Metaproteome Analysis Of Soybean Paste

Nowadays many families still remain the culture of making soybean paste in winter by using natural fermentation while the produces from natural homemade fermentation show different variations in nutritional ingredients, appearance and taste. Furthermore, the soybean paste is easy to be contaminated by microorganism and it is hard to maintain the stability of nutritional ingredients and the quality of homemade soybean paste. The production of soybean paste has already expand into industrial scale. Compared with traditional method, the industrial method could guarantee the high level of stability and yield of the produce. However, various challenges, such as limited types of zymophytes, insufficient fermentation period and high level of salt content, still need to be improved during the industrial product in order to cater for customer further needs and keep the quality of product.By now, few researches regarding to using Metaproteomics technology to study soybean paste have been reported. In the research, the Metaproteomics of the traditional fermented soybean paste are investigated in detail. Two parts of the Metaproteomics of soybean paste are involved into this research, including intracellular Metaproteome and extracellular Metaproteome.Dodecyl sulfate-polyacrylamide gel electrophoresis were used to exact both intracellular Metaproteome and extracellular Metaproteome from four types of soybean paste, followed by the exacted Metaproteome were identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MC/MS). Finally, based on the methods of proteome bioinformatics, the differences of Metaproteome function and source of microorganism from four types of home-made soybean paste were analyzed.The results are as follows:(1) The centrifugal rate of purification, bacteria collection and the ultrasonic power of bacterial cell break wereinvestigated and optimized. The results analyzed by SDS-PAGE suggested that centrifugal rate is under 2000r/min and 6000r/min is suitable for purification and bacteria collection respectively. The optimum process of bacterial cell break:liquid nitrogen grinding, TCA-Acetone sedimentation, acetone washing, ultrasonic power of bacterial cell break is 26w.(2)The extraction method of extracellular Metaproteome is estsablished by SDS-PAGE: 2000r/min and 6000r/min is suitable for purification and bacteria removion,12000r/min is suitable for extracellular Metaproteome collection. TCA-Acetone sedimentation, acetone washing, ultrasonic power of bacterial cell break is 26w.(3) The intracellular and extracellular protein expression maps of four different types of soybean paste were built by using the results from SDS-PAGE and LC-MS/MS. Most of microbial intracellular proteins from soybean paste are involved into the processes of protein synthesis, sugar metabolism and nucleic acid synthesis. The percentage of intracellular involved into functional reaction is under 10% of the total amount of intracellular protein. Compared with intracellular proteins, most of extracellular proteins make contributions to protein synthesis and sugar metabolism. The percentage of extracellular proteins involved into nucleic acid synthesis is under 6.7% of the total amount of extracellular proteins. Furthermore, proteins which are associated with nucleic acid synthesis are not detected in the sample S2 and S4. The amount of extracellular proteins involved into other functional reaction is larger than that of intracellular proteins are relative to other functional reaction. These proteins include outer membrane protein and heat shock protein, The common sources of microorganisms at the species level of four kinds of soybean pastes including Leuconostoc, Leuconostoc, Enterococcus faecalis, Lactococcus lactis, Acinetobacter baumannii, Bacillus amyloliquefaciens, Lactobacillus sake, Lactobacillus plantarum, Lactobacillus bulgaricus deshi bacteria, Lactobacillus casei, Saccharomyces cerevisiae and S. pombe. The common sources of microorganisms in the project level including Bacilli and Dikarya. Specific microbial including Betaproteobacteria, delta/epsilon, subdivisions, Oscillatoriophycideae, Nostocales, Thermotogales, Mollicutes and Thiotrichales. Specific microbial origin of S3 including Hydrogenophilales.