响应面法优化低聚木糖诱导大豆抗毒素合成条件

doi:10.13304/j.nykjdb.2022.0073

摘要/Abstract

摘要：

为研究大豆抗毒素（glyceollins，GLYs）合成的诱导条件，以低聚木糖（xylooligosaccharides，XOS）诱导黑豆合成的GLYs含量为考察目标，以XOS诱导质量浓度、诱导时间、培养温度为考察单因素，在此基础上采用中心组合设计-响应面法优化GLYs合成量的诱导条件。结果显示，建立的模型适合度显著（F=13.780），GLYs实际合成量与预测值拟合良好（R²=0.925 4）。优化后的最佳诱导条件为XOS诱导质量浓度4.0 g·100 mL^-1，培养温度24 ℃，诱导时间4 d。在此条件下，GLYs合成量为1.376 5 mg·g^-1 DW，与预测值（1.411 8 mg·g^-1 DW）相比，相对误差较小。培养温度对GLYs合成量影响极显著（P<0.05）；XOS诱导质量浓度和诱导时间对GLYs合成量影响不显著；三因素交互作用对GLYs合成量影响均不显著。由此表明，该方法合理可行，为大豆抗毒素制备及功能产品开发提供了理论依据。

关键词: 黑豆, 大豆抗毒素, 诱导, 低聚木糖, 响应面法

Abstract:

To study condition for inducible synthesis of glyceollins （GLYs）， the content of GLYs synthesized by xylooligosaccharides（XOS） in black soybean was used as an index. And the induction time， XOS mass concentration and temperature were optimized as independent variables. Based on the single factor experiment， central composite design-response surface methodology was employed to optimize inducible condition for GLYs synthesis. The results indicated that the established model fitted significantly well （F=13.780）， and produced GLYs content was matched well with predicted value （R²=0.925 4）. The optimal conditions subjected to optimization were XOS mass concentration 4.0 g·100 mL^-1， temperature 24 ℃， induction time 4 d. Under this condition， the content of accumulated GLYs reached 1.376 5 mg·g^-1 DW. Effect of temperature factor on producing GLYs content （P<0.05） was significant， but XOS mass concentration， induction time and interactions among factors were not significant on GLYs content. Hence， the present method was reasonable and feasible， which provided theoretical basis for glyceollin preparation and functional products development.

Key words: black soybean, glyceollins, induction, xylooligosaccharides, response surface methodology

中图分类号:

王凯强, 杨雪, 李常风, 段晓, 彭晴, 乔宇, 石波. 响应面法优化低聚木糖诱导大豆抗毒素合成条件[J]. 中国农业科技导报, 2022, 24(10): 208-217.

Kaiqiang WANG, Xue YANG, Changfeng LI, Xiao DUAN, Qing PENG, Yu QIAO, Bo SHI. Optimization of Glyceollins Synthesis Condition Induced by Xylooligosaccharides Based on Response Surface Methodology[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 208-217.

图/表 9

表1 HPLC梯度洗脱程序

Table 1 HPLC gradient elution procedure

时间Time/min	流速Rate/（mL·min^-1）	A/%	B/%
0	1.0	100	0
17	1.0	55	45
27	1.0	10	90
33	1.0	10	90
34	1.0	0	100
49	1.0	0	100
50	1.0	100	0
65	1.0	100	0

表2 中心组合设计因素与水平

Table 2 Factors and levels for central composite design

水平Level	因素Factor
水平Level	X₁：诱导时间 Induction time/d	X₂：XOS诱导质量浓度 XOS concentration/（g·100 mL^-1）	X₃：培养温度 Temperature/℃
-1.682	2	2.0	17
-1.000	3	3.0	21
0.000	4	4.0	25
+1.000	5	5.0	29
+1.682	6	6.0	33

表3 诱导时间、诱导浓度和培养温度的中心组合设计及GLYs含量

Table 3 CCD of induction time， XOS concentration and temperature and the GLYs content

运行 Run	X₁：时间 Time/d	X₂：质量浓度 Mass concentration/（g·100 mL^-1）	X₃：温度 Temperature/℃	大豆抗毒素含量 GLYs content/（mg·g^-1 DW）
运行 Run	X₁：时间 Time/d	X₂：质量浓度 Mass concentration/（g·100 mL^-1）	X₃：温度 Temperature/℃	实际值 Actual value	预测值 Predicted value
1	3	3	21	0.935 8	1.00
2	5	3	21	0.932 7	1.00
3	3	5	21	0.949 0	0.96
4	5	5	21	1.047 5	1.00
5	3	3	29	0.526 8	0.61
6	5	3	29	0.594 0	0.62
7	3	5	29	0.807 9	0.78
8	5	5	29	0.850 7	0.82
9	2	4	25	1.012 5	0.95
10	6	4	25	0.964 1	0.98
11	4	2	25	1.017 4	0.89
12	4	6	25	0.948 3	1.02
13	4	4	17	1.002 8	0.96
14	4	4	33	0.489 9	0.48
15	4	4	25	1.631 6	1.39
16	4	4	25	1.350 2	1.39
17	4	4	25	1.318 1	1.39
18	4	4	25	1.326 0	1.39
19	4	4	25	1.308 9	1.39
20	4	4	25	1.390 9	1.39

