Study On Optimizing Enzyme System By Using Multi-strains During Koji Making And Improving Quality Of Soy Sauce

Soy sauce is a traditional fermented condiment commonly consumed in China, Korea and Japan. And it is a popular condiment all over the word in recent years. In 2006, the production in China is about 5 million tons. But Japanese soy sauce takes the main role in international market, though China is chasing after Japan. With the price increase of raw material in recent years, it is more urgent need to improve the protein utilization rate. It is useful and important to improve the protein utilization rate and flavor of soy sauce. The strains is more important in soy sauce fermentation, but little report on koji making by multi-strain, this research focus on improving the protein utilization rate and flavor by using multi - strains in koji making and the low-salt solid-state-fermented technology. The main research contents of this thesis are as follows.1) The character of enzyme system were studied during koji making, by considering the activity of neutral, acid and alkaline proteinase, amino peptidase, amylase, cellulase and xylanase. Then the combination strains in koji making were carried out. It was found that the activities of neutral and acid proteinase and amino peptidase were increased significantly. The activity of proteinase in C₂B₃ was 4.48 and 3.78 times respectively to C₂ and B₃.The protein utilization was improved about 4.87% compared with industrial strains in 60 L pilot test.2) Based on the four factors and three levels orthogonal experiment, the RSM was carried out. The optimum conditions were wheat bran 42%, the addition ratio of water 100 %, the time for raw material cooking 21 min and for koji making 42 h. It is helpful to proteinase and amylase production, and to leucine amino peptidase activity increasing significantly.3) There is no different between low-salt solide-state fermented soy sauce and high-salt liquid-state fermented soy sauce in physical and chemical properties. Acids, phenols and heterocyclic compounds were higher in high-salt liquid-state fermented soy sauce than that in low-salt solide-state fermented soy sauce. So did the content of total free amino acid and MSG-like amino acid.4) Compare the characters of physical and chemical properties between soy sauces which fermented by combination strains and by single strains. Esters in sample fermented by combination strains was higher, acids in sample fermented by single strain was higher. The important flavor compounds heterocyclic compounds was higher in sample fermented by multi-strain. Content of total free amino acids and MSG-like amino acids in soy sauce fermented by combination strains were higher than that fermented by single strain. Compared with C₂ and B₂, the content of Glu was increased 30.04% and 41.88% in B₂C₂. Compared with B₃ and C₂, the content of Glu was increased 26.52% and 23.31% in B₃C₂.5) Result of physical and chemical properties and free amino acids and mode identification of soy sauces suggested that the six physical and chemical indexs could be replaced by three principal components. Total nitrogen, formol titration nitrogen and total acid can be expressioned by F1. There was a close relationship between total sugar and reducing sugar content. The third principal component indicated the concentration of NaCl. Clustering analysis and discriminant analysis were take out, discriminant equation was D=7.415 x₁-5.883 x₂+2.63 x₃+48.146 x₄-4.685 x₅+15.457 x₆-138.224. 94.12% of original cases were correctly classified according to the equation. Free amino acids index could be replaced by four principal components. Asp,Val,Ser,His,Gly,Glu,Ala and Leu can be expressioned by F₁. F₂ were Glu,Ser,Cys,Phe,Ilu and Leu. The third principal component indicated the Asp,Arg,Tyr,Pro,Ilu and Lys. The F4 was Met. Clustering analysis and discriminant analysis were take out, discriminant equation was D=11.65x₁-40.159 x₂-12.934 x₃-22.76 x₄+56.347 x₅-79.137 x₆-17.507 x₇ +50.227 x₈+15.998 x₉-28.615 x₁₀+28.48 x₁₁+9.633 x₁₂-286.486. 100% of original cases were correctly classified according to the equation.In conclusion, the results indicated that combined multi-strains of Aspergillus oryzae in koji making could improve the enzyme system composition and protein utilization and flavor of low-salt solide-state fermented soy sauce effectively. Meanwhile,it was suggested that the discriminant equation could be useful tools for monitoring the quality of soy sauce.