碘比色法测定高粱中直链淀粉和支链淀粉的方法探讨

唐云; 闫海彦; 赵亚雄; 郇丹; 宗文文; 宋菲红

doi:10.13386/j.issn1002-0306.2022070293

碘比色法测定高粱中直链淀粉和支链淀粉的方法探讨

doi: 10.13386/j.issn1002-0306.2022070293

金徽酒股份有限公司，甘肃陇南 742308

详细信息

作者简介:
唐云（1974−），男，本科，高级工程师，研究方向：酿酒生产工艺与白酒质量管控，E-mail：ty@jinhuijiu.com

通讯作者:
闫海彦（1994−），女，硕士，研究方向：食品检测分析，E-mail：767928507@qq.com

中图分类号: O656.31
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- 文章访问数: 29
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出版历程
- 收稿日期: 2022-08-02
- 网络出版日期: 2023-05-22
- 刊出日期: 2023-07-01

Determination of Amylose and Amylopectin in Sorghum by Iodine Colorimetric Method

Jinhui Liquor Co. Ltd., Longnan 742308, China

摘要

摘要: 目的：高粱中淀粉含量与其衍生物的品质密切相关，对其中直链淀粉（Am）和支链淀粉（Ap）含量的测定很有必要。本文采用碘比色法对高粱籽粒中淀粉（Am、Ap）含量的进行测定，探讨最佳预处理与检测方法。方法：以高粱籽粒中的Am与Ap为主要研究对象，比较碘比色法中的三种检测方法，即：单波长混标法（I）、双波长单标法（II）及双波长混标法（III），通过精密度、检出限、定量限等对三种测定方法进行了一系列的方法学验证，并对预处理及测定过程中NaOH和KOH分散剂、糊化温度、糊化时间、乙酸和盐酸pH调节剂、碘试剂用量和显色时间进行了比较优化，最后通过重复性验证、回收率验证和常见谷物样品的测定验证了最佳方法的准确性。结果：三种方法的检出限和定量限均能满足实际样品的测定范围需求，其中检出限：Am均≤0.55 mg/L，Ap均≤2.75 mg/L，定量限：Am均≤1.65 mg/L，Ap均≤8.33 mg/L；样品准确率实验发现，混标溶液标曲得到的误差绝对值较小（0.13%），但单标溶液标曲误差较大（7.47%），因此需要选用混标溶液配制标曲；高粱籽粒中淀粉的最佳预处理与测定条件是：以NaOH作分散剂，以乙酸或盐酸为pH调节剂，70 ℃糊化10~20 min，采用标准碘试剂浓度，显色20~30 min内测定结果较为稳定、准确；测定方法中，双波长混标法（III）所测定的Am、Ap及总淀粉含量准确，且与理论粗淀粉含量吻合，重复性和回收率（80%~120%）高。结论：碘比色双波长混标法是测定高粱籽粒中直链淀粉和支链淀粉含量的最佳测定方法，可用于实际样品的测定。
- 直链淀粉 /
- 支链淀粉 /
- 碘比色法 /
- 高粱 /
- 分析检测
Abstract: Objective: The content of starch was closely related to the derivative products of sorghum. It was very important to determination of the content of starch, including amylose (Am) and amylopectin (Ap) in sorghum. In this study, the content of Am and Ap was determined by the iodine colorimetry. And conditions of pretreatment and determination were optimized. Method: Using Am and Ap in sorghum as the main object, the content of starch was measured by the iodine colorimetry with different detection methods, including the single wavelength mixed standard method (I), the dual wavelength single standard method (II), and the dual wavelength mixed standard method (III). A series of methodological verifications, such as the precision, detection limit and quantification limit, were compared. And the conditions of pretreatment, such as NaOH and KOH dispersant, gelatinization temperature and time, acetic acid and hydrochloric acid pH regulator, dosage of iodine reagent and color rendering time were optimized. The repeatability verification recovery rate and the determination of common grain samples of the obtained method was also tested, which verified the accuracy of the method. Result: The detection limits and quantification limits of the three methods could meet the requirements of the actual sample measurement range, with detection limits of Am≤0.55 mg/L, Ap≤2.75 mg/L, quantification limits of Am≤1.65 mg/L, Ap≤8.33 mg/L. The absolute error of the accuracy experiments was 0.13% from the mixed standard solution calibration curve, the single standard solution calibration curve error was relatively large (7.47%), thus the mixed standard solution was better to prepare the calibration curve. The optimal preprocessing of sorghum: Using NaOH as the dispersant, the gelatinization temperature at 70 ℃ for 10~20 min, with acetic acid or hydrochloric acid as pH regulator, using standard iodine reagent concentration, controlling the coloring time at 20~30 min, the determination results were relatively stable and accurate. The total content of starch was consistent with the starch content in theory, and it had a good repeatability and recovery (80%~120%). Conclusion: In summary, the dual wavelength mixed standard method of iodine colorimetry was a best method for measuring the content of Am and Ap in sorghum, and it was more suitable for the determination of actual samples in crops.
- amylose /
- amylopectin /
- iodine colorimetry /
- sorghum /
- analytical detection

