Screening And Optimization Of Producing Enzyme Condition Of Excellent Rhizopus In Chinese Koji

Rhizopus,fungus of the Mucoraceae family at the Phycomycetes Class,can produce lots of amylase,proteinase,zymase with their growth.It is the main saccharification fungus in Chinese koji.Be rich in Chinese koji,China has a huge library of Microorganisms,which contains a lot of quality fungus.Because of this,many savants are interested in separating,purifying and screening the superior Rhizopus.In this thesis,selected superior strains from the superior tranditional Chinese koji,and got two superior Rhizopus:ERh203 and ERh505.At the same time,the characters of Enzyme Production are studied and the Rhizopus identification are completed.All of these can give the theoretical basis and reference data for the actual production.The main results are as follows:(1) Through analysis of the 8 samples of tranditional Chinese koji,sample 2 has the highest glucoamylase and liquefication enzyme activities with higher protease enzyme activities.Sample 5 has the hightest protease enzyme activities and glucoamylase and liquefication enzyme activities.Therefore,Sample 2 and Sample5 are chosen for further investigation.By seperating and purifying these two samples,here gets 12 strains.Comparing 12 stains with the characters of enzyme Production and the rice fermentation test which were isolated from sample 2 and sample 5,two superior strains have been selected for further studies:ERh203 and ERh505.Additionally,both of them can produce zymases,which is the most important character of being a superior strains.(2) Through the Single factor experiment,cultivation time has more influence in Enzyme Production.It will improve the growth of the two strains and the activities of Enzyme by 3 to 4 days.Meanwhile,temperture also influences the Enzyme Production. Culturing the two strains at 30℃,the activities of s glucoamylase and liquefication enzyme are much higher,but the activities of protease enzyme get the max at 26℃.In the course of production,controlling the temperature with the requirement is important. The initial pH value does not have much more influence in the two strains.Between pH5.2 to pH7.0,the activities of enzyme Production will be more stable.Putting some corn flour in bran culture medium can improve the ability of enzyme production.Adding the corn flour at a concentration of 8%-12%to the two trains,the activities of glucoamylase,liquefication enzyme and protease enzyme increase in varying degrees. For ERh203,when the adding com flour concentration is 8%,the glucoamylase and liquefication enzyme activities get the max,919.37 mg/h·g^-1 and 648.64 mg/h·g^-1. comparing without adding corn flour,it increases 6.7%and 5.4%.The activities of protease enzyme when adding 12%corn flour is 860.74μg/min·g^-1 that increased 5.9%.When adding corn flour beyond the concentration,the activities of Enzyme will be down.Here is the reason,the more corn flour,the more of Viscosity of culture medium,which reduces the activities of enzyme.The reason effects the growth of Rhizopus.(3) From the results of orthogonal tests,the results show that the temperature and the cultivation time and the concentration of corn flour affect the livingbess of activities of enzyme obviously.The influence of activities of glucoamylase is temperature＞cultivation time＞concentration of corn flour with the best cultivation temperature of 30℃,the best cultivation time of 3 days,at the conceration of 8%of corn flour.The influence of activities of liquefication enzyme is cultivation time＞temperature＞concentration with the best culturing temperature of 30℃,the best culturing time of 3 days,at the conceration of 8%of corn flour.The influence of activities of protease enzyme is cultivation time＞temperature＞concentration with the best culturing temperature of 28℃,the best culturing time of 4 days,at the conceration of 12%of corn flour.(4) Through the observation of colony,the obervation and the measure of hypha, rhizoid,sporangium,Spore,Chlamydospore etc.by microscope,here is the result. ERh203 is Rhizopus oryzae Went et Prinsen Geerlings Species of Rhizopus Ehrenberg Genus of Mucoraceae Family at Mucorales Orderat Phycomycetes Class,and ERh505 is Rhizopus tonkinensis Vuill Species of Rhizopus Ehrenberg Genus of Mucoraceae Family at Mucorales Orderat Phycomycetes Class.