苯乳酸抑制链格孢霉作用靶位的分析

以从自然腐败的樱桃上分离的链格孢霉（Alternaria sp.）LD3.0086为指示菌，研究苯乳酸对链格孢霉的主要抑制作用靶位。应用分光光度法测定苯乳酸对链格孢霉的最小抑菌浓度，通过卡尔科弗卢尔荧光增白剂染液（calcofluor white，CFW）染色观察苯乳酸对菌丝顶端生长的破坏作用，利用扫描电子显微镜和透射电子显微镜观察链格孢霉的超微结构变化，通过测定苯乳酸作用前后链格孢霉上清液中N-乙酰葡萄糖胺质量浓度变化研究苯乳酸对菌丝细胞壁的破坏作用，应用荧光双染色法观察苯乳酸对链格孢霉菌丝细胞膜的损伤作用。结果表明，12.5 mmol/L的苯乳酸能有效抑制链格孢霉的生长；与对照组（无菌水处理）相比，苯乳酸处理后链格孢霉顶端生长细胞无明显形变，经12.5 mmol/L苯乳酸处理的链格孢霉上清液中N-乙酰葡萄糖胺质量浓度基本不变；苯乳酸处理24 h，链格孢霉菌丝细胞壁表面无明显损伤，细胞内结构发生明显变化；苯乳酸短时间（4 h）处理链格孢霉，菌丝细胞膜仍较为完整，加入苯乳酸较长时间（8 h）后细胞膜发生破裂。综合分析可知，苯乳酸对链格孢霉的主要作用靶位应不是菌丝体的细胞壁和细胞膜，而是在菌丝体内部，通过破坏菌丝内部细胞器结构或引起细胞内的生化反应，从而抑制链格孢霉的生长和繁殖，发挥抑菌活性。

Analysis of Sites of Action of Phenyllactic Acid in Inhibiting Alternaria sp.

The major sites of action of phenyllactic acid (PLA) against Alternaria sp. LD3.0086, isolated from naturally decayed cherries, were investigated. The minimum inhibitory concentration (MIC) was determined by spectrophotometry. The damage of PLA to the growing hyphal tip was observed by calcofluor white (CFW) staining. The ultrastructural changes of Alternaria sp. were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). The damage to the mycelial cell wall was evaluated by measuring the change of N-acetylglucosamine concentration in the culture supernatant of Alternaria sp. before and after exposure to PLA, and the damaging effect of PLA on the mycelial cell membrane was observed by the fluorescein diacetate (FDA)/propidium iodide (PI) fluorescence double staining method. The results showed that 12.5 mmol/L PLA effectively inhibited the growth of Alternaria sp. Compared with the control group treated with sterile water, PLA treatment did not cause the growing hyphal tip to deform. The concentration of N-acetylglucosamine in the culture supernatant of Alternaria sp. treated with 12.5 mmol/L PLA was basically unchanged; there was no obvious damage to the cell wall surface of Alternaria sp. treated with 12.5 mmol/L PLA for 24 h, but the intracellular structure changed obviously. After PLA treatment for a short time (4 h), the mycelial cell membrane was still relatively intact, while it was ruptured after PLA treatment for a long time (8 h). Collectively, we concluded that the major site of action of PLA against Alternaria sp. cannot be the cell wall and cell membrane of the mycelium, but inside the cells, and that PLA can enter into the cells to destroy the internal organelle structure of the hyphae or trigger a biochemical reaction, thereby inhibiting the growth and reproduction of Alternaria sp. and exerting antimicrobial activity.