肉桂醛抑制干辣椒中黑曲霉生长及其产毒合成机制研究

【作者】 陈江；

【导师】 万婧；

【作者基本信息】 贵州大学 ， 生物学， 2023， 硕士

【摘要】 干辣椒作为全世界非常受欢迎香辛料,市场的需求量巨大。中国作为世界上第二大辣椒生产国,其中贵州省辣椒种植面积已达36.2万公顷,年产量约770万吨。由于辣椒干制后极易吸潮受到霉菌侵染进而导致真菌毒素的产生,如赭曲霉毒素A(OTA)。本课题首先对贵州市售干辣椒进行OTA检测和产OTA真菌的分离、纯化和鉴定。近年来食品级的肉桂醛已被证明在食品安全和保存中得到广泛应用。本文通过研究肉桂醛对干辣椒中分离纯化的OTA高产菌Aspergillus niger DTZ-12的抑制作用和机理,为肉桂醛进一步在干辣椒的储藏防控应用中提供理论依据。主要结果如下:(1)对贵州干辣椒的皮和籽分别进行(n=49)OTA含量检测,发现22.45%的辣椒皮和34.70%的辣椒籽超过了欧盟规定的OTA最高水平(20μg/kg),而干辣椒皮和辣椒籽中OTA的最高值分别达到54.12μg/kg和36.67μg/kg,平均值分别为16.19±8.54μg/kg和18.73±6.09μg/kg。从干辣椒中共分离纯化34株真菌,9株具有产OTA的能力,分别为Penicillium chrysogenum(4株)、P.ruben(3株)、A.niger(1株)和A.fumigatus(1株)。其中,A.niger DTZ-12的产OTA能力最强,其次是P.chrysogenum DL-8。A.niger DTZ-12和P.chrysogenum DL-8在温度低于15℃时,均生长缓慢,在温度高于30℃,P.chrysogenum DL-8生长受到显著抑制,而A.niger DTZ-12生长则加快,具有各自的生长特征。(2)肉桂醛对A.niger DTZ-12半最大效应浓度(EC50)为0.049 mg/m L。肉桂醛可以有效地抑制A.niger DTZ-12的菌丝生长、孢子萌发。显微结构显示菌株孢子细胞壁变薄并形成沉淀物,细胞膜破裂,细胞质缺失,细胞质无组织聚集,核膜破裂和线粒体等重要细胞器消失。此外,肉桂醛能够抑制A.niger DTZ-12在干辣椒上的生长和OTA的生物合成。在生化水平上,肉桂醛抑制麦角甾醇生物合成,增加细胞膜通透性,降低ATP含量和ATP酶活性,促进细胞内活性氧(ROS)积累和脂质过氧化。(3)通过转录组学技术进一步发现,肉桂醛在EC50浓度处理后,与CK相比,A.niger DTZ-12中293个基因表达上调,220个基因表达下调。差异表达基因上调最丰富的分别是类固醇生物合成,甘氨酸、丝氨酸和苏氨酸代谢。差异表达基因下调最丰富的为真核生物核糖体生物合成途径,RNA聚合酶。蛋白质组学结果中,共筛选出100个上调差异表达蛋白,80个下调差异表达蛋白。上调蛋白主要涉及蛋白质靶向线粒体、线粒体蛋白质定位、蛋白质跨膜转运、氧化应激反应、细胞内稳态。在下调差异表达蛋白中,主要涉及细胞形态发生、细胞生长、单细胞生物的繁殖、生物过程调节以及肌动蛋白结合。综上,肉桂醛抑制菌株能量代谢、孢子萌发和OTA合成酶PKS、NRPS和b ZIP相关基因的表达,从而抑制A.niger DTZ-12生长和OTA生物合成。与此同时,肉桂醛还通过抑制染色体、核孔结构蛋白的组成、翻译和转录相关蛋白表达,使细胞出现衰老代谢特征。更多还原

