山苍子精油提取工艺优化及抑菌活性

伍楷文; 阮学瑞; 孙玮玮; 贝加炎; 潘镇泽; 徐会有; 倪林

doi:10.19303/j.issn.1008-0384.2023.08.011

山苍子精油提取工艺优化及抑菌活性

doi: 10.19303/j.issn.1008-0384.2023.08.011

1.
福建农林大学植物保护学院，福建　福州　350002
2.
宁德市林业科研与技术推广中心，福建　宁德　352100
3.
福建省屏南古峰国有林场，福建　宁德　352399

基金项目: 中央财政林业科技推广项目（闽〔2022〕TG09号）

详细信息

作者简介:
伍楷文（2000 —），男，硕士研究生，主要从事活性天然产物的开发与利用研究，E-mail：kevinwu2233@163.com

通讯作者:
倪林（1986 —），博士，副教授，硕士生导师，主要从事活性天然产物的研究与开发研究，E-mail：nilin_fjau@126.com

中图分类号: R284
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出版历程
- 收稿日期: 2023-02-27
- 修回日期: 2023-05-25
- 刊出日期: 2023-08-28

Study on process optimization and antibacterial activity of Litsea cubeba essential oil

1.
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Ningde Forestry Research and Technology Extension Center, Ningde, Fujian　352100, China
3.
Fujian Pingnan Gufeng State-owned Forest Farm, Ningde, Fujian　352399, China

摘要

摘要: 目的优化山苍子精油提取工艺，探究精油抑菌活性，为山苍子精油的加工及应用提供参考数据。方法通过响应面法优化山苍子精油提取过程中NaCl体积分数、液料比、蒸馏时间的工艺条件。采用生长速率法探究山苍子精油对瓜果腐霉菌的抑制作用，通过测定精油对菌丝干重、丙二醛含量、还原糖含量、过氧化氢酶、过氧化物酶和超氧化物歧化酶等保护酶活性的影响，探讨了其抑制植物病原真菌的生理生化作用。结果确定了最佳工艺条件：NaCl体积分数为2.39%，液料比[V（mL）∶m（g）]为4.8∶1，蒸馏时间4.94 h。在该条件下，精油得率可达4.43%。山苍子精油对瓜果腐霉具有较好抑制效果，EC₅₀值为224.4 μg· mL⁻¹。瓜果腐霉菌经山苍子精油处理后，菌丝干重减少，细胞膜通透性增加，菌体内还原糖含量减少，保护酶含量增多。结论经响应面优化后的工艺稳定可行，与其他的各类提取方法相比成本低消耗低与得率高。山苍子精油可以通过抑制菌丝生长、破坏细胞膜结构以及降低菌丝体保护酶活性等发挥抑菌活性。
- 山苍子 /
- 抑菌活性 /
- EC₅₀ /
- 生理生化 /
- 瓜果腐霉 /
- 响应面
Abstract: Objective This study aimed to optimize the extraction process of Litsea cubeba essential oil and investigate its antibacterial activity and mechanism of action against plant pathogenic fungi. Methods Response surface methodology was employed to optimize the extraction conditions of Litsea cubeba essential oil, including NaCl volume fraction, liquid-solid ratio, and distillation time. The inhibitory effect of Litsea cubeba essential oil on Pythium aphanidermatum was evaluated by the growth rate method, while its physiological and biochemical effects were assessed by measuring fungal mycelium dry weight, MDA content, reducing sugar content, and protective enzyme activities such as catalase, peroxidase, and superoxide dismutase. Results The optimal extraction conditions for Litsea cubeba essential oil were determined as NaCl volume fraction of 2.39%, liquid-solid ratio of 4.8:1 (mL·g⁻¹), and distillation time of 4.94 hours, resulting in an extraction yield of 4.43%. Litsea cubeba essential oil exhibited a potent inhibitory effect on Pythium aphanidermatum, with an EC₅₀ value of 224.4 μg·mL⁻¹. Treatment with Litsea cubeba essential oil reduced fungal mycelium dry weight, increased cell membrane permeability, decreased reducing sugar content in the fungal body, and increased protective enzyme activities. Conclusion The optimized extraction process was stable, feasible, and cost-effective compared to other methods. Litsea cubeba essential oil exerted antibacterial activity by inhibiting mycelial growth, disrupting cell membrane structure, and decreasing protective enzyme activities in the mycelia.
- Litsea cubeba /
- Antibacterial activity /
- EC₅₀ /
- physiology and biochemistry /
- Pythium aphanidermatum /
- Response surface

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图 1 不同条件对山苍子精油得率的影响

Figure 1. Effect of different conditions on extraction yield of Litsea cubeba essential oil

下载: 全尺寸图片幻灯片

图 2 两因素交互作用对山苍子精油得率的影响

Figure 2. Effect of interaction of two factors on yield of Litsea cubeba essential oil

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图 3 山苍子精油对瓜果腐霉菌菌丝形态影响

A为生理盐水对照组，B为山苍子精油处理组。

Figure 3. Effect of Litsea cubeba essential oil on hyphae morphology of Pythium aphanidermatum

A is the control group using physiological saline solution, and B is the group for the processing of Shancangzi essential oil.

