| The unique feature of Chinese liquor is Solid-State-Fentation (SSF), in whichsaccharification and fermentation simultaneously occurred. Besides various microbes andbio-enzymes, SSF also involve complex interactions among microbes, bio-enzymes, andsubstrates. Among various microbes, Aspergillus play a central role in the brewing process,because it could not only provide the process with saccharifying enzyme, amylase and otherfunctional enzymes, which are employed to degrade the macromolecules of substrates andsupport the growth of other functional microbes, but also promote the formation andaccumulation of alcohol and many flavor compounds. Additionally, the acid environment ofSSF is also suitable for the growth of Aspergillus strain and the functioning of otherbio-enzymes. Therefore, to carry out research on Aspergillus resource from the SSF ofChinese liquor and their effects on this process would enrich our knowledge on themechanism of fermentation and upgrade the technology of Solid State Fermentation.Due to the central position of Aspergillus in SSF of China white-liquor, this research firstfocused on the ecological environment where Motain-flavor liquor was made. Subsequently,an Aspergillus strain with high ability to produce saccharifying enzyme and protease wasachieved. By modelling SSF, the strain and its product: saccharifying enzyme and proteasewere tested on their tolerance to the stressed condition in SSF. With an aim of determining theeffect of different fermentation conditions on its ability to produce enzymes, the extracellularproteins produced by the strain growing in different enironments were analyzed. Additionally,the extracellular acid protease was purified and characterized. Finally, the function of thepurified protease was confirmed in SSF with an aim of studying its effects on the formation offlavor compounds. The main conclusions are as follows:(1) In light of the specific property of brewing environment, screening of microbesproducing high levels of menfentation functional enzymes was carried out. Consequently,103Aspergillus strains were obtained from the environment where Moutai-flavor liquor was made.Those strains could be classified into10species: A. niger, A. oryzae, A. terreus, A.tubingensis, A. flavus, A. hennebergii, A. fumigatus, A. niveus, A. candidus and Monascus.Among them, A. hennebergii was identified as the dominant strain, which could produce highlevels of saccharifying enzymes and proteases. Their highest concentration was2173.51U/gand368.59U/g, respectively. Besides, this strains could tolerate extreme environment, suchas low pH (2.5), high temperature (50oC), and high levels of alcohol (8%). Together, thisstrain was an excellent Aspergillus with white-liquor brewing function.(2) In order to investigate the difference of extracellular proteins produced by A.hennebergii grown in solid and liquid medium, the technology of2-DE combined withMALDI-TOF MS/MS was employed to analyze its extracellular proteome. As a result,147protein dots were successfully identified. These proteins shared the same sequences with thesefrom A. niger, A. orizae, and other5species of Aspergillus. Additionally,47proteins of theseidentified proteins were differentiated proteins, which were implicated in30proteins. Theseresults suggested SSF was not only good at the production of saccharifying enzyme and protease, but also suitable for the secretion of other enzymes, which could prompt theformation of flavor compounds.(3) According to the analysis on proteome of the strain, the extracellular acid proteasewas purified and characterized. The purified proteases were AP2and AP3, responding to the15and274dot on2-D electrophoresis. The molecular weight of AP2and AP3were48kDaand33kDa, respectively. Studies on their kinetics indicated that AP2and AP3displayedhigher hydrolysis activity to proteins from wheat. However, the catalytic efficiency of AP3was superior to that of AP2. Additionally, studies on the optimum pH and pH stability ofpurified enzyme also suggested AP3was superior to that AP2. The optimum temperature ofAP2and AP3were50oC and45oC, respectively. The thermostability of AP3was superior tothat of AP2in the range of30-60oC. AP3kept its normal activity when alcohol concentrationwas less than10%. These results indicated purified protease has advantage against acid,temperature and alcohol. Its advantage was important for the existence of wheat and soy inSSF.(4) Solid-state-fermentation was conducted with the aim to confirm the applicationproperty of the purified protease. The results suggested that the addition of AP3in SSF couldsignificantly enhance the concentration of flavor compounds. The quantitative analysis of theflavor compound indicated the total amount of flavor compounds in fermented grains. Thesecompounds included alcohol, acid, ester, and aromatic compounds. Compared with thecontrol group, the application of AP3could obviously improve the hydrolysis degree of wheatproteins, the content of amino acid nitrogen, the cell biomass of S.cerevisae, the total acid,and the alcohol content. Their improvement was28.26%,34.21%,37.09%,36.17%, and38.29%, respectively. These results suggested the addition of AP3could prompt thehydrolysis of proteins, increasing the content of amino acid nitrogen, total acids, and alcoholin the system of SSF.In summary, the obtained A. hennebergii was good at secreting saccharifying enzymeand protease of brewing function and other enzymes producing flavor compounds.Additionally, the microbe could also coordinate several processes such as saccharification,fermentation, and flavor production. This work not only provided modern brewing technologywith excellent microbial resource, but also enriched the theory and practice of basic researchon SSF. |
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