Studies On Screening Of Mould Producing Debitterness Protease And Properties Of The Protease

Peptide has well nutritional and sanitarian function. It can beobtained by hydrolyzing protein with acid, alkali and enzyme, among whichenzymolysis is the best one. Because of the bitterness in the proteolysis,which influence the product taste, removing bitter taste fromproteolysis has become a urgent problem in modern industry. Since thereare some advantages by using specific microorganisms to debitter, theseeking for a microorganism to debitter successfully has become one ofthe urgent task.This thesis deals with four parts:1 In order to find out a highest protease-producing mould strain,mucor, aspergillus niger, Aspergillus terricola 3.942 and M1, M2, M3which were separated from the beans sauce fermented naturally wereincubated in their solid and liquid mediums, their protease-producingactivities had been tested. With their thick enzyme liquids to act onthe bitter-peptide, thick enzyme liquid of M3 and mucor could reducethe bitterness obviously, but the aspergillus niger and Aspergillusterricola 3.942's had few effect on the bitterness. So M3 was screenedfor its excellent protease producing and the ability of bitternessremoval. M3 belonged to the Aspergillus genera according to its morphacharacters of colonies and filamentous.2 The culture conditions of Aspergillus M3 were studied. The results showed that the best fermentation medium was bran: glucose: water=9:1:5,6% inoculation amount, 28℃for 72hr, and the neutral protease activityis up to 12608.52 unit pre gram dry moldy bran after extraction bydistilled water. The protease powder could be purified using ammoniumsulfate precipitations, and its properties were also studied. The resultsshowed: the activity of the enzyme powder was 159781.1u/g, the optimumtemperature and pH were 50℃and 7.5 respectively; the enzyme was ratherstable at 30～40℃and at pH 6.5～7.5, it was activated by NH₄¹⁺ andK¹⁺, while Fe³⁺ strongly inhabited its activity.3 Using the Aspergillus M3 protease to hydrolyze soy protein isolate(SPI), the best condition of pretreatment and enzymic hydrolysis wasdetermined. Experiment showed the best pretreatment condition washeating for 10min in 80℃water; the best condition of enzymic hydrolysiswas 5%(W/V) substrate concentration, 4200u/g(protein) enzyme, pH7.5, 50℃. Under this condition, the degrees of hydrolysis at different timewere determined and their bitterness were tasted, the study showed thatthe degree of Hydrolysis increased with time, and there wash'tbitterness at any time.4 Aspergillus M3 was mutated by UV and NTG, and a mutant of N43was screened. Its protease activity raised by 146% Contrast toAspergillus M3, moreover, its genetic stability was very good. Thereis no bitterness in the proteolysis of SPI hydrolyzed by N43 protease.