图1 低聚木糖诱导子叶组织中大豆抗毒素的合成确认A：XOS诱导子叶组织的HPLC谱图；B：未处理子叶组织的HPLC谱图；C和D：分离纯化GLYs的HPLC谱图与质谱图

Fig. 1 Confirmation of the glyceollins synthesis from XOS-induced soybean cotyledon tissueA： HPLC chromatogram of XOS-treated cotyledon tissue； B： HPLC chromatogram of untreated cotyledon tissue； C and D： HPLC chromatogram and MS spectrum of separated and purified GLYs

图2 3个产地黑豆子叶中GLYs的诱导合成量注：不同小写字母表示不同品种间在P<0.05水平差异显著。

Fig. 2 Synthesized GLYs content from three origins of black soybean under inductionNote：Different lowercase letters indicate significantly differences between different treatments at P<0.05 level.

图3 不同因素下临沂大粒黑豆中大豆抗毒素的诱导合成量注：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 Synthesized GLYs contents from LL under induction of different factorsNote：Different lowercase letters indicate significantly differences between different treatments at P<0.05 level.

表4 诱导时间、诱导质量浓度和培养温度的中心组合设计试验模型的可信度

Table 4 Credibility for CCD model of induction time， XOS concentration and temperature

变异来源 Source of variation	平方和 Sum of squares	自由度 df	均方 Mean square	F值 F value	P值 P value
模型Model	1.570	9	0.170	13.780	0.000 2
X₁	1.126×10^-3	1	1.126×10^-3	0.089	0.771 5
X₂	0.022	1	0.022	1.750	0.215 5
X₃	0.280	1	0.280	21.980	0.000 9
X₁X₂	7.450×10^-4	1	7.450×10^-4	0.059	0.813 1
X₁X₃	2.664×10^-5	1	2.664×10^-5	2.107×10^-3	0.964 3
X₂X₃	0.021	1	0.021	1.660	0.226 6
X $12$	0.330	1	0.330	25.950	0.000 5
X $22$	0.340	1	0.340	26.620	0.000 4
X $32$	0.810	1	0.810	63.710	<0.000 1
残差Residual	0.130	10	0.013	—	—
失拟项Lack of fit	0.051	5	0.010	0.670	0.665 0
纯误差Pure error	0.076	5	0.015	—	—
总误差Total error	1.700	19	—	—	—

表4 诱导时间、诱导质量浓度和培养温度的中心组合设计试验模型的可信度

Table 4 Credibility for CCD model of induction time， XOS concentration and temperature

变异来源 Source of variation	平方和 Sum of squares	自由度 df	均方 Mean square	F值 F value	P值 P value
模型Model	1.570	9	0.170	13.780	0.000 2
X₁	1.126×10^-3	1	1.126×10^-3	0.089	0.771 5
X₂	0.022	1	0.022	1.750	0.215 5
X₃	0.280	1	0.280	21.980	0.000 9
X₁X₂	7.450×10^-4	1	7.450×10^-4	0.059	0.813 1
X₁X₃	2.664×10^-5	1	2.664×10^-5	2.107×10^-3	0.964 3
X₂X₃	0.021	1	0.021	1.660	0.226 6
X $12$	0.330	1	0.330	25.950	0.000 5
X $22$	0.340	1	0.340	26.620	0.000 4
X $32$	0.810	1	0.810	63.710	<0.000 1
残差Residual	0.130	10	0.013	—	—
失拟项Lack of fit	0.051	5	0.010	0.670	0.665 0
纯误差Pure error	0.076	5	0.015	—	—
总误差Total error	1.700	19	—	—	—

图4 拟合模型的适应性分析

Fig. 4 Adaptability analysis of the fitting model

图5 诱导时间、XOS诱导质量浓度、培养温度交互效应对GLYs累积量的等高线图和曲面图A和B： XOS诱导浓度和诱导时间的交互效应；C和D：培养温度和XOS诱导质量浓度的交互效应；E和F：培养温度和诱导时间的交互效应。

Fig. 5 Contour and response surface plots for the interactive effects of induction time， XOS mass concentrationA and B： Interactive effect of induction time and XOS concentration； C and D： Interactive effect of temperature and XOS mass concentration； E and F： Interactive effect of temperature and induction time.and temperature on GLYs content

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