响应面法优化酶解辅助-超声提取菥蓂中的黑芥子苷

doi:10.13304/j.nykjdb.2022.0964

摘要/Abstract

摘要：

为从菥蓂种子中高效提取高得率的目的活性成分黑芥子苷，采用酶解辅助-超声提取法，并利用高效液相色谱测定其含量。在单因素试验的基础上，以酶添加量、酶解温度和酶解时间为关键因素，以黑芥子苷得率为响应值，应用Design-Expert V11软件，Box-Behnken Design响应面法优化菥蓂种子中黑芥子苷酶解辅助-超声提取工艺；并与单独超声、索氏提取2种提取法的黑芥子苷得率进行比较。结果表明，采用Agilent HC-C18柱（25 mm×4.6 mm×5 μm），流动相A和B分别为甲醇和0.5%磷酸水溶液，检测波长215 nm，柱温25 ℃，流速1 mL·min^-1，进样量10 μL，在等度洗脱10 min下，获得的分离效果和峰型较好。以筛选出的碱性蛋白酶为酶制剂，最佳提取工艺条件为：酶解温度49 ℃，酶用量2.90%，酶解时间18 min；在此条件下，菥蓂种子中黑芥子苷得率为2.679 mg·g^-1，经方差分析与理论值（2.687 mg·g^-1）差异不显著，但显著高于单独超声提取（2.036 mg·g^-1）和索氏提取（1.702 mg·g^-1）（P<0.05）。优化得到的菥蓂中黑芥子苷酶解辅助-超声提取工艺，方法简单、节能、环保、高效，目的活性成分得率高，可应用于菥蓂中黑芥子苷的生产实践，为菥蓂的深度开发奠定了理论基础。

关键词: 菥蓂, 黑芥子苷, 酶解辅助-超声提取, 响应面法

Abstract:

In order to obtain active ingredient sinigrin with high efficiency and high yield from seeds of Thlaspi arvense， the enzymolysis assisted-ultrasonic extraction method was used. The content of sinigrin was determined by high performance liquid chromatography （HPLC）. Based on the single factor experiment， enzymolysis time， enzymolysis temperature and enzyme addition amount were the key factors， and the yield of sinigrin was taken as the response value. The Design-Expert 11 software was applied and the Both-Behnken Design response surface methodology was used to optimize the enzymolysis assisted ultrasonic extraction process of sinigrin. The yield of sinigrin was compared with that of ultrasonic and Soxhlet extraction. The results showed that Agilent HC-C18 column （25 mm×4.6 mm×5 μm）， mobile phase A methanol， mobile phase B 0.5% phosphoric acid aqueous solution， detection wavelength 215 nm， column temperature 25 ℃， flow rate 1 mL·min^-1， sample size 10 μL， and the same degree elution for 10 min， the separation effect and peak shape were better. Using the screened alkaline protease as enzyme preparation， the optimal extraction conditions were that the enzymolysis temperature was 49 ℃， the enzyme dosage was 2.90%， the enzymolysis time was 18 min， the yield of sinigrin was 2.679 mg·g^-1 under optimal conditions， which was no significant difference with the theoretical value （2.687 mg·g^-1）. The yield of sinigrin was significantly higher than that of ultrasonic extraction （2.036 mg·g^-1） and Soxhlet extraction （1.702 mg·g^-1）（P<0.05）. The enzymolysis assisted-ultrasonic extraction process of sinigrin from T. arvense was simple， energy saving， eco-friendly， high efficiency and high yield， and it could be applied to the production and utilization of sinigrin from T. arvense， which laid a theoretical foundation for the in-depth development of T. arvense.

Key words: Thlaspi arvense, sinigrin, enzyme assisted-ultrasonic extraction, response surface methodology

中图分类号:

S377

刘东玲, 司皓, 郑宝江, 张玉红. 响应面法优化酶解辅助-超声提取菥蓂中的黑芥子苷[J]. 中国农业科技导报, 2024, 26(4): 225-233.

Dongling LIU, Hao SI, Baojiang ZHENG, Yuhong ZHANG. Optimization of Enzyme Assisted-ultrasonic Extraction of Sinigrin in Thlaspi arvense Seeds by Response Surface Methodology[J]. Journal of Agricultural Science and Technology, 2024, 26(4): 225-233.

