Metaproteomic Research Of The Extract Of Rice Wine Wheat Qu

To completely understand all soluble proteins (mainly including raw material wheat enzymes and microbe exocellular enzymes) in the extract of rice wine wheat Qu, this paper introduced the theory and ideology of metaproteomics for the first time, using the typical two-dimensional electrophoresis (2-DE) technology and the mass spectrometry analysis for the research of the metaproteome of the extract of rice wine wheat Qu. In this thesis, metaproteome referd to all soluble proteins in the extract of rice wine wheat Qu.By groping and comparing different 2-DE conditions (isoelectric focusing electrophoresis parameters, sample loading quantity and strip length), the best isoelectric focusing electrophoresis programs and sample loading quantities (7 cm: ~110μg, 18 cm: ~700μg) for strips of different lengths were established. By comparing the affects to the results of 2-DE under different pH of buffer solution, combining the actual condition of rice wine fermentation, established the pH of buffer solution for the preparation of 2-DE samples as 4.2. According to the comparison of different sample preparation methods (TCA-acetone precipitation, acetone precipitation and 2-D Cleanup Kit), established to use 2-D Cleanup Kit for the sample preparation of different wheat Qu. Then, the suitable two-dimensional electrophoresis technology for the metaproteome of rice wine wheat Qu extract was established.Using the Shenyonghe machine-made wheat Qu (Zhejiang Guyuelongshan Shaoxing Wine Co., Ltd) as object of study, made metaproteome sample for 2-DE experiment, and selected all megascopic pritein spots (145) for mass spectrometry analysis and database retrieval, then a reference map of the metaproteome of Shaoxing rice wine wheat Qu extract was established. Among the 124 protein spots which were successfully identified, there were only 11 protein spots from microbes (four kinds of fungi, two kinds of bacteria and one kind of actinomycete), including glucose 6-phosphate isomerase, carboxypeptidase T precursor, malate dehydrogenase, chitin deacetylase, Cu/Zn superoxide dismutase and other five kinds of proteins. The other 113 spots were mainly from wheat (Triticum aestivum), rice (Oryza sativa) and some weeds. They were mainly enzyme inhibitor, hydrolysis enzyme, dehydrogenase, dismutase, isomerase, reduction enzyme, et al. There were also some other kinds of proteins.They were total 44 kinds of proteins.Using two-dimensional electrophoresis, compared the metaproteome of the extract of different rice wine wheat Qu, including the differences between machine-made and manual wheat Qu which were made of different modes of production, and the differences between machine-made Qu produced in different regions. Through the analysis of 2-DE analysis software, some protein points which had differences were found. And these points were conjectured preliminarily combining the reference map which had been established.