【Abstract】 Dried red chili is a very popular spice worldwide witha huge demand in the market.China is the second largest chili producer in the world,of which Guizhou province has planted 362,000 hectares of chilies with an annual output of 7.7 million tones.As dried red chilies are highly susceptible to contamination by toxin-producing fungi,leading to the production of ochratoxin A(OTA).Therefore,studies of OTA pollution and isolation of OTA-producing fungi were carried out in dried red chilies in Guizhou Province.In recent years,food grade cinnamaldehyde has proven to be widely used in food safety and preservation.This paper investigated the antifungal mechanisms of cinnamaldehyde against Aspergillus niger DTZ-12,a high yielding OTA-producing fungi isolated from dried red chilies,to provide a theoretical basis for the application of cinnamaldehyde in the storage prevention and control of dried red chilies.The main results showed:(1)The current study detected OTA contents in both chili peels(n=49)and seeds(n=49)of Guizhou dried red chilies.Results showed that 22.45 % of chili peels and34.70 % of chili seeds exceeded the EU regulatory limit of OTA content(20 μg/kg),whereas the highest OTA level was up to 54.12 μg/kg in chili peels and 36.67 μg/kg in chili seeds.In total,34 fungal strains were isolated from Guizhou dried red chilies with9 of them had OTA-producing abilities,belonging to Penicillium chrysogenum(4strains),P.rubens(3 strains),A.niger(1 strain)and A.fumigatus(1 strain)by morphology and molecular techniques.In vitro and in vivo experiments identified A.niger DTZ-12 and P.chrysogenum DL-8 displayed the top 2 OTA-producing capacities with their own growth characteristics.The growth of A.niger DTZ-12 and P.chrysogenum DL-8 were slow at temperatures below 15°C,and that of P.chrysogenum DL-8 was significantly inhibited at temperatures beyond 30°C,while the growth of A.niger DTZ-12 was accelerated by their respective growth characteristics.(2)The half maximum effect concentration(EC50)of cinnamaldehyde on A.niger DTZ-12 was 0.049 mg/m L.cinnamaldehyde inhibited mycelial growth and spore germination of A.niger DTZ-12,and the microstructure revealed thinning of the cell wall,formation of precipitates,rupture of the cell membrane,absence of cytoplasm,disorganized aggregation of cytoplasm,rupture of the nuclear membrane and loss of important organelles such as mitochondria.In addition,cinnamaldehyde was able to inhibit the growth and OTA biosynthesis of A.niger DTZ-12 on dried red chilies.At the biochemical level,cinnamaldehyde could inhibit ergosterol biosynthesis,increase cell membrane permeability,reduce ATP content and ATPase activity,and promote intracellular ROS accumulation and lipid peroxidation.(3)According to the transcriptome study,293 genes in A.niger DTZ-12 were upregulated and 220 genes were down-regulated after the treatment of cinnamaldehyde at EC50 concentration.The most abundant differentially expressed genes were upregulated in steroid biosynthesis,glycine,serine and threonine metabolism,respectively.Differentially expressed genes were most abundantly down-regulated in the eukaryotic ribosomal biosynthetic pathway,and RNA polymerase.A total of 100 up-regulated and80 down-regulated differentially expressed proteins were screened as a result of the proteomics study.The up-regulated proteins were mainly involved in protein targeting to mitochondria,mitochondrial protein localization,protein translocation across membranes,oxidative stress response,and intracellular homeostasis.Among the downregulated differentially expressed proteins,they are mainly involved in cell morphogenesis,cell growth,reproduction of single-celled organisms,regulation of biological processes and actin binding.In summary,cinnamaldehyde could inhibit the expression of genes related to energy metabolism,spore germination and the substrates of OTA synthesis,included PKS,NRPS and b ZIP,thereby suppressing A.niger DTZ-12 growth and OTA biosynthesis.Cinnamaldehyde also caused A.niger DTZ-12 to develop a senescence metabolic profile by inhibiting chromosome,nuclear pore structure protein composition,translation and transcription-related protein expression.更多还原