激光共聚焦显微法测定面团面筋网络结构的影响因素

doi:10.3864/j.issn.0578-1752.2023.14.013

摘要/Abstract

摘要：

【目的】面团样品切片、混合染色剂浓度以及染色时间是激光共聚焦显微镜测定面团面筋网络结构的关键影响因素。本研究旨在分析这些因素对面团面筋网络结构测定结果的影响规律，进而优化出最佳的切片厚度和染色条件，为面团面筋网络结构及面制品品质高质量研究提供技术支撑。【方法】选取面筋筋力不同的两个小麦品种郑麦366（Z366）和小偃22（X22）为试验材料，磨粉制作成面团，并分别冷冻0 d和1 d；用冷冻切片机将面团分别切成10、14和20 μm厚度的薄片，并配制不同浓度罗丹明B（RDB）和异硫氰酸荧光素（FITC）混合染液，分别染色1、10和20 min，分析这些因素对激光共聚焦显微法（CLSM）测定面团面筋网络结构的影响。【结果】切片厚度为10 μm时，郑麦366（Z366）和小偃22（X22）面团的面筋网络结构均最清晰，且在此切片厚度下，两个品种冷冻0 d和冷冻1 d的面团面筋网络结构之间的差异均最显著（P<0.01）。染色条件研究结果表明，低浓度染色剂（0.001%+0.01%）染色时间越长，面筋蛋白网络结构呈现越清晰；高浓度染色剂（0.025%+0.25%）在染色1 min时，面筋网络结构呈现最佳，染色10 min时，面筋网络结构最差。【结论】面团切片厚度10 μm，不同筋力和不同冷冻处理的面团网络结构差异最大，即面筋网络结构区分效果最明显；在此切片厚度条件下，当不考虑时间成本时，运用低浓度混合染色剂染色20 min面筋网络结构呈现效果最佳；当需要节省时间并观察淀粉颗粒细微结构时，使用高浓度染色剂染色1 min效果最好。

关键词: 激光共聚焦显微法, 面筋网络结构, 切片厚度, 混合染色剂

Abstract:

【Objective】The thickness of sample slices, the concentration of staining solution and the staining time are the key factors influencing the results of gluten network structure analysis using confocal laser scanning microscopy (CLSM). The aim of this study was to investigate the effects of the slice thickness, the staining solution concentration and the staining time on the CLSM results of gluten network structure, and the optimal slice thickness and staining conditions for the dough structure analysis were selected, so as to provide a technical support for the studies on dough structure and/or its final products. 【Method】Two wheat varieties, i.e., Zhengmai 366 (Z366) and Xiaoyan 22 (X22) with different wet gluten indexes, were selected as raw materials, which were milled and made into dough, respectively. Then, the dough samples were frozen for 0 and 1 day. The dough slice thickness was set as 10, 14 and 20 μm, respectively. Meanwhile, two concentrations of staining solution (0.001%+0.01%, 0.025%+0.25%) using Rhodamine B (RDB) and fluorescein isothiocyanate (FITC) were prepared. The staining time was set for 1, 10 and 20 min, respectively. 【Result】The sample slice with 10 μm thickness showed the best and clearest image for both doughs of Z366 and X22. Besides, under this slice thickness, the gluten network of the dough frozen stored for 0 day showed the most obvious difference compared with that for 1 day (P<0.01). Additionally, under low-concentration staining agent (0.001%+0.01%), the clearer the results of gluten network structure were obtained with longer staining time. Oppositely, under the high-concentration staining agent (0.025%+0.25%), the slice with a staining time of 1 min showed the most rational results on the gluten network structure, whereas the result on the slice with a staining time of 10 min was the worst. 【Conclusion】The gluten network structure of dough with different protein content and freezing treatments demonstrated more differences when the thickness of dough slices was 10 μm, indicating the gluten network structure of different samples could be distinguished obviously. Under the selected thickness, the gluten network structure of 20 min stained with low-concentration mixed staining agent showed the best effect if time-consuming was not considered. Moreover, the high-concentration staining for 1 min was time-saving and better for observing the structure of starch granules.

Key words: confocal laser scanning microscopy, gluten network structure, slice thickness, mixed staining agent

姜继凯, 张影全, 郭波莉, 杨静洁, 黄璐瑶, 李明, 钟耕. 激光共聚焦显微法测定面团面筋网络结构的影响因素[J]. 中国农业科学, 2023, 56(14): 2787-2797.

