山苍子精油抑制黄曲霉菌的生长和产毒作用研究

黄晓霞; 彭伟斌; 李振宇; 杨东润; 杨世锟

doi:10.13386/j.issn1002-0306.2022060269

山苍子精油抑制黄曲霉菌的生长和产毒作用研究

doi: 10.13386/j.issn1002-0306.2022060269

1.
嘉应学院化学与环境学院，广东梅州 514015
2.
广东省山区特色农业资源保护与精准利用重点实验室，广东梅州 514015

基金项目: 2020年广东省科技专项资金项目（2020A0104003）；2022年广东省普通高校重点领域专项（2022ZDZX4050）；广东省大学生创新创业训练计划项目（S202110582023）。

详细信息

作者简介:
黄晓霞（1976−），女，本科，高级实验师，研究方向：微生物的防治与利用，E-mail：huangxiaoxiamm@163.com

中图分类号: S188
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出版历程
- 收稿日期: 2022-06-28
- 刊出日期: 2023-05-01

Inhibitory Effect of Litsea cubeba Essential Oil on Aspergillus flavus Growth and Aflatoxin Production

1.
School of Chemistry and Environment, Jiaying University, Meizhou 514015, China
2.
Guangdong Provincial Key Laboratory of Conservation and Precision Utilization of Characteristic Agricultural Resources in Mountainous Areas, Meizhou 514015, China

摘要

摘要: 山苍子精油是一种纯天然植物精油，本文研究了其对黄曲霉生长、代谢和毒素产生的抑制作用，探讨了山苍子精油对黄曲霉菌的抑菌能力和作用机理。本研究将花生放置于自然环境染菌并分离纯化目标菌，采用形态学并结合ITS序列法进行菌株分类鉴定；结合抑菌圈、抑菌率和最低抑菌浓度（MIC）的测定探讨山苍子精油对黄曲霉菌的抑制能力；进行了山苍子精油影响黄曲霉孢子萌发率、生长曲线和黄曲霉毒素B1产生的实验研究；从细胞膜渗透性、细胞酶活性的变化探讨了山苍子精油抑制黄曲霉的作用机理。实验结果表明：从腐败花生中分离筛选出菌株HB₂，经ITS序列法鉴定为黄曲霉（Aspergillus flavus）；黄曲霉素测定结果显示其含有黄曲霉素B1（AFB1），质量浓度为3.4×10³ μg·kg⁻¹（纯湿菌体）；抑菌圈随精油浓度的增大明显变大，对黄曲霉的最低抑菌体积分数（MIC）为0.800 μL·mL⁻¹；孢子萌发率、牙管长度、黄曲霉菌体的生长量和AFB1的浓度随培养液中精油浓度的增大呈显著下降趋势，当山苍子精油浓度为0.100 μL·mL⁻¹时，肉眼看不到菌体生长；随精油浓度的增大，培养液电导率增大、还原糖利用率和菌体蛋白质含量减少、菌体内苹果酸脱氢酶和琥珀酸脱氢酶活性降低。这些结果表明，山苍子精油对黄曲霉产生不可逆的破坏，推测山苍子精油破坏细胞壁和细胞膜，影响细胞的生长和代谢，并最终导致细胞死亡。因此，山苍子精油对黄曲霉具有良好的抑制作用，可广泛用于粮食储藏、食品防霉等方面。
- 山苍子精油 /
- 黄曲霉 /
- 黄曲霉素B1 /
- 抑制作用
Abstract: Litsea cubeba essential oil is a pure natural plant essential oil. In this paper, the inhibitory effect of Litsea cubeba essential oil on the growth, metabolism and toxin production of Aspergillus flavus was investigated. In this study, peanuts were placed in a natural environment to infect bacteria, and the target bacteria were isolated and purified. Morphology and ITS sequence methods were used to classify and identify the strains. The inhibition ability of essential oil on Aspergillus flavus was discussed by the determination of inhibition zone, inhibition rate and minimum inhibitory concentration (MIC). The effects of essential oil on the germination rate, growth curve and aflatoxin B1 production of Aspergillus flavus, as well as the effects of cell membrane permeability and cellular enzyme activity, were tested. The results showed that strain HB₂ was isolated and screened from spoilage peanuts and was identified as Aspergillus flavus by the ITS sequence method. The results of aflatoxin determination showed that aflatoxin B1 (AFB1) had a mass concentration of 3.4×10³ μg·kg⁻¹ (pure wet bacteria). The bacteriostatic zone significantly increased with increasing essential oil concentration, and the minimum inhibitory concentration (MIC) for Aspergillus flavus was 0.800 μL·mL⁻¹. The germination rate, the length of the dental tube, the growth of Aspergillus flavus, and the concentration of AFB1 showed a significant decreasing trend with increasing concentrations of essential oil in the culture medium. When the concentration of Litsea cubeba essential oil was 0.100 μL·mL⁻¹, the growth of the bacteria was invisible to the naked eye. With the increase in the concentration of essential oil, the conductivity of the culture medium increased, the utilization rate of reducing sugars and the protein content of the bacteria decreased, and the activities of malate dehydrogenase and succinate dehydrogenase in the bacteria decreased. These results indicated that Litsea cubeba essential oil caused irreversible damage to Aspergillus flavus. It is speculated that Litsea cubeba essential oil damaging the cell wall and cell membrane, affecting cell growth and metabolism and ultimately leading to cell death. Therefore, Litsea cubeba essential oil has a good inhibitory effect on Aspergillus flavus and can be widely used in grain storage and food antimolding.
- Litsea cubeba essential oil /
- Aspergillus flavus /
- aflatoxin B1 /
- inhibitory effect

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图 1 菌落形态

注：a：正面；b：反面。

Figure 1. Colony morphology

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图 2 分生孢子（a）和分生孢子梗（b）

Figure 2. Condidium (a) and conidiophore (b)

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图 3 菌株HB₂基于ITS序列邻接法构建系统发育树

Figure 3. Phylogenetic tree derived from ITS domain sequences and neighbor-joining analysis of strain HB₂

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图 4 山苍子精油浓度对黄曲霉生长和黄曲霉素B1产生的影响

Figure 4. Effects of Litsea cubeba essential oil concentration on the growth and the production of aflatoxin B1 of Aspergillus flavus

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图 5 山苍子精油浓度对培养液电导率的影响

Figure 5. Effect of Litsea cubeba essential oil concentration on conductivity of culture medium

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图 6 山苍子精油浓度对菌体蛋白质的影响

Figure 6. Effect of Litsea cubeba essential oil concentration on protein content

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图 7 山苍子精油浓度对菌体还原糖利用率的影响

Figure 7. Effect of Litsea cubeba essential oil concentration on reducing sugar utilization

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表 1 山苍子精油浓度对黄曲霉抑菌圈的影响

Table 1. The bacteriostasis diameters of Litsea cubeba essential oil concentration on Aspergillus flavus

体积分数（μL·mL⁻¹）	0	3.13	6.25	12.50	25.00	50.00	80.00	100.00
抑菌圈直径（mm）	6.00±0.25	10.23±1.42	12.34±2.51	14.67±2.60	15.00±3.36	17.17±2.64	−	−
注：“−”表示无菌生长；抑菌圈直径>15 mm为最敏感；10~15 mm为中等程度敏感；7~9 mm时为低等程度敏感；无抑菌者为不敏感^[24]。

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表 2 平板法测山苍子精油浓度对黄曲霉的抑制效果

Table 2. Inhibitory effect of Litsea cubeba essential oil concentration on Aspergillus flavus by plate method

体积分数（μL·mL⁻¹）	0.038	0.075	0.150	0.300	0.600	0.800	1.000	1.200
抑菌率（%）	29.21±2.03	40.45±2.11	58.43±1.68	70.79±5.21	92.13±2.74	100±0	100±0	100±0

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表 3 山苍子精油浓度对黄曲霉孢子萌发率和芽管长度的影响

Table 3. Effects of Litsea cubeba essential oil concentration on spore germination and germ tube elongation of Aspergillus flavus

体积分数（μL·mL⁻¹）	0	0.013	0.025	0.030	0.040	0.050	0.060	0.080
萌发率（%）	85.22±2.95	71.50±2.30	49.76±3.48	28.79±3.78	25.87±0.75	6.19±1.66	1.90±4.23	0
芽管长度（μm）	123.80±3.74	108.90±1.25	56.70±2.63	35.50±5.01	24.30±3.22	15.20±0.43	8.04±2.98	−

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表 4 山苍子精油对菌体酶活性的影响

Table 4. Effect of Litsea cubeba essential oil on bacterial enzyme activity

山苍子油浓度（μL·mL⁻¹）	0	0.03	0.05
NADP-MDH（nmol/min/g鲜重）	4565.3±40.05	4051.0±29.65	727.0±31.54
SDH（nmol/min/g鲜重）	27.4±2.02	12.5±2.98	3.1±0.53

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