动物营养学报 >

2021 , Vol. 33 >Issue 10: 5838 - 5848

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.10.043

实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

鸡蛋叶酸含量检测方法优化及其富集规律研究

  • 白彦 ,
  • 王瑞 ,
  • 杨玉 ,
  • 李锐瑞 ,
  • 武笑天
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  • 1. 山西农业大学动物科技学院, 太谷 030801;
    2. 吕梁学院生命科学系, 吕梁 033000
白彦(1985-),女,山西吕梁人,博士研究生,动物营养与饲料科学专业。E-mail:byshanxind@163.com

收稿日期: 2021-03-18

  网络出版日期: 2021-10-16

基金资助

山西功能农业共性关键技术研究与示范（201703D211001-05）；山西省"1331"工程畜牧学重点学科建设（J202011315）；山西省现代农业鸡产业技术体系（20211102）；山西省高等学校科技创新项目（2019L0959）

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Optimization of Method for Folate Content Determination in Eggs and Study on Its Enrichment Rule

  • BAI Yan ,
  • WANG Rui ,
  • YANG Yu ,
  • LI Ruirui ,
  • WU Xiaotian
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  • 1. College of Animal Science, Shanxi Agricultural University, Taigu 030801, China;
    2. Department of Life Sciences, Lyuliang University, Lyuliang 033000, China

Received date: 2021-03-18

  Online published: 2021-10-16

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摘要

本试验旨在建立简单、准确且较快速检测鸡蛋中叶酸含量的方法，并研究饲粮中添加叶酸对鸡蛋叶酸含量的影响，以及1个生物学产蛋年叶酸在鸡蛋中的富集规律。试验方法：1）基于高效液相色谱法，比较荧光检测器和紫外检测器的性能，比较超声、高温、酶解等提取方法及其组合对蛋黄叶酸检测值的影响，确定最佳检测方法。2）选取30周龄京红蛋鸡648只，随机分为6组，每组6个重复，每个重复18只，分别饲喂在基础饲粮中添加0（对照组）、1、3、6、12和24 mg/kg叶酸的试验饲粮，试验期35 d，筛选生产富叶酸鸡蛋饲粮中叶酸适宜添加量。3）依据筛选结果，在16 000只蛋鸡舍选10个监测点，于20、21、22、23、24、25、27、29、33、37、41、46、50、55、60周龄检测鸡蛋叶酸含量。结果表明：1）荧光检测器的检出限和定量限低于紫外检测器，峰面积和峰高极显著高于紫外检测器（P<0.01）。高温和超声5 min+高温提取的蛋黄叶酸含量检测值显著高于超声5 min+酶解和高温+酶解（P<0.05）。检测方法确定为超声5 min后90℃水浴5 min提取、荧光检测器检测，其精密度和稳定性的相对标准偏差（RSD）分别为2.15%和0.57%，回收率平均为98.01%。2）6和12 mg/kg组鸡蛋叶酸含量极显著高于对照组和1 mg/kg组（P<0.01），与3和24 mg/kg组无显著差异（P>0.05）。回归分析显示饲粮叶酸添加量与全蛋叶酸含量存在极显著三次曲线关系（决定系数=0.679；P<0.001）。3）20~60周龄饲喂添加7.5 mg/kg叶酸的饲粮，蛋黄叶酸含量在23周龄趋于饱和。20~33周龄，全蛋叶酸含量从36.9 μg/枚逐渐增加到87.7 μg/枚，之后基本稳定。综上所述：1）本试验优化改进的检测鸡蛋中叶酸含量的方法操作简单，准确性高，较快速。2）从20周龄起在京红蛋鸡饲粮中添加7.5 mg/kg叶酸，23周龄后蛋黄叶酸含量保持在5 μg/g以上，33周龄后全蛋叶酸含量保持在85 μg/枚以上。

关键词： 蛋黄; 5-甲基四氢叶酸; 高效液相色谱; 叶酸富集

本文引用格式

白彦 , 王瑞 , 杨玉 , 李锐瑞 , 武笑天 . 鸡蛋叶酸含量检测方法优化及其富集规律研究[J]. 动物营养学报, 2021 , 33(10) : 5838 -5848 . DOI: 10.3969/j.issn.1006-267x.2021.10.043

Abstract

This experiment aimed to develop a simple, accurate and relatively fast determination method for folate in eggs, to study the effect of dietary folate supplementation on the folate content in eggs, and to study the enrichment rules of folate in eggs during the period of egg-laying. Experimental methods:1) The optimal detection method was determined by comparing the performance of fluorescence detector and ultraviolet detector of high-performance liquid chromatography, and comparing the detection value of folate in yolks extracted by sonicate, heat treat (water-bath at 90℃), enzymatic hydrolysis and their combination. 2) The suitable dietary folic acid supplemental level for folate-enriched eggs production was screened under the feeding experiment lasted for 35 days. Six hundred and forty-eight Jinghong laying hens (30 weeks of age) were randomly divided into 6 groups with 6 replicates in each group and 18 hens in each replicate. Laying hens in one group (control group) was fed a basal diet, while those in the other five groups were fed the basal diet supplemented with 1, 3, 6, 12 and 24 mg/kg folic acid, respectively. 3) Given these results of screening test, a long-term experiment was conducted by feeding 16 000 laying hens (20 to 60 weeks of age) with 7.5 mg/kg folic acid supplementation in the diet, and detecting the content of folate in 10 egg monitoring points at 20, 21, 22, 23, 24, 25, 27, 29, 33, 37, 41, 46, 50, 55 and 60 weeks of age. The results showed as follows:1) the limit of detection (LOD) and the limit of quantitation (LOQ) of fluorescence detector were lower, and the peak area and peak height of fluorescence detector were extremely significantly higher than those of ultraviolet (UV) detector (P<0.01). The detected value of folate content in yolk extracted by heat treatment and sonicate for 5 min+heat treatment was significantly higher than that extracted by sonicate for 5 min+enzymolysis and heat treat treatment+enzymolysis (P<0.05). The best condition for extracting folate from yolk was 5 min of sonicate followed by 5 min of 90℃ water bath, and the fluorescence detector had good detection performance. The relative standard deviation (RSD) of precision and stability were 2.15% and 0.57%, respectively; the spiked recoveries averaged 98.01%. 2) The folate content in eggs of 6 and 12 mg/kg groups were extremely significantly higher than those of control group and 1 mg/kg group (P<0.01), and there was no significant difference with 3 and 24 mg/kg groups (P>0.05). Regression analysis showed that there was an extremely significant cubic linear relationship between dietary folic acid supplemental level and whole egg folate content (determinate coefficient=0.679; P<0.001). 3) Adding 7.5 mg/kg folic acid to laying hen diet from 20 to 60 weeks of age, the content of folate in yolk tended to be saturated at 23 weeks of age, and the content of folate in whole egg gradually increased from 36.9 μg/egg at 20 weeks of age to 87.7 μg/egg at 33 weeks of age, then remained stable. In conclusion:1) the egg folate detection method determined in this experiment is simple to operate, high accuracy accurate and rapid. 2) Due to add 7.5 mg/kg folic acid to diet from 20 weeks of age, the folate content in yolk remains at more than 5 μg/g from 23 weeks of age, and the folate content in whole egg remains at more than 85 μg/egg from 33 weeks of age.

Key words： yolk; 5-methyltetrahydrofolate; high performance liquid chromatography; folate enrichment

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