| Patulin (PAT) is a toxic fungal secondary metabolite, and is one of the mycotoxins that can cause most serious health problems to human beings. Patulin has been identified in various fruits and fruit products throughout the world, especially in apples and its derived products. Control and degradation of patulin in fruits and fruit products using antagonistic yeasts has a promising future. However, researches to control patulin biosynthesis using antagonistic yeasts are still at the rudimentary stage especially with the mechanisms involved in the control and/or degradation. In this study, Rhodotorula mucilaginosa which was isolated by our research team was used.The primary objective of this study was to figure out the mechanisms involved in the control and biodegradation of patulin by antagonistic yeast (R. mucilaginosa). The specific objectives were to determine: (1) the physiological mechanisms of controlling the growth of Penicillium expansum and patulin synthesis in apples by R.mucilaginosa, (2) the mode of biodegradation of patulin by R.mucilaginosa in vitro test; (3) the degradation product and detection the toxicity of product; (4) change in expression of transcriptome and proteomes of R. mucilaginosa involved in degradation of patulin. The main research findings were summarized as follows:1. R. mucilaginosa controlled blue mold of apples caused by P. expansum and decrease PAT concentration of apples. Moreover, R. mucilaginosa decreased the P.expansum biomass through RT-qPCR analysis. The results indicated that R.mucilaginosa reduced the overall level of patulin contamination by inhibiting the growth of P. expansum in apple wounds.2. There were several physiological mechanisms involved in control of postharvest decay of apples by R. mucilaginosa, which included: R. mucilaginosa colonized and increased rapidly in apple surface, R. mucilaginosa had ability to induce defense-related enzymes such as Chitinase, β-1,3-glucanase and Phenylalanine Ammonia Lyase to enhance resistance of apples, moreover, R.mucilaginosa could adhere to the mycelia of P. expansum and inhibit its growth. In vitro test, the mechanism of patulin degradation by R. mucilaginosa was explored,our results showed that viable R. mucilaginosa cells are essential and the presence of patulin can induce R. mucilaginosa to produce associated intracellular enzymes,which had the ability to degrade patulin.3. An iTRAQ-based proteomic analysis revealed that the responses of R.mucilaginosa to patulin were complex. There were 131 up-regulated proteins and 101 down-regulated proteins. The up-regulated proteins and down-regulated proteins were mainly involved in single-organism process, metabolic process and cellular process.The classification showed that the largest differentially proteins were associated with the catalytic activity and binding. The enriched domain of up-regulated proteins was mainly related to the binding function of NAD(P), and the enriched domain of down-regulated proteins was related to the binding function of FAD/NAD(P).4. The global change of lysine acetylation (Kac) involved in patulin degradation by R. mucilaginosa was analyzed. Among the acetylation, 54 acetylation sites in 46 lysine acetylation proteins were up-regulated and 81 acetylation sites in 60 lysine acetylation proteins were down-regulated. For the up-regulated proteins, most of the acetylated proteins were classified under binding, catalytic activity and oxidoreductase activity. Under the biological process, the tricarboxylic acid metabolic process and metabolic process occupied most of the up-regulated Kac proteins,followed by cellular protein metabolic process, nucleic acid metabolic process and cofactor biosynthetic process. The classification of the down-regulated Kac proteins showed that the largest acetyl proteins were associated with the binding,oxidoreductase activity and catalytic activity. Under the biological process, the cellular protein metabolic process, metabolic process and response to oxidative stress occupied the most Kac proteins. The metabolic pathways involved in the differentially expressed acetylated proteins include tricarboxylic acid cycle, glycolytic pathway and pentose phosphate pathway.5. Quantitative lysine crotonylome analysis of R. mucilaginosa in response to the addition of PAT in vitro indicated that 102 crotonylation sites in 79 crotonylation proteins were up-regulated and 67 crotonylation sites in 46 lysine crotonylation proteins were down-regulated in the presence of PAT. Among the assigned processes,the top three categories were metabolic process, cellular process and single-organism process in up-regulation or down-regulation proteins respectively. The three function(catalytic activity, binding and structural molecular activity) were the most important molecular function either in up-regulation or down-regulation proteins. From the results, the largest group of the up-regulated proteins was associated with mitochondria, the up-regulated proteins were related to ROS activity and tricarboxylic acid cycle.6. A large number of transcription information of R. mucilaginosa genome was obtained by RNA-Sequencing technology, and there were 182 differentially expressed genes selected of which 104 were up-regulated and 78 were down-regulated. Most of them may be related to metabolic process and glycolysis process.7. The degradation product of patulin by R. mucilaginosa was identified as ascladiol through HPLC and HPLC-PDA-MS analysis, and it was proved none toxic by HepG2 cells by a certain concentration. |
PDF Full Text Request