JIANG JiKai, ZHANG YingQuan, GUO BoLi, YANG JingJie, HUANG LuYao, LI Ming, ZHONG Geng. Influencing Factors of Gluten Network Structure Analysis Using Confocal Laser Scanning Microscopy[J]. Scientia Agricultura Sinica, 2023, 56(14): 2787-2797.

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样品 Sample	灰分（干基） Ash (dry basis, %)	水分 Moisture (%)	蛋白含量（干基） Protein content (dry basis, %)	湿面筋含量（湿基，含水量14%） Wet Gluten (wet basis, moisture content 14%)
Z366	0.47	12.55	13.19	30.14
X22	0.45	11.16	13.57	31.21

样品 Sample	吸水率 Water absorption (%)	形成时间 Development time (min)	稳定时间 Stability time (min)	弱化度 Degree of softening (FU)	粉质质量指数 Farinograph quality number (mm)	高分子谷蛋白亚基 HMW glutenin subunit
Z366	68.50	9.2	10.5	66	141	1,7+8,5+10
X22	69.20	2.7	1.3	139	37	N,7+9,2+12

品种 Variety	切片厚度 Thickness of section (μm)	冷冻时间 Freezing time (d)	面筋百分比面积Gluten percentage area (%)	t值（显著性） t-value (significance)	分支率Branching rate (×10^-3)	t值（显著性） t-value (significance)	节点密度Junctions density (×10^-4)	t值（显著性） t-value (significance)	平均面筋长度Average gluten length (×10²μm)	t值（显著性） t -value (significance)	端点率End-point rate (×10^-3)	t值（显著性） t-value (significance)	空隙度Lacunarity（×10^-2）	t值（显著性） t-value (significance)
Z366	10	0	40.91±1.62	7.920**	2.54±1.28	6.044**	10.40±0.82	8.881**	2.52±0.14	4.059**	2.40±0.11	1.435	7.57±0.70	-7.517**
	10	1	35.64±1.66	7.920**	2.14±0.17	6.044**	7.64±0.75	8.881**	2.19±0.34	4.059**	2.33±0.13	1.435	10.41±0.99	-7.517**
	14	0	38.08±1.40	3.048**	2.00±0.81	-1.264	7.61±0.29	0.325	2.13±0.13	1.390	1.92±0.21	-5.771**	10.08±0.37	-2.345*
	14	1	35.61±1.47	3.048**	2.10±0.15	-1.264	7.48±0.74	0.325	1.91±0.31	1.390	2.41±0.14	-5.771**	10.79±0.98	-2.345*
	20	0	35.11±2.26	1.859	2.11±1.49	2.532*	7.40±0.77	2.781*	1.95±0.34	2.458*	2.38±0.16	-2.056	11.02±1.49	-1.640
	20	1	33.18±1.96	1.859	1.94±0.12	2.532*	6.45±0.63	2.781*	1.58±0.28	2.458*	2.61±0.23	-2.056	12.06±1.16	-1.640
X22	10	0	34.64±1.35	3.141**	2.09±0.14	6.413**	7.25±0.72	5.652**	9.74±0.20	4.295**	2.57±0.20	-2.470*	10.39±0.89	-4.951**
	10	1	32.34±1.50	3.141**	1.81±0.08	6.413**	5.98±0.45	5.652**	10.41±0.37	4.295**	2.77±0.24	-2.470*	12.32±1.13	-4.951**
	14	0	35.20±1.49	2.920*	2.02±0.16	2.635*	7.10±0.52	4.947*	10.04±0.54	4.025**	2.44±0.21	-2.077	10.54±0.76	-3.106**
	14	1	33.02±1.58	2.920*	1.82±0.16	2.635*	5.98±0.43	4.947*	10.52±0.64	4.025**	2.64±0.20	-2.077	12.04±1.13	-3.106**
	20	0	33.05±1.70	1.427	2.05±0.13	6.442**	6.77±0.60	5.475**	9.76±0.24	5.193**	2.72±0.21	-2.086	11.46±1.11	-2.189*
	20	1	31.99±1.70	1.427	1.72±0.11	6.442**	5.50±0.46	5.475**	10.58±0.42	5.193**	2.93±0.27	-2.086	12.53±1.13	-2.189*

