| Protease is one of the most important enzymes in high-salt liquid-state soy saucefermentation. It is mainly responsible for the degradation of raw materials and the generationof the amino nitrogen which directly determines the quality of the soy sauce. High saltenvironment of moromi in high-salt liquid-state soy sauce fermentation inhibit the proteaseactivity with the fermentation phase of up to six months, greatly restricting the developmentof the soy sauce industry. Therefore, catalytic mechanism of protease at high salt environmentis of great significance to the improvement of the soy sauce brewing process.Aspergillus oryzae CICIM F0899was used in this thesis. The catalytic properties andstability of protease was investigated at high salt environment. Finally, the Aspergillus oryzaeand its salt-tolerant protease were used in high-salt liquid-state soy sauce production. Themajor research contributions are shown as follows:(1) A strain of Aspergillus oryzae capable of producing neutral protease was used in thisresearch. The enzymatic properties and water activity of protease secretion by Aspergillusoryzae were investigated. In addition, properties of the protease secreted by this strain weretested and compared with profiles of an industrial strain Aspergillus oryzae3042. The resultsindicate that the optimal temperature for the extracellular protease from F0899was60°C,with the stable temperature range between20～40°C. The optimal pH was8.0, with the stablepH range between6.0～8.0. It can withstand the presence of18%sodium chloride and stillretains20%activity, which is as high as2.11times of that from3042. Three water activitycontrol agents had impact on growth and protease production, among which NaCl had themost significant inhibition.(2) A two factorial design of experiments was applied to study the simultaneous effect oftemperature and water content on Aspergillus oryzae growth and protease production in thesolid-state fermentation. The logistic model was used to calculate growth kinetic parametersat different levels of temperature and water content in solid-state fermentation. Ratkowskyequation was used to express the effect of temperature on specific growth rate. Quadraticpolynomials express relationship between water content and growth parameters of Aspergillusoryzae inoculated on wheat bran. The highest value of μmand Xmwere0.1671/h and211mg/g biomass at30°C and50%, respectively. Protease secretion from Aspergillus oryzae iscoupled with the cell growth. The experiment results showed that this model could predict thegrowth pattern and protease production very well. The protease fermentation was carried outin solid-state bioreactor and reached to1560U/g.(3) The kinetic model of salt-tolerant protease was esatablished at hypertonicenvironment and compared to papain. The protease was purified to homogeneity throughammonium sulfate precipitation, ion-exchange chromatography and gel filtrationchromatography. Its molecular weight was estimated to be50kDa by sodiumdodecylsulfate-polyacrylamide gel electrophoresis. A kinetic model incorporating theDebye-Hückel limiting law was proposed for protease at hypertonic environment. It wasfound that, with the higher ionic strength, the Michaelis constant of the protease monotonically increases while the turnover number kcatdecreases in accordance with the firstkinetic model. The high salt environment affects the papain kinetics in a different way. Bycomparing the binding site, active site and amino acid sequence between two proteases, wefound that high salt environment had impact on enzyme activity for the binding site, activesite and amino acid sequence. The result was consistent with the kinetic analysis.(4) The mechanism protecting protease at hypertonic environment was revealed bythermodynamics changes. The protease activity at various temperatures and NaClconcentrations were investigated. The half-life of the protease was significantly prolonged asNaCl concentration increased at temperatures ranged from45to70°C. When the protease wasperformed at24%NaCl and60°C, its half-life was increased to30min,211%longer than thatof without NaCl and at60°C. At24%NaCl concentration, the Gibbs free energy andactivation energy increased by2.1and4.75kJ/mol, respectively. Results of circular dichroismspectroscopy analysis at various NaCl concentrations revealed that α helix conformation inthe protease increased at higher NaCl concentrations. The α helix conformation increasedfrom3.2%to9.3%and31.7%when the NaCl concentration increased from0%to6%and18%, respectively. Adding18%NaCl at pH4.0, the half-life of the protease was prolongedfrom4.3d to7.2d. When the NaCl concentration reached18%at pH5.0, the half-life alsoextended by3d. The stability of protease was increased by adding high concentration of NaClat acidic environment. The protease exhibited higher thermal stability in high salt conditions.This feature is beneficial to soybean sauce fermentation by enhancing the proteaseperformance and taste of the product.(5) Aspergillus oryzae F0899and its salt-tolerant protease were used in high-saltliquid-state soy sauce fermentation. A novel inculation method using salt-tolerant protease toincrease amino nitrogen content was utilized. It shows that the organic acids were producedby microorganisms in the moromi fermentation stage and caused excessive reduction0f pH.The organic acids also influenced the protease activity and led the changes of amino nitrogenand volatile compounds. The amino nitrogen content was reached to0.62g/100ml, was11.11%higher than3042. HPME and GC-MS were applied to analyze the flavour compoundsof the soy sauce. Totally49compounds were identified including7categories, which thecontent of esters and heterocyclic compounds were higher than3042and aldehydes werelower. Aspergillus oryzae CICIM F0899has more advantage on soy sauce fermentation.Adding salt-tolerant protease in the early stage of moromi fermentation could improve aminonitrogen content and free amino acid significantly. After two months of fermentation, aminonitrogen content increased by5.05%and4.55%, respectively. |
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