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图 4 山苍子精油对瓜果腐霉菌干重的影响

不同小写字母表示差异显著（P＜0.05）。

Figure 4. The effect of Litsea cubeba essential oil on the dry weight of Pythium aphanidermatum

Data with different lowercase letters indicate significant differences (P＜0.05).

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图 5 山苍子精油对瓜果腐酶生理生化各项数值影响

图A~F分别表示山苍子精油对瓜果腐酶的MDA（A）、还原糖（B）、CAT（C）、POD（D）、SOD（E）、相对电导率（F）的影响，LC为处理组，CK为空白对照组。不同小写字母表示同一时间不同处理之间差异显著（P＜0.05）。

Figure 5. Effects of litsea cubeba essential oil on the physiological and biochemical values of Pythium aphanidermatum

The letters a-f in the figure indicate the effects of litsea cubeba essential oil on MDA（A）, reducing Glucose（B） , CAT（V）, POD（D）, SOD （E）and relative electrical conductivity（F）of melon and fruit rot enzymes, respectively,"LC" is the treatment group and "CK" is the blank control group.Different lowercase letters indicate significant differences between different treatments at the same time point (P＜0.05).

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表 1 响应面设计方案与结果

Table 1. The program and experimental results of response surface methodology

编号 Serial number	A NaCl体积分数 A NaCl volume fraction/ %	B蒸馏时间 B Distillation time/h	C液料比 C liquid-material ratio /[ V （mL）∶ m （g）]	精油得率 Yield of Litsea cubeba essential oil/%
1	3.00	4.00	4.00	4.37
2	4.00	5.00	4.00	4.23
3	3.00	3.00	3.00	4.06
4	4.00	4.00	3.00	4.05
5	3.00	4.00	4.00	4.38
6	3.00	4.00	4.00	4.37
7	2.00	4.00	5.00	4.48
8	3.00	5.00	5.00	4.43
9	4.00	3.00	4.00	4.25
10	4.00	4.00	5.00	4.44
11	3.00	5.00	3.00	4.04
12	2.00	5.00	4.00	4.24
13	3.00	3.00	5.00	4.46
14	2.00	4.00	3.00	4.07
15	3.00	4.00	4.00	4.41
16	2.00	3.00	4.00	4.29
17	3.00	4.00	4.00	4.41

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表 2 回归方程方差分析表

Table 2. Variance analysis of mathematical regression model

方差来源 Soruce of variation	平方和 Sum of squares	自由度 df	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型 Model	0.38	9	0.043	170.18	＜0.01	**
A	1.51×10⁻³	1	1.51×10⁻³	6.03	4.37×10⁻²	*
B	0.32	1	0.32	12.60.45	＜0.01	**
C	1.80×10⁻³	1	1.80×10⁻³	7.18	3.16×10⁻²	*
AB	1.00×10⁻²	1	1.00×10⁻²	0.40	5.48×10⁻¹
AC	2.25×10⁻⁴	1	2.25×10⁻⁴	0.90	3.75×10⁻¹
BC	2.50×10⁻⁵	1	2.50×10⁻⁵	0.10	7.61×10⁻¹
A²	1.60×10⁻²	1	1.60×10⁻²	63.52	＜0.01	**
B²	1.90×10⁻²	1	1.90×10⁻²	74.27	＜0.01	**
C²	2.30×10⁻²	1	2.30×10⁻²	91.96	＜0.01	**
残差 Residual	1.76×10⁻³	7	2.51×10⁻⁴
失拟 Lack of fit	7.50×10⁻⁵	3	2.50×10⁻⁵	0.060	9.79×10⁻¹
纯误差 Pure error	1.68×10⁻³	4	4.20×10⁻⁴
总计 Aggregate	0.39	16
：差异显著（P＜0.05）;：差异极显著（P＜0.01）。 indicates significance difference（P＜0.05）; ** indicates extremely significance difference（P ＜0.01）.

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表 3 山苍子精油对病原菌的抑制率测定

Table 3. Determination of inhibition rate of Litsea cubeba essential oil against pathogenic bacteria

供试病原菌 Pathogenic bacteria for test	抑菌率 Bacteriostasis rate/%
瓜果腐霉菌 Pythium aphanidermatum	86.31±0.25 a
苹果黑腐皮壳病菌 Valsa mali Miyabe et Yamada	82.52±0.12 a
番茄灰霉病菌 Botrytis cinerea	81.60±0.48 a
禾谷镰刀病菌 Fusarium graminearum	77.09±0.63 b
燕麦镰孢菌 FusaHum graminearum Sehw	74.66±0.72 b
西瓜尖孢镰孢菌 Fusarium oxysporumf	57.96±0.61 c
不同小写字母表示差异显著（P＜0.05）。 Data with different lowercase letters indicate significant differences (P＜0.05).

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表 4 山苍子精油与阳性对照对瓜果腐霉菌的毒力测定

Table 4. Determination of the virulence of Litsea cubeba essential oil against Pythium aphanidermatum

样品 Sample	毒力曲线 Virulence curve	相关系数 Correlation coefficient (R²)	EC₅₀ / (μg·mL⁻¹）
山苍子精油 Litsea cubeba essential oil	y=1.896x+0.345	0.989	224.4
70%甲基硫菌灵 WP（对照） Thiophanate- Methyl （control）	y=0.428x−1.141	0.946	466.9

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