of yellow pigment content measured by the two methods

变异来源 Source	自由度 DF	传统AACC 14-50法 AACC 14-50 method		改良方法 Modified method
		平方和 SS	F值 F value	平方和 SS	F值 F value
重复 Replication	2	0.174	1.54	0.007	1.31
品种 Cultivars	11	29.480	385.44**	4.370	1425.50**

**表示在 0.001水平差异显著
** Significant at P=0.001

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2.3 传统AACC 14-50法与改良方法的差异性比较

如表2所示,改良方法面粉和水饱和正丁醇所需量仅为传统AACC 14-50法的1/16,大大节约检测成本。该方法应用96孔酶标板和酶标仪同时可检测多个样本的黄色素含量,显著提高检测效率。

Table 2
表2
表22种黄色素含量测定方法比较
Table 2Comparison of two methods for detecting yellow pigment content

项目Item	传统AACC 14-50法 AACC 14-50 method	改良方法 Modified method
面粉用量 Flour consumption (g)	8.0	0.5
水饱和正丁醇用量 Amount of n-butanol saturated with water (mL)	40	2.5
检测方法 Detection method	分光光度计 Spectrophotometer	酶标仪 Multiskan spectrum microplate reader
上样容器 Container	比色皿 Cuvette	96孔酶标板 96-well microtiter plate
上样量 Loading quantity	1 mL	200 μL
检测效率 Detection efficiency	一次只能检测单个样本,1个样品/分钟 Only one sample can be tested at a time, one sample/min	同时检测多个样品,12—15个样品/分钟 Multiple samples can be tested simultaneously, 12-15 samples/min

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2.4 改良方法测定黄色素含量稳定性分析

为了分析改良方法测定黄色素含量的稳定性和可靠性,应用此方法对144份中国黄淮麦区代表性品种黄色素含量进行重复测定（图2）,3次重复两两间黄色素含量相关系数分别为0.997、0.998和0.998（P<0.001）,表明改良方法测定黄色素含量重复性好,稳定性高。

图2

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图2改良方法测定黄色素含量重复间的相关性

Fig. 2The correlation of yellow pigment content between three replications measured by the modified method

2.5 不同提取时间对黄色素含量的影响

利用水饱和正丁醇对藁优5218、冀师02-2和郑麦366面粉分别进行15、30、45、60、75、90、105和120 min的提取,黄色素含量测定结果如图3所示。振荡提取15 min时,黄色素含量较低,藁优5218、冀师02-2和郑麦366的黄色素含量分别为0.93、1.24和1.53 μg·g^-1。提取时间为30 min时,黄色素含量达到最大值,藁优5218、冀师02-2和郑麦366的黄色素含量分别为0.97、1.33和1.62 μg·g^-1。提取时间为30—120 min时,黄色素含量保持在稳定水平（0.94—0.97、1.30—1.33和1.59—1.62 μg·g^-1）。

图3

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图3提取时间对面粉黄色素含量的影响

Fig. 3Effect of extraction time on yellow pigment content of flour

2.6 保存时间对黄色素含量的影响

利用改良方法,对室温放置0—20 d的藁优5218、冀师02-2和郑麦366面粉进行黄色素含量测定（图4）,发现其黄色素含量差异不显著（0.93—0.97、1.29—1.33和1.60—1.64 μg·g^-1;P>0.05）。

图4

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图4保存时间对面粉黄色素含量的影响

Fig. 4Effect of preservation time on yellow pigment content of flour

2.7 283份国内外小麦品种黄色素含量分析

由表3可知,283份国内外小麦品种籽粒黄色素含量变异范围广,遗传基础丰富。其中,黄淮麦区小麦品种黄色素含量平均值为1.15 μg·g^-1,变异范围为0.51—2.42 μg·g^-1;北部冬麦区平均值为1.57 μg·g^-1,变异范围为0.90—2.52 μg·g^-1;长江中下游麦区平均值为1.07 μg·g^-1,变异范围为0.56—2.54 μg·g^-1;国外品种平均值为1.61 μg·g^-1,变异范围为0.94—2.48 μg·g^-1。

