Studies On The Screening And Identification Of Strain With High Naringinase And Its Enzyme Producing Characteristics

This paper considers the utilization of debittering enzyme to reduce pomelo's bitterness without destroying nutritional composition.No effect or alteration to the natural good flavor of pomelo juice or pomelo wine is caused.It is the best method of industralization application foreground.In addition,it is also a focus research in this field.This topic uses the strains isolated from nature as the original strains.This research focues on one of the main enzymes of debittering,enzyme-naringinase.This research entails screening of strains with high productivity,strain identification,cyclic fermentation rules,a comparison of different enzyme activity's detection methods,and basic properties of crude enzymes.This study screened a total of 60 fungin strains which were isolated by our laboratory from moldy pomelo's peels and soils with decayed pomelo fruits.Naringinase is one species of induced enzyme.Medium contained naringin powder or acetone extraction fluid of bitterness matter is the best induced producing enzyme medium.Using method of of transparent zones and plat streaking,strains experienced prliminary purified separation.Inoculated the purified strain to the selective solid plate medium,which contained 0.5%naringin powder as an inducer of producing enzyme for cell growth.This was cultivation at a temperature of 28℃,over 5 days.Performed the primary screen to strain through observation on its growth situation.Then inoculated the primary screened strains by a liquid fermentation medium,which contained 0.5%naringin powder as the as an inducer of producing enzyme for cell growth.This was done at a temperature of 28℃,a rotation speed of 160 rmp,and a culture time of 5 days.We utilized a desktop medium hypothermia centrifuge(centrifugal speed is 3500rmp and time is 15 minutes) to obtained the crude enzyme.To determine the enzyme activity we used the Davis Method.The result showed that strain P-61,so named by our laboratory,had the highest enzyme activity of 310.64.Thus decided to choose this strain as the target strain for further study via sexual observation,apperance observation and microscopic observa -tion.This lead us to the conclusion that we couldn't observe this strain's hallucal.The conidial was spike spherical and the colour was purplebrown or black.It is coincident wih the description of Moniliaceae,Amerosporoideae of Moniliaceae,which belongs to the Aspergilleae.Aspergillus Mich.ex Fr.A.niger.Therefore,this strain was identified as the aspergillus niger.This study aslo researched the fermentation regularity of aspergillus niger P-61 and compared the advantages and disadvantages of UV Spectrophotometry(Davis Method) and High-Performance Liquid Chromatography.Under the same cultural condition,through the observation to the cultural medium's variation trend following cultural time increased.Included the observation of reducing sugar content,naringin content,trihydroxyflavanone and naringinase activity.This allowed us to conclude that:A) the medium which contained naringin solution concentration at 1.5g/L as an inducer of producing enzyme was the most stable,B) Davis Method is fast,but likely due to the interference by naringin degradation intermediate,the accuracy was poor,and C) using High-Performance Liquid Chromatography,although operating the machine during pretreatment was complex,the resulting accuracy was good and only had minor interference.Finally,it conducted a basic study of crude enzyme liquid at a temperature of 28℃and a rotation speed of 160rmp for 5 days,followed by using a hypothermia centrifuge to centrifugal(centrifugal speed of 5000g and time of 15 minutes).It was determined to use a two-step deposition would be appropriate and 30%ammonium sulfate was used first for eