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

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

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。

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.