Table 3
表3
表3283份国内外小麦品种黄色素含量基本信息
Table 3Information of yellow pigment content in 283 domestic and foreign wheat varieties

麦区 Wheat region	平均值 Mean	标准差 SD	最小值 Minimum	最大值 Maximum	变异系数 CV
黄淮麦区Huang-Huai wheat region
2013-AY	1.17	0.44	0.51	2.42	0.38
2013-SX	1.13	0.40	0.53	2.24	0.36
2014-AY	1.26	0.43	0.61	2.38	0.35
2014-SX	1.07	0.38	0.52	2.16	0.36
平均值Mean	1.15	0.41	0.57	2.21	0.35
北部冬麦区Northern winter wheat region
2013-SJZ	1.58	0.37	1.02	2.42	0.23
2013-BJ	1.66	0.38	1.08	2.52	0.23
2014-SJZ	1.43	0.36	0.90	2.32	0.25
2014-BJ	1.61	0.36	1.07	2.40	0.23
平均值Mean	1.57	0.36	1.02	2.33	0.23
长江中下游麦区Middle-Lower reaches of the Yangtze River wheat region
2013-CD	0.99	0.27	0.56	1.77	0.28
2013-SX	0.98	0.26	0.64	1.71	0.27
2014-CD	1.30	0.38	0.81	2.54	0.29
2014-SX	1.03	0.27	0.66	1.86	0.26
平均值Mean	1.07	0.25	0.73	1.82	0.23
国外品种Foreign wheat varieties
2013-SJZ	1.72	0.35	1.22	2.48	0.20
2013-BJ	1.64	0.35	1.06	2.31	0.22
2014-SJZ	1.54	0.36	0.94	2.36	0.23
2014-BJ	1.54	0.28	1.07	2.23	0.18
平均值Mean	1.61	0.31	1.11	2.20	0.19

AY：安阳;SX：濉溪;SJZ：石家庄;BJ：北京;CD：成都
AY: Anyang; SX: Suixi; SJZ: Shijiazhuang; BJ: Beijing; CD: Chengdu

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同一品种不同环境间黄色素含量的相关系数为0.73—0.98。比较各个品种在不同环境下黄色素含量,筛选出环境间稳定存在的黄色素含量较低的品种24个和黄色素含量较高的品种26个（表4）,可作为面制品颜色性状和营养品质遗传改良的优异资源。

Table 4
表4
表4籽粒黄色素含量较低和较高小麦品种
Table 4The wheat varieties with lower and higher yellow pigment content in grain

麦区 Wheat region	低黄色素含量小麦品种 Varieties with lower yellow pigment content	高黄色素含量小麦品种 Varieties with higher yellow pigment content
黄淮麦区 Huang-Huai wheat region	丰产3号、临旱2号、陕农981、小偃54、鲁麦23、西农979-005 Fengchan 3 hao, Linhan 2 hao, Shaannong 981, Xiaoyan 54, Lumai 23, Xinong 979-005	鲁麦14、淄选2号、陕麦509、豫麦7号、中麦895、鲁麦11、烟农18 Lumai 14, Zixuan 2 hao, Shaanmai 509, Yumai 7 hao, Zhongmai 895, Lumai 11, Yannong 18
北部冬麦区 Northern winter wheat region	中优9507、CA1055、秦农151、CA0548、CA1133 Zhongyou 9507, CA1055, Qinnong 151, CA0548, CA1133	京冬22、轮选987、科衡6654、京411、中麦415、北京841、新麦37 Jingdong 22, Lunxuan 987, Keheng 6654, Jing 411, Zhongmai 415, Beijing 841, Xinmai 37
长江中下游麦区 Middle-lower reaches of the Yangtze river wheat region	鄂麦14、川麦107、扬麦10号、川麦42、鄂麦23、鄂麦21 Emai 14, Chunmai 107, Yangmai 10 hao, Chunmai 42, Emai 23, Emai 21	徐州25、宁麦9号、中国春、鄂恩5号、蓉麦4号 Xuzhou 25, Ningmai 9 hao, Chinese Spring, Een 5 hao, Rongmai 4 hao
国外品种 Foreign wheat varieties	Jagger、Salmone、WGRC10、Mesofold、F98047G14-2INC、RE714、Fr03711	YANA、Lovrin10、Fr03732、Lovrin13、E、SELYANKA、C39

