Study On Proteinase Which Microbe Secreted From Traditional Fermented Soybean (Douchi) In Yunnan Province

Fermented soybean is the one of the most ancient ferment food that originated from China. It is a tradition ferment seasoning make from the black bean and soybean fermented by microbe. It is welcome by consumer because it's sweet smell and special flavor. Fermented soybean have most value of nutrition and rich in soluble sugar, soluble nitrogen, vitamin A, vitamin E, vitamin B and so on. It contains many functional factor such as soy isoflavones, soybean oligosaccharides, soybean saponin, soybean peptides, Fermented soybean fibrinolytic enzyme. Fermented soybean not only have great nutritious buy also have many function such as detoxication, remove worry and melancholy, cure season fever, headache, feel cold and heat, depress, worry and not sleep, loss of appetite, indigestion and so on. Fermented soybean can prevent frequently old disease such as alzheimer's disease, osteoporosis, hypertension, diabetes. Fermented soybean fibrinolytic enzyme is a hot research in our country, it is a new fibrinolytic enzyme can dissolve fibrin directly found in fermented soybean. And it has a high fibrinolytic activity, the use of safe and reliable. So it has been deep attention by domestic and foreign scholars.The most famous fermented soybean in our country main includes yongchuan fermented soybean in Sichuan province, liuyang fermented soybean in hunan province, linqi fermented soybean in Shandong province. Few studies on the fermented soybean in Yunnan, Yunnan is China's most ethnically diverse province, the nation still retains the traditional fermented food technology, so more types of fermented soybean. In this study four cities in Yunnan Province (Kunming, Yuxi, Zhaotong, yuan yang) Douchi sample collection, the types of micro-organisms in fermented soybean a preliminary study, by SDS-Zymography and other experiments on the fermented soybean contains a preliminary analysis of protease, and the right of the fibrinolytic activity in fermented soybean make a preliminary determination.The purpose of this study is use different selective solid medium and some biological and chemical experiments in the microbial diversity of douche and make preliminary study on the microorganisms in douchi samples for a preliminary identification. At the same time to establish an effective system for DNA extraction and use 16SrDNA universal primer for PCR amplification, use the sequence alignment compare microorganisms in samples for species identification and establish phylogenetic trees, discuss and analysis the diversity of microorganisms in douche in Yunnan province. And make a preliminary study on protease activity in douchi, use the detecte method of protease activity and SDS-PAGE-Zymography experiments and analysis the species and activity of protease in sample, then sum up species and activity of protease in douchi in different area. Collecte the sample and do related experiments in different parts of Yunnan province for sum up the diversity of microorganisms in douchi in different aera, and discuss the activity and species of protease in samples in order to discover useful microorganisms resources.Use the SDS-Zymography and other experiments for preliminary analysis the protease in douchi, according the number of bands and brightness on the plastic sheet to identified the type of protease, the result is No.9 bacterial strain in Kunming have high protease activity, secretion the largest number of protease molecules, it is about nine kinds of molecular weight of protease, molecular weight is distribution in 14KDa-116KDa, inference it may contain a variety of proteases such as gelatin, collagen protease, fibrinolytic enzyme and so on. No.11â‘ bacterial strain in Yuanyang is similar to No.9 bacterial strain in the Kunming, secretion the largest number of protease molecules, it is about eleven kinds of molecular weight of protease. The No 1,3 and 9 such as six strains in Yuxi secreted protease have the most of molecules weight. And about five kinds of molecular weight of protease. The No.25 bacterial strain in Zhaotong secretion the largest number of protease molecules, it is about four kinds of molecular weight of protease. In summary, the analysis the activity and species of protease in strains in four aera, the most prominent strains is No.9 in Kunming and No.11â‘ , and these strains secreted the number of molecules and protease activity is highest.Use the improved method of fibrin plate test, calculate the size of soluble protein ring. And base on SDS-fibrin-PAGE and protease activity of ninhydrin assay experimental results in No.YX-1 strain in Yuxi of protease activity reached 96.22U/mL, No.YX-11 of protease activity reached 93.88U/mL, No.YX-18 strain of protease activity reached 96.66U/mL, the No.ZT-13 strain in Zhaotong of protease activity reached 97.09U/mL, No.ZT-15 strain of protease activity reached 94.61U/mL, then speculated that the fibrinolytic enzyme activity in samples of douche in Yuxi was highest in four regions, and the follow is Zhaotong region. The results of experimental for make a foundation for the depth research in high plasmin activity in the sample of douche in future.