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图 1 双波长法测定Am、Ap的全谱图

Figure 1. Full spectrum of Am and Ap determined by dual-wavelength method

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图 2 KOH（左）和NaOH（右）分散高粱溶液与碘络合后的颜色

Figure 2. Color of KOH (left) and NaOH (right) dispersed sorghum solution complexed with iodine

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图 3 糊化温度对吸光度/St含量的影响

Figure 3. Influence of gelatinization temperature on absorbance/total starch content

注：a：方法I；b：方法II；c：方法III；d：方法II-St质量；e：方法III-St质量。

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图 4 糊化时间对高粱淀粉吸光度值的影响

Figure 4. Effect of gelatinization time on absorbance value of sorghum starch

注：a：方法I；b：方法II；c：方法III；图5、图6同。

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5 碘试剂用量对高粱淀粉吸光度值的影响

5. Effect of iodine reagent dosage on absorbance values of sorghum starch

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图 6 显色时间对高粱淀粉吸光度值的影响

Figure 6. Influence of color development time on absorbance value of sorghum starch

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表 1 三种方法绘制标曲的Am和Ap用量

Table 1. Amounts of Am and Ap for plotting standard curves by three methods

序号	方法
	I		II		III
	Am（mL）	Ap（mL）	Am（mL）	Ap（mL）	Am（mL）	Ap（mL）
1	0	5.00	0.30	2.00	0	5.00
2	0.25	4.75	0.50	2.50	0.50	4.50
3	0.50	4.50	0.70	3.00	1.00	4.00
4	1.00	4.00	0.90	3.50	1.50	3.50
5	1.50	3.50	1.10	4.00	2.00	3.00
6	2.00	3.00	1.30	4.50	2.50	2.50
7	/	/	/	5.00	3.00	2.00
8	/	/	/	/	3.50	1.50

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表 2 三种方法测得的标准曲线

Table 2. Standard curves measured by three methods

方法	Am		Ap
I	y=0.02x+0.1586	R²=0.9973	/	/
II	y=0.0186x−0.00254	R²=0.9999	y=0.01835x+0.0372	R²=0.9968
III	y=0.00258x+0.0385	R²=0.9963	y=0.00275x−0.00302	R²=0.9987

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表 3 三种方法测定标准样品的准确性分析

Table 3. Accuracy analysis of standard samples determined by three methods

配制样品	方法I （mg/L）	准确率（%）	方法II （mg/L）	准确率（%）	方法III （mg/L）	准确率（%）
30% Am	29.302	97.67	33.458	111.52	29.782	99.27
20% Am	19.677	98.39	23.193	115.97	19.936	99.68
10% Am	10.339	103.39	12.283	122.83	10.021	100.21
70% Ap	70.698	100.98	67.28	96.11	70.056	100.08
80% Ap	80.323	100.40	78.553	98.19	80.015	100.02
90% Ap	89.661	99.62	90.189	100.21	89.948	99.94
平均值	/	100.08	/	107.47	/	99.87
误差（%）	/	0.08	/	7.47	/	-0.13

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表 4 三种方法对10份高粱样品淀粉含量的测定比较（%）

Table 4. Comparison of starch content of 10 sorghum samples determined by three methods (%)