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3 讨论

3.1 改良方法测定黄色素含量的准确稳定性和高效经济性分析

小麦是中国主要的粮食作物,随着人们生活水平提高,对小麦品质提出更高的要求。面粉中黄色素含量直接影响面制品的色泽和营养品质,已成为小麦品质改良的重要研究内容^[31,32,33]。缺乏准确、稳定、高效测定小麦籽粒黄色素含量的方法,严重制约了面制品颜色性状和营养品质遗传改良。

本研究对传统AACC 14-50法进行改良,发现2种方法测定所得黄色素含量呈极显著正相关,相关系数达0.994,表明改良方法与传统方法在测定小麦籽粒黄色素含量准确度上无显著差异。且改良方法所测黄色素含量的相对标准偏差小于传统AACC 14-50法（0.01%—6.47% VS 0.09%—11.13%）,表明改良方法测定黄色素含量的精度更高。应用改良方法重复测定144份小麦品种黄色素含量,3次重复两两间的相关系数分别为0.997、0.998和0.998,表明该方法重复性好,稳定性高。此外,改良方法应用96孔酶标板和酶标仪测定黄色素含量,操作更加简便,无需更换清洗比色皿,大大减少工作量,同时可检测多个样本,缩短检测时间,避免因测定时间过长而导致的误差,适用于大样本测定分析,检测效率提高12—15倍。同时,样本试剂用量小,仅为传统AACC 14-50法用量的1/16,大大降低试验成本。因此,本研究改良方法具有操作简便、准确可靠、精密度高、重复性好、经济高效等优点,可以代替传统方法用于小麦籽粒黄色素含量高通量检测,为小麦品种资源筛选、面制品颜色和营养品质改良提供技术支撑。

3.2 不同提取时间和保存时间对样本黄色素含量影响

应用水饱和正丁醇,对藁优5218、冀师02-2和郑麦366面粉振荡提取15 min时,黄色素含量较低,表明提取时间过短黄色素提取不充分。当提取时间为30 min时,黄色素含量达到最大值,且提取时间在30—120 min时,黄色素含量保持在稳定水平。因此,提取时间为30 min时,能较快、较好地反映面粉黄色素含量真实水平。

室温存放0—20 d的藁优5218、冀师02-2和郑麦366面粉,其黄色素含量差异不显著,表明面粉黄色素相对较稳定,为大批量样本检测提供可能。

3.3 面粉及其制品色泽和营养品质遗传改良

小麦籽粒黄色素含量对面条和馒头等面制品的表观色泽和营养品质具有重要影响,已成为小麦品质改良的重要内容^[31,32,33]。缺乏优异种质资源成为小麦面粉及其制品品质改良的“瓶颈”,严重影响中国小麦品种面制品颜色和营养品质的遗传改良。

本研究对283份国内外小麦品种籽粒黄色素含量进行检测,结果表明,黄色素含量变异范围广,遗传基础丰富,环境间较稳定。共筛选出不同环境下黄色素含量均较低品种24个和黄色素含量均较高品种26个,可作为面制品颜色性状和营养品质遗传改良的优异资源。此外,在面粉及其制品颜色和营养品质遗传改良工作中,还应利用本研究改良方法对高代育种材料进行黄色素含量测定和筛选,以提高育种选择的精准度。

4 结论

建立了一个准确稳定、经济高效、操作简便的小麦籽粒黄色素含量检测方法。与传统方法相比,改良方法显著降低了工作量,缩短检测时间,效率提高12—15倍,且样本、试剂用量仅为原来的1/16,大大节约成本,可代替传统AACC 14-50法用于大规模样本黄色素含量测定。利用新改良方法对283份国内外小麦品种黄色素含量进行检测,筛选出不同环境下稳定存在的黄色素含量较低品种24个和黄色素含量较高品种26个。

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