品种 Variety	浓度（罗丹明B+异硫氰酸荧光素） Concentration (RDB+FITC, %)	时间 Time (min)	面筋百分比面积 Gluten percentage area (%)	分支率 Branching rate (×10^-3)	节点密度 Junctions density (×10^-4)	平均面筋长度 Average gluten length (×10² μm)	端点率 End-point rate (×10^-3)	空隙度 Lacunarity (×10^-2)
Z366	0.001+0.01	1	30.59±1.04bC	1.93±0.08cD	5.89±0.36bD	1.67±0.14bC	2.50±0.12aB	14.17±1.16aB
		10	29.69±1.83bCD	2.13±0.09bC	6.34±0.66bCD	1.74±0.31bC	2.55±0.25aB	15.53±1.30aA
		20	34.63±0.72aB	2.50±0.05aB	8.64±0.12aB	2.47±0.24aB	2.15±0.07bC	11.38±0.72bC
	0.025+0.25	1	37.53±1.64aA	2.67±0.10aA	10.01±0.72aA	3.58±0.86aA	2.06±0.20bD	8.86±0.71cD
		10	28.60±1.46cD	1.71±0.15cE	4.89±0.60cE	1.10±0.19bD	2.90±0.39aA	15.64±0.87aA
		20	30.94±0.91bC	2.16±0.11bC	6.68±0.50bC	1.45±0.20bCD	2.70±0.21aAB	13.30±0.88bB
X22	0.001+0.01	1	33.12±1.65aA	1.83±0.15bB	6.05±0.59bB	1.34±0.26abBC	2.81±0.24abBC	11.87±1.03bCD
		10	29.17±1.25bB	1.72±0.14bB	5.01±0.43cC	1.12±0.14bCD	2.89±0.22aBC	15.59±1.17aB
		20	33.61±1.87aA	2.06±0.12aA	6.93±0.78aA	1.64±0.36aA	2.53±0.20bD	11.91±1.23bCD
	0.025+0.25	1	33.75±2.09aA	1.87±0.13aB	6.29±0.47aB	1.47±0.20aAB	2.65±0.19bCD	11.17±1.10cD
		10	27.65±1.30cB	1.48±0.07cC	4.09±0.30cD	0.99±0.12bD	2.93±0.19aA	17.22±1.75aA
		20	31.72±2.22bA	1.79±0.13bB	5.67±0.63bB	1.32±0.16aBC	2.87±0.16aBC	12.87±1.88bC

品种 Variety	浓度（罗丹明B+异硫氰酸荧光素） Concentration (RDB+FITC, %)	时间 Time (min)	面筋百分比面积 Gluten percentage area (%)	分支率 Branching rate (×10^-3)	节点密度 Junctions density (×10^-4)	平均面筋长度 Average gluten length (×10²μm)	端点率 End-point rate (×10^-3)	空隙度 Lacunarity (×10^-2)
Z366	0.001+0.01	1	31.69±0.51bB	1.92±0.04cC	6.09±0.19cC	1.51±0.27bB	2.79±0.18aB	12.21±0.26aB
		10	33.40±1.54aA	2.04±0.07bBC	6.81±0.49bB	1.89±0.24aA	2.39±0.20bC	11.72±1.00aBC
		20	32.84±0.81abAB	2.43±0.10aA	7.98±0.42aA	1.88±0.19aA	2.53±0.22bC	12.00±0.74aBC
	0.025+0.25	1	34.24±1.15aA	2.13±0.12aB	7.29±0.53aB	1.89±0.19aA	2.39±0.14bC	10.92±0.66bC
		10	29.56±0.94cC	1.69±0.15cD	5.00±0.50cD	1.10±0.17bC	2.88±0.28aA	14.10±0.78aA
		20	31.59±1.75bB	1.90±0.11bC	6.02±0.54bC	1.69±0.32aAB	2.35±0.28bC	13.64±1.90aB
X22	0.001+0.01	1	29.36±1.35cD	1.54±0.07cD	4.51±0.14cD	1.00±0.06cD	3.05±0.13aA	14.57±1.53aA
		10	32.94±0.75bBC	1.85±0.13bB	6.11±0.52bBC	1.54±0.22bB	2.67±0.12bB	11.53±0.69bC
		20	36.36±1.06aA	2.08±0.09aA	7.59±0.48aA	2.02±0.30aA	2.41±0.16cC	9.60±0.77cD
	0.025+0.25	1	32.57±2.56aC	1.87±0.16aB	6.12±0.96abBC	1.49±0.35aB	2.66±0.24aB	12.73±1.69aB
		10	32.20±1.35aC	1.71±0.08bC	5.52±0.43bC	1.17±0.15bCD	2.93±0.22aA	11.78±0.78bBC
		20	34.33±1.80aB	1.85±0.07aB	6.36±0.51aB	1.43±0.33abBC	2.82±0.25aAB	10.34±1.02cD