基于R5 ELISA和RP-HPLC法的小麦发芽过程中主要致敏蛋白含量变化

doi:10.3864/j.issn.0578-1752.2020.06.016

摘要/Abstract

摘要：

【目的】探索不同萌发状态小麦麸质蛋白、醇溶蛋白和谷蛋白等主要致敏蛋白含量的动态变化规律,以及醇溶蛋白、谷蛋白亚基的含量变化,为无麸质食品的研发和发芽小麦的利用提供科学依据。【方法】控制发芽条件获得7种不同萌发状态的小麦,采用SDS-PAGE分析不同萌发状态小麦醇溶蛋白（Gliadins）、谷蛋白（Glutenin）的亚基组成变化,通过R5 ELISA和RP-HPLC进一步测定小麦发芽过程中麸质蛋白、醇溶蛋白和谷蛋白亚基的含量变化。【结果】以R5 ELISA法和RP-HPLC法两种方法可有效测定小麦中麸质蛋白、醇溶蛋白和谷蛋白亚基的含量,发芽处理对上述过敏蛋白及亚基有不同程度的影响;麸质蛋白含量在发芽初期变化不大,后期显著减少;ω-醇溶蛋白相对含量变化不显著;α-/β-醇溶蛋白的相对含量在发芽过程中大幅度降低（从未处理小麦籽粒的41.85%降低到发芽处理后的31.51%—35.35%,P＜0.01）,γ-醇溶蛋白相对含量从31.37%显著增加到发芽处理后的36.69%—39.02%（P＜0.05）;高分子量麦谷蛋白亚基（high molecular weight glutenin subunit,HMW-GS）和低分子量麦谷蛋白亚基（low molecular weight glutenin subunit,LMW-GS）的相对含量变化不显著,HMW-GS略有减少,从8.66%（未处理组）降低到5.94%（芽长1/4）,再回升到7.28%（芽长=籽粒长）;LMW-GS略有增加,从8.30%（未处理组）增加到10.45%（芽长=籽粒长）。【结论】R5 ELISA法和RP-HPLC法两种方法都可以有效进行小麦致敏蛋白定量分析;发芽过程中小麦的致敏蛋白含量总体呈下降趋势,特别是在发芽芽长达籽粒长1/2时,含有最多致敏肽的α-/β-醇溶蛋白下降尤为显著,提示小麦进行适度发芽处理可以降低致敏性。

关键词: 乳糜泻, 醇溶蛋白, 谷蛋白, 麸质蛋白, R5 ELISA, RP-HPLC

Abstract:

【Objective】 The current study was conducted to determine the dynamic changes of gluten, gliadins and glutenin contents in different germination states of wheat, so as to provide a scientific basis for the development of gluten-free food and utilization of germinated wheat. 【Method】Seven wheat grains with different germination states were obtained by controlling the germination conditions. Changes of composition of gliadins and glutenin were analyzed by sodium dedecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and changes of gluten, gliadins and glutenin subunits during wheat germination were further determined by enzyme-linked immunosorbent assay based on R5 antibody (R5 ELISA) and reversed-phase high-performance liquid chromatography (RP-HPLC). 【Result】The content of gluten, gliadins and glutenin subunits in wheat could be determined by R5 ELISA and RP-HPLC. Germination treatment had different effects on the allergic proteins and subunits mentioned above. The content of gluten changed little at the early stage of germination, but decreased significantly at the later stage. The relative content of ω-gliadins did not change significantly. During the germination process, the relative content of α-/β-gliadins was significantly reduced, with the percentage of untreated wheat seeds decreased from 41.85% to 31.51%-35.35% after germination (P＜0.01). The relative content of γ-gliadins increased significantly from 31.37% to 36.69%-39.02% after germination (P＜0.05). The relative content changes of high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS) were not significant. HMW-GS decreased slightly from 8.66% (untreated group) to 5.94% (1/4 bud length) and then to 7.28% (bud length=grain length). LMW-GS increased slightly from 8.30% (untreated group) to 10.45% (bud length=grain length).【Conclusion】Both of the methods R5 ELISA and RP-HPLC could be used for quantitative analysis of wheat sensitized proteins. The content of allergenic protein in wheat decreased during germination. In particular, when the germinating bud was up to 1/2 long, the α-/β-gliadin which containing the most sensitive peptide decreased significantly. It was suggested that moderate germination treatment could reduce sensitization of wheat.

Key words: celiac disease, gliadin, glutenin, gluten, R5 ELISA, RP-HPLC

胡慧敏,潘雪峰,杨恒,陈晨,陈银基. 基于R5 ELISA和RP-HPLC法的小麦发芽过程中主要致敏蛋白含量变化[J]. 中国农业科学, 2020, 53(6): 1247-1255.

HuiMin HU,XueFeng PAN,Heng YANG,Chen CHEN,YinJi CHEN. Wheat Gluten, Gliadins and Glutenin Content Changes During Germination Based on the Methods of R5 ELISA and RP-HPLC[J]. Scientia Agricultura Sinica, 2020, 53(6): 1247-1255.

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亚基 Subunit	发芽状态Germination state
亚基 Subunit	A	B	C	D	E	F	G
ω	9.48±1.06a	9.81±0.40a	9.69±0.84a	11.30±1.00a	10.20±0.37a	12.03±0.47a	10.57±0.88a
α+β	41.85±0.63A	33.81±1.65B	34.36±1.83B	35.35±0.77B	33.19±1.35B	31.51±0.32B	33.52±0.77B
γ	31.37±0.76a	36.69±0.37b	39.02±1.04b	37.62±2.40b	38.70±2.27b	37.28±1.29b	36.91±1.10b
HMW	8.66±0.61a	8.98±1.33a	8.12±1.11a	6.32±1.15a	5.94±0.34a	8.00±0.20a	7.28±0.19a
LMW	8.30±0.74a	9.80±0.61a	8.81±0.40a	8.73±0.80a	10.13±1.03a	10.49±0.33a	10.45±0.43a