The Select Of The Microbe Excreting Tannase And The Study Of The Tannase Property Produced By It

The persimmon is a fruit that is rich in nutritious component and containsmany kinds of physiologically active factor and food value is very high. It containssugar, protein, necessary amino acid of human body, and various microelements,vitamin, organic acids, flavones etc. The persimmon resources in Our country is veryrich, The plantation area of Chinese persimmon has increased more than 293.8 ha, withan annual output of 3.47 million t, accounting for over 70% of the total world output. Inour country, Approximately over 80% of persimmons are the astringent persimmons, thepersimmon fruit that isn't astringent will appears the phenomenon that it returnsastringent after the processing process or in the processing.Until now it does not havethe effective method to solve the problem in the producing practice and it has seriouslyrestricted the development of industrialization in persimmon processing.The tradition ways that remove astringency of astringent persimmon doesn't makethe tannin vanished, only because the solubility of tannin reduces, and the combiningability of the tannin with the protein in the oral reduces, then the person can't feel theastringency.It has the important practice instruction significance to impels thedevelopment of persimmon industry and increase the persimmon fruit's attachmentvalue, enhances the economic efficiencyThis paper made the astringent persimmon as the micro environment, let themicroorganism grow naturally, scoring the long-base on the culture medium plate toseparate the microorganism, obtaining the purebred strain.By the culture medium whichcontains Bromophenol blue and making use of COD taking off rate as a index tomeasure the ability of a microorganism secreting tannase, four strains wereobtained. Then tolerance to the tannin was studies, in order to selecte the bacterial strainbeing able to bear high tannin concentration and to producing more tannase. Afteridentifyed, it was proved that the strain is Aspergillus niger.Preparing the protoplast of the target bacterial strain, that was dealed withultraviolet light. Choosing four mutating strains that the tannase produced by them hashigher activity than the original strain, and the mutating strains were proved that substrains had inheritance stability.The strain was selected that can produce tannasewhose activity was 35.6% higher than those of Original strains.The A. niger was cultivated in flask on rotary shaker, and the fermentationtechnological parameter to the produce enzyme was studied. It concluded cultivatvingtime, the temperature, initial pH, glucose thickness, inoculating amounts, tanninconcentration and volume of the fermentation liquid.Adopting L₂₇(3¹³) orthogonality experiment design, the above seven factors wereoptimized. Properties of tannase secreted by Aspergillus niger was studies, the Km andthe Vm value in Michaelis-Menten parameters through the curve of Linewear-Burk wasascertained,the optimum PH and temperature, the stability of tannase to PH andtemperature, as well as the impact of ions on the tannase. The results as follows:(1) It is for 72 hours that AspergiIlus niger can obtain bigger biomass and themaximal enzyme activity in shaking flask (160r/min).(2) With Shaking (160r/min) for 72h, it is 28～32℃that is available for Aspergillusniger to product tannase. At 30℃the tannase activity is the highest, 142.9U/mL.(3) With Shaking (160r/min) at 30℃for 72h, the optimal tannin concentration is2-5%.When tannin thickness is 4%, it may gain maximal enzyme activity: 156.7 U/mL.(4) With Shaking (160r/min) at 30℃for 72h, when pH is 5～7, Aspergillus nigergrows well and enzyme vigor is higher.When pH is 5, the biomass is biggest, 356.8mg;When pH is 6, the enzyme vigor is highest, 160.3U/mL(5) With Shaking (160r/min) at 30℃for 72h, glucose concentration range from 0.5to 1.0%, the A. niger can produce more tannase.When the glucose concentration is 0.8%enzyme vigor is highest.(6) With Shaking (160r/min) at 30℃for 72h, the optimal inoculating amount is1.5%～2.5%. Whileit is 2%, the enzyme vigor is highest.(7) With Shaking (160r/min) at 30℃for 72h, adding 40～60mL fermentationliquid to 250mL flask can obtain higher tannase activity, when it is 50mL the enzymevigor is highest.(8) Through the orthogonal experiment, we may conclude that the order from priorfactor to inferior factors is the time, the temperature the volume, tannin concentration,sugar thickness, initial pH, inoculating amount. The optimum assembling was: culturetime 72 hours, culture temperature 28℃, volume of culture medium 40mL, tanninconcentration 3%, sugar thickness 0.8%,initial pH 7.0, inoculating amount 2.5%. (9) When PG selected as the substrate, the research of dynamics indicate that theKm=0.595mmol/L,Vm=0.363mol/L.min(10) The optimal pH value of tannase is 6.0. In the pH range of 5.0～7.0, thetannase activity is stabile.(11) The most suitable reaction temperature is 55℃. The tannase activity remainsmajority when temperature is lower than or equal 60℃.(12) Effect of mental ion on tannase activity is that:Ca²⁺has little effect on tannaseactivity; while Al³⁺,Sn²⁺and Mn²⁺have no effect on it; Cu²⁺,Zn²⁺,Fe²⁺,Al³⁺, Co²⁺could inhibited the enzyme activity obviously.