烟草秸秆绿原酸提取工艺优化及其抑菌效果研究

doi:10.13304/j.nykjdb.2021.0888

摘要/Abstract

摘要：

为扩展烟草秸秆的利用途径，探究烟草秸秆绿原酸对烟草疫霉的抑菌效果，采用单因素试验和响应面分析法优化烟草秸秆绿原酸微波辅助提取工艺，并通过离体培养探讨烟草秸秆绿原酸对烟草疫霉菌的抑制作用。单因素及响应面结果表明，烟草秸秆绿原酸最佳提取工艺为料液比1∶15（g·mL^-1），提取温度50.03 ℃，乙醇体积分数50%，此时粗提物中绿原酸含量为3.48 mg·g^-1；离体条件下，烟草秸秆绿原酸的添加均抑制了疫霉菌的生长，其中175 mg·L^-1烟草秸秆绿原酸对菌落直径的抑制效果最为显著，抑菌率高达32%。此外，碘化丙啶（PI）染色结果表明，175 mg·L^-1的烟草秸秆绿原酸可通过破坏烟草疫霉菌细胞膜的完整性抑制菌丝生长。研究结果为烟草秸秆有效利用和新型天然植物源杀菌剂开发奠定了基础。

关键词: 烟草秸秆, 绿原酸, 响应面优化, 抑菌效果

Abstract:

In order to expand the utilization approach to tobacco stalk， and explore the antibacterial effect of tobacco stalk chlorogenic acid on tobacco Phytophthora， single factor test and response surface analysis were used to optimize the microwave-assisted extraction process of chlorogenic acid from tobacco stalk and isolated culture was applied to explore the antifungal effect of chlorogenic acid on Phytophthora nicotianae. The single factor and response surface results showed that the optimal extraction process conditions were： solid-liquid ratio （g·mL^-1）1∶15， extraction temperature 50.03 ℃， ethanol volume fraction 50%. Under these conditions， the extraction amount of chlorogenic acid in the crude extract reached 3.48 mg·g^-1. In the vitro experiment， the addition tobacco stalk of chlorogenic acid could inhibit the growth of Phytophthora. 175 mg·L^-1 tobacco stalk chlorogenic acid had a significant inhibitory effect on the colony diameter， which reached to 32% of antifungal rate. In addition， propidium iodie （PI） staining showed that 175 mg·L^-1 tobacco stalk chlorogenic acid could inhibit mycelial growth by destroying the integrity of cell membrane of Phytophthora nicotianae. Therefore， above results provided basis for effective utilization of tobacco straw and development of new natural plant fungicides.

Key words: tobacco stalk, chlorogenic acid, response surface optimization, antifungal effect

中图分类号:

张豫丹, 王卫民, 倪博, 马晓寒, 李俊领, 许自成, 贾玮, 史久长. 烟草秸秆绿原酸提取工艺优化及其抑菌效果研究[J]. 中国农业科技导报, 2023, 25(1): 119-127.

Yudan ZHANG, Weimin WANG, Bo NI, Xiaohan MA, Junling LI, Zicheng XU, Wei JIA, Jiuchang SHI. Extraction Optimization of Chlorogenic Acid from Tobacco Stalk and Its Antifungal Effect[J]. Journal of Agricultural Science and Technology, 2023, 25(1): 119-127.

图/表 12

表1 响应面试验因素水平设计

Table 1 Factors and levels of response surface design

水平 Level	因素 Factor
水平 Level	A：料液比 Solid-liquid ratio/（g·mL^-1）	B：乙醇体积分数 Ethanol volume fraction/%	C：提取温度 Extraction teperature/℃
-1	1∶15	50	30
0	1∶20	60	40
1	1∶25	70	50

图1 绿原酸标准曲线

Fig. 1 Standard curve of chlorogenic acid

图2 料液比对绿原酸含量的影响

Fig. 2 Effect of solid-liquid ratio on extraction yield of chlorogenic acid

图3 提取温度对绿原酸含量的影响

Fig. 3 Effect of extraction temperature on extraction yield of chlorogenic acid

图4 提取时间对绿原酸含量的影响

Fig. 4 Effect of extraction time on extraction yield of chlorogenic acid

图5 乙醇体积分数对绿原酸含量的影响

Fig. 5 Effect of ethanol volume fraction on extraction yield of chlorogenic acid

表2 响应面试验设计及结果

Table 2 Experimental results of response surface design

编号 No.	因素 Factor			绿原酸含量 CGA content/（mg·g^-1）
编号 No.	A	B	C	绿原酸含量 CGA content/（mg·g^-1）
1	-1	-1	0	1.51
2	1	-1	0	2.54
3	-1	1	0	1.46
4	1	1	0	2.49
5	-1	0	-1	1.45
6	1	0	-1	2.58
7	-1	0	1	1.58
8	1	0	1	2.65
9	0	-1	-1	2.21
10	0	1	-1	2.18
11	0	-1	1	2.33
12	0	1	1	2.27
13	0	0	0	2.08
14	0	0	0	2.11
15	0	0	0	2.14
16	0	0	0	2.06
17	0	0	0	2.16

表3 回归模型方差分析结果

Table 3 Variance analysis results of regression model

来源 Source	平方和 Sun of squares	自由度 DF	均方 Mean freedom	F值 F value	P值 P value	显著性 Statistical significance
模型 Model	2.42	9	0.27	215.52	<0.000 1	***
A	2.26	1	2.26	1812.80	<0.000 1	***
B	0.004 5	1	0.004 5	3.58	0.100 3
C	0.02	1	0.02	16.36	0.004 9	**
AB	0.004 6	1	0.004 6	0.037	0.853 8
AC	0.001 2	1	0.001 2	0.94	0.364 1
BC	0.001 7	1	0.001 6	0.14	0.724 0
A²	0.092	1	0.092	73.53	<0.000 1	***
B²	0.005	1	0.005	4.00	0.085 6
C²	0.042	1	0.042	33.84	0.000 7	**
残差 Residuel	0.008 8	7	0.001 2	—	—
失拟项 Lack of fit	0.002 2	3	0.007 2	0.44	0.736 2
纯误差 Pure error	0.006 6	4	0.001 6	—	—
总差 Cor total	2.43	16	—	—	—

图6 各因素对绿原酸含量影响的响应面和等高线图

Fig. 6 Response surface and contour plots showing the pairwise interactive effects of the factors on extraction yield of chlorogenic acid

表4 次绿原酸含量验证

Table 4 Extraction yield of chlorogenic acid in validation experiments

编号

No.

料液比 Material ratio/（g·mL^-1）

提取温度

Extraction temperature/℃

乙醇体积分数

Ethanol volume fraction/%

绿原酸含量

Chlorogenic acid content/（mg·g^-1）

图7 绿原酸对烟草疫霉菌菌落直径的影响注：图中不同小写字母表示差异显著。

Fig. 7 Effect of chlorogenic acid on the diameter of P. nicotianaeNote：Different small letters in the figure indicate significant difference at P<0.05 level.

图8 绿原酸对烟草疫霉菌丝细胞死亡的影响

Fig. 8 Effect of chlorogenic acid on cell death of P. nicotianae

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