序号	方法I			方法II			方法III
序号	Am	Ap	St	Am	Ap	St	Am	Ap	St
1	12.83	87.17	100	16.05	83.95	93.58	22.73	77.27	66.09
2	13.43	86.57	100	15.61	84.39	94.82	19.57	80.43	75.64
3	13.23	86.77	100	12.62	87.38	95.73	17.69	82.31	68.27
4	13.13	86.87	100	15.57	84.43	90.49	21.81	78.19	62.89
5	12.83	87.17	100	15.49	84.51	95.55	21.80	78.20	67.89
6	12.83	87.17	100	15.73	84.27	94.09	22.31	77.69	66.34
7	12.53	87.47	100	15.54	84.46	93.87	22.06	77.94	66.13
8	12.63	87.37	100	15.14	84.86	93.44	21.54	78.46	65.69
9	13.23	86.77	100	13.89	86.11	86.17	19.28	80.72	62.09
10	13.03	86.97	100	16.53	83.47	88.89	22.25	77.75	66.03
平均值	12.97	87.03	100	15.22	84.78	92.66	21.10	78.90	66.71
SD	0.29	0.29	0.00	1.14	1.14	3.13	1.66	1.66	3.68
RSD	2.22	0.33	0.00	7.48	1.34	3.38	7.87	2.11	5.52
检出限（mg/L）	0.49	/	/	0.55	2.75	/	0.53	2.35	/
定量限（mg/L）	1.47	/	/	1.65	8.33	/	1.61	7.13	/
注：Am和Ap表示占淀粉总量的百分比；St表示占试样的百分比；表9同。

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表 5 NaOH和KOH分散高粱样品测定Am，Ap的含量

Table 5. Determination of Am and Ap in NaOH and KOH dispersed sorghum sample

方法	分散剂种类
	NaOH		KOH
	Am（%）	Ap（%）	Am（%）	Ap（%）
方法I	15.54	84.46	6.24	93.76
方法II	16.90	83.10	3.75	96.25
方法III	15.75	84.25	6.25	93.75

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表 6 溶液pH对吸光度值的影响

Table 6. Influence of solution pH value on absorbance value

名称	pH	方法I	方法II （Am）	方法II （Ap）	方法III （Am）	方法III （Ap）
参比溶液	3.27	0	0	0	0	0
样品溶液	11.65	/	/	/	/	/
乙酸（1 mL，1 mol/L）	4.65	0.337	0.170	0.164	0.171	0.164
乙酸（4 mL, 1 mol/L）	3.84	0.340	0.163	0.172	0.163	0.172
盐酸（0.5 mL, 1 mol/L）	3.78	0.339	0.163	0.171	0.163	0.169

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表 7 高粱样品中淀粉含量的重复性测定

Table 7. Repeatability of starch content in sorghum samples

样品名称	重复次数	Am（mg）	Ap（mg）	St（mg）	Ap/Am
1号样	1	12.19±0.28	55.42±0.76	67.6±0.47	4.55±0.33
	2	10.88±0.47	60.07±0.54	70.95±0.63	5.52±0.58
	3	10.99±0.35	56.97±0.38	67.95±0.57	5.18±0.40
2号样	1	12.84±0.23	67.82±0.47	80.66±0.55	5.28±0.26
	2	12.08±0.15	68.59±0.59	80.89±0.62	5.68±0.24
	3	12.3±0.18	62.39±0.39	74.47±0.73	5.07±0.21
3号样	1	13.28±0.32	65.49±0.44	78.77±0.58	4.93±0.30
	2	12.19±0.44	71.69±0.56	83.88±0.25	5.88±0.52
	3	12.30±0.37	71.62±0.63	83.92±0.31	5.82±0.45

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表 8 高粱样品的加标回收率验证

Table 8. Verification of spiked recovery rate of sorghum samples

样品	加入量（mg/L）	实测值（mg/L）	回收率（%）
Am	0.25	0.271	108.40
	1.0	0.872	87.20
	6.0	5.443	90.72
Ap	3.5	3.871	110.60
	20	19.745	98.73
	70	69.253	98.93

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表 9 不同谷物样品中Am、Ap和St含量

Table 9. Am, Ap and total starch contents in different grain samples

样品	Am（%）	Ap（%）	St（%）	Ap/Am
高粱	14.01±0.42	85.99±0.18	69.86±0.47	6.14±0.32
小麦	18.85±0.29	81.15±0.32	63.51±0.26	4.31±0.25
玉米	12.14±0.46	87.86±0.42	83.36±0.39	7.24±0.35
大米	15.43±0.28	84.57±0.52	93.75±0.27	5.48±0.44

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