图/表 10

图1 黑芥子苷标准品溶液和样品液的色谱图A：黑芥子苷标准品；B：样品；1为黑芥子苷

Fig. 1 HPLC chromatograms diagramof standardand sampleA：Standard of sinigrin； B：Sample； 1 is sinigrin

表1 响应面试验因素水平表

Table 1 Response surface experimental factor level table

水平Level	因素Factor
水平Level	A：酶添加量 Amount of enzyme added/%	B：酶解时间 Enzymatic hydrolysis time/min	C：酶解温度 Enzymatic hydrolysis temperature/℃
-1	2	15	45
0	3	20	50
1	4	25	55

图2 不同酶制剂下菥蓂中黑芥子苷的得率注：不同小写字母表示不同酶间在P<0.05水平差异显著。

Fig. 2 Yield of sinigrin in T. arvense underdifferent enzymesNote： Different lowercase letters indicate significant differences between different enzymes at P<0.05 level.

图3 不同单因素下菥蓂中黑芥子苷的得率注：不同小写字母表示不同处理间在P<0.05水平差异显著。

Fig. 3 Yield of sinigrin in T. arvense under different factorsNote：Different lowercase letters indicate significant differences between different treatments at P<0.05 level.

表2 中心组合优化方案及结果

Table 2 Response surface design and results

编号 Code	因素 Factor			Y：黑芥子苷得率 Sinigrin yield/（mg·g^-1）
编号 Code	A：酶添加量 Amount of enzyme added/%	B：酶解时间 Enzymatic hydrolysis time/min	C：酶解温度 Enzymatic hydrolysis temperature/℃	实际值 Actual value	预测值 Predicted value
1	2	15	50	2.50	2.52
2	3	15	45	2.61	2.60
3	2	25	50	2.36	2.37
4	4	15	50	2.45	2.44
5	3	20	50	2.63	2.67
6	4	25	50	2.30	2.34
7	3	20	50	2.69	2.67
8	4	20	55	2.34	2.35
9	3	20	50	2.70	2.67
10	3	20	50	2.67	2.67
11	3	20	50	2.65	2.67
12	3	15	55	2.46	2.47
13	4	20	45	2.35	2.38
14	2	20	45	2.45	2.45
15	3	25	55	2.41	2.43
16	2	20	55	2.41	2.39
17	3	25	45	2.39	2.39

表3 响应面回归方程方差分析结果

Table 3 Results of variance analysis of response surface regression equation

来源 Source	自由度 Degree of freedom	平方和 Sum of squares	均方 Mean square	F值 F valve	P值 P valve
模型 Model	9	0.270	0.030	34.84	<0.000 1
A	1	6.050×10^-3	6.050×10^-3	6.97	0.033 5
B	1	0.031	0.031	35.98	0.000 5
C	1	4.050×10^-3	4.050×10^-3	4.66	0.067 7
AB	1	6.250×10^-4	6.250×10^-4	0.72	0.424 3
AC	1	2.250×10^-4	2.250×10^-4	0.26	0.626 4
BC	1	7.225×10^-3	7.225×10^-3	8.32	0.023 5
A²	1	0.110	0.110	132.38	<0.000 1
B²	1	0.031	0.031	35.23	0.000 6
C²	1	0.056	0.056	64.39	<0.000 1
残差Residual	7	6.080×10^-3	8.686×10^-4
失拟项Lack of fit	3	2.800×10^-3	9.333×10^-4	1.14	0.434 6
误差Error	4	3.280×10^-3	8.200×10^-4
总离差Total deviation	16	0.280

表4 置信分析

Table 4 Confidence analysis

指标Index	响应值 Response value
标准偏差Standard deviation	0.029 0
变异系数Coefficient of variable/%	1.180 0
拟合系数R²	0.978 2
调整系数 R²_Adj	0.950 1
预测系数 R²_Pred	0.820 7

图4 黑芥子苷得率残差的正态分布及预测值与实际值的关系

Fig. 4 Normal distribution of residuals and relationship between predicted and actual values of sinigrin yield

图5 不同因素交互作用的响应面

Fig. 5 Response surface of interaction of different factors

图6 不同提取方法的黑芥子苷得率注：不同小写字母表示不同方法间在P<0.05水平差异显著。

Fig. 6 Yield of sinigrin by different extraction methodsNote：Different lowercase letters indicate significant differences between different methods at P<0.05 level.

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