【作者】 李红娟；

【导师】 吕加平；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2014， 博士

【摘要】 Lb. casei AST18是本实验室筛选到的一株对食品腐败真菌具有良好抑制作用的乳酸菌菌株。在获得此菌株基础上，本论文对发酵液中成分进行了初步分离纯化，利用液相质谱对分离纯化部分进行了鉴定；结合不同时间段发酵液的抑菌效果，采用代谢组学技术和思路，利用核磁共振及液质联用对发酵液中主要差异代谢物进行分析；并以产黄青霉为指示菌株，研究Lb. casei AST18对产黄青霉的抑制作用，推测其在产黄青霉体内的作用方式；利用N+注入对Lb. casei AST18进行诱变，以期获得具有更加良好抗真菌效果的乳酸菌菌株；最后利用诱变后的正突变菌株，将其作为干酪附属发酵剂应用到干酪制作中，并考察其对干酪贮藏期内主要理化指标及感官品质的影响，对Lb. casei AST18（1-3）作为附属发酵剂在干酪中抑菌情况进行了研究，论证了其应用在干酪防霉上的可行性，为其在干酪中的应用打下理论基础。本研究主要结论如下：（1）发酵液中除乳酸外，存在大量其它分子量小于1000Da的抑真菌物质。本实验初步分离纯化具有抑菌性能的5部分物质，并通过液质联用对其进行分析，检测到潜在抗真菌物质主要为：乳酸、乙酸等小分子有机酸、小分子酯类、芳香杂环类物质、生物碱以及多种含苯环的小分子有机物。实验证明，乳酸菌发酵液抑真菌性能是由多种小分子化合物协同作用的效果。（2）通过不同时间发酵液抑菌效果及结合代谢组学技术分析发现，在乳酸菌发酵初期抑菌效果增长主要是由于乳酸、乙酸的大量产生对霉菌造成的抑制；在乳酸菌稳定期之后，通过液质代谢组学手段结合第二章实验分析结果发现，对抑菌效果的增长起较大贡献作用的主要物质有：肉桂酸、羟基肉桂酸（香豆酸）、L-3-苯乳酸、苯乳酸、苯乙酸、对香豆酸、壬二酸、水杨酸、4-羟基苯甲酸、(R)2,3-二羟基-3-甲基戊酸、甲羟戊酸、cyclo(L-Leu-L-Pro)等。（3）Lb. casei AST18对青霉、曲霉、镰刀霉均有抑制效果，可以抑制菌丝生长及孢子萌发，同时导致青霉属菌落生长形态异常；Lb. casei AST18引起了P. chrysogenum自噬性死亡，观察到的主要变化过程为：液泡增大，同时部分细胞质凝集颜色加深；在液泡边缘出现包裹大量细胞质的自噬小体并慢慢进入液泡，在液泡中被消化；在衰亡后期的菌体中，细胞中只剩下大量的双层膜，观察不到任何细胞器结构。（4）Lb. casei AST18（1-3）作为辅助发酵剂添加到切达干酪中，对切达干酪感官品质影响较小，能够增加干酪水解度，增加干酪中乳杆菌数，且能够使干酪具有良好的防霉效果。更多还原

【Abstract】 Lb. casei AST18was screened in our laboratory because of its excellent antifungal activity. In thisresearch, we used a series of separation and purification methods and obtained several antifungalfermentation parts. Then we used UPLC-Q-TOF to detect the antifungal substances in the separatedparts. On this basis, we use the metabolomics technology to collect the data of the culture with differentfermrntation time. Then we used multivariate statistical analysis to analyze the main difference betweenthe fermentation culture, in order to obtain the information of the antifungal substances. We analyzedthe antifungal spectrum and the effect of Lb. casei AST18to the growth morphology of P.chrysogenum, which was used as indictor fungals. Double flat antagonize method was used to study theantifungal activities of Lb. casei AST18. SEM and TEM were used to study the ultrastructure inside thefungal and to speculate the action mode of Lb. casei AST18on P. chrysogenum. We used N+Implantation Mutation Breeding (IMB) to process Lb. casei AST18in order to obtain a stain with betterantifungal effect and use the strain in the actual production industry. At last, we used the mutant strainas adjunct fermentation culture in Cheddar. We analyzed the main physicochemical and sensoryqualities of the cheese during the storage period. The antifungal activities of Lb. casei AST18（1-3）as aadjunct culture in Cheddar was evaluated. This experiment lay a theoretical foundation of antifungalLAB in the cheese production industry.The main conclution were as follows:(1) There are serveal low molecular substance exist in fermentation culture of LAB except lacticacid; the main antifungal substance in the culture detected by UPLC-Q-TOF was organic acid,nitrogen-containing small molecule compounds and small molecule esters. The antifungal activities ofLAB was a synergistic effect of all this substances. The antifungal substances detected in thisexperiment which have been reported was: Lactic acid, acetic acid, citric acid, benzoic acid, cinnamicacid, salicylic acid, azelaic acid, succinic acid, sialic acid and phenyllactic Acid.(2) Through the analysis of antifungal activities and the metabolic footprinting, the increasedantifungal activities of the fermentation culture during the prophase was caused by lactic acid; after thestable period, the increased antifungal activities was caused by Nitrogen-containing small moleculecompounds and other orginic acid..(3) Lb. casei AST18caused the autophagic death of P. chrysogenum. The main changes weobserved during the process was as follows:The vacoule enlarged to the entire cell; part of the cytoplasmic aggregated and the color wasdeepened; autophagosomes contained cytoplasm entered into the vacoule; and then the autophagosomeswas digested in the vacoule; when the color of colony was grayish brown, there was no organelles left inthe cell, only bilayer structure left; the autophagic death should be verified by genetic level.(4) The use of Lb. casei AST18（1-3）as adjunct culture in Cheddar has little effect on the sensoryquality of the cheese. It can increase the degree of hydrolysis and Lactobacillus count, and also it has a good antifungal effect in the cheese.更多还原