油茶籽拮抗黄曲霉侵染及其毒素积累的机制研究

Aflatoxins(AFT) are toxic metabolic product that are produced by Aspergillus flavus and Aspergillus parasiticus from contaminated food, such as peanuts, corn, peanut oil, etc, which is currently among the most carcinogenic substances known. Our previous study found that Camellia oil seed is not contaminated by Aspergillus flavus easily, and the content of aflatoxins are decreased gradually even artificial inoculated with Aspergillus flavus with time extended. Based on above findings，the research aim to investigate effects of the seed crude extracts from Camellia oil seed on growth of Aspergillus flavus and production capacity of aflatoxins, and determine the active ingredient of resistance to Aspergillus flavus. By in vitro antibacterial test, to compare the different Camellia oil seed extracts effects on the mycelial growth and quantification of aflatoxins production, preliminary determine the type of antibacterial substances; to identify the active composition of crude extract by column chromatography, GC-MS-MS, LC-MS-MS combined with nuclear magnetic resonance(NMR), and to isolate and determine the single active composition by in vivo and in vitro test, and therefore clarify the mechanism involved in inhibitory effect of Aspergillus flavus infection and toxin accumulation of camellia oil seed. This study will provide the basis for further study of camellia oil seed against Aspergillus flavus growth and aflatoxin production，and provides the evidence and guidance for the development of anti-fungal agents to other oil seeds being susceptible to Aspergillus flavus.

黄曲霉毒素是黄曲霉和寄生曲霉的有毒代谢产物，主要来自于受污染的粮油食品，如花生、玉米、花生油等，是目前发现的最强的化学致癌物。本课题组在前期研究中发现油茶籽不易感染黄曲霉，且在人为接种黄曲霉菌后，随培养时间延长，黄曲霉毒素含量逐渐下降。基于这一研究发现，本项目拟开展油茶籽提取物对黄曲霉生长及产毒能力的影响研究，明确其中拮抗黄曲霉的活性成分。通过体外抑菌试验，对比不同油茶籽粗提物对黄曲霉生长和产毒的影响，初步确定抑菌物质的类型；再运用柱层析、GC-MS-MS、LC-MS-MS和核磁共振技术，分离鉴定粗提物中的活性组成，结合体内抑菌试验和体外在易感黄曲霉油料（花生、玉米）上的验证试验，确定油茶籽中拮抗黄曲霉的活性成分，阐明油茶籽抑制黄曲霉侵染及其毒素积累的机制。本研究为进一步阐明油茶籽中的抑菌成分对黄曲霉菌生长及黄曲霉产毒的作用机制提供依据，也为其他易感黄曲霉油料的抑菌剂研发提供借鉴和指导。

本项目开展了油茶籽及其饼粕不同溶剂提取物对黄曲霉菌菌丝生长及产毒的影响研究，选用水、甲醇、乙醇和乙酸乙酯作为溶剂，对比抑菌效果。综合抑菌圈直径、菌丝干重和黄曲霉毒素含量结果看，80%甲醇提取物浓度为20mg/ml时，对黄曲霉菌的生长没有明显的抑制作用，40 mg/ml时出现明显抑菌圈，菌丝干重也出现显著下降，黄曲霉毒素B1也出现了明显下降。可见，甲醇提取物的最小抑菌浓度为40 mg/ml。.将80%甲醇的提取物依次用乙酸乙酯、正丁醇萃取，将各萃取液及萃余水相浓缩，真空干燥，测定抑菌活性。正丁醇相液体培养的菌丝量显著低于乙酸乙酯相和水相，且随着浓度增大对菌丝干重明显减少，100mg/ml时菌丝干重从25mg/ml时的0.121g减少到了0.156g，比对照减少了42.88%，可见，正丁醇萃取相浓度越大，对菌丝的抑制效果越好。.采用柱色谱法纯化正丁醇萃取相，依次用蒸馏水、25%乙醇、50%乙醇、75%乙醇、无水乙醇洗脱，75%乙醇洗脱物对黄曲霉菌有明显的抑制效果，在浓度为50 mg/ml和100 mg/ml时，抑菌圈直径分别为146.6mm和163.7mm，在25 mg/ml时未出现抑菌圈，可见其最小抑菌浓度为50 mg/ml。其余洗脱物对黄曲霉菌均无抑菌效果。75%乙醇洗脱物使得黄曲霉菌的菌丝形态发生扭曲变形，有些出现明显的断裂、折叠和扭曲现象，有些菌丝顶端膨大，菌丝内部原生质体分布不均匀，而对照组菌丝较为平滑清晰，形状规则。在其他油料的体外试验上验证了75%乙醇洗脱物对黄曲霉菌有明显的抑制作用。.基本阐明了油茶籽抑制黄曲霉侵染和产毒的机制，当黄曲霉菌侵染时通过油茶籽中所含的75%乙醇洗脱物这种物质激发了PPO、POD、PAL等酶的活性，从而起到抑制菌丝生长和产毒的效果。本研究为进一步阐明油茶籽中的抑菌成分对黄曲霉菌生长及黄曲霉产毒的作用机制提供了理论依据，也为其他易感黄曲霉油料的抑菌剂研发提供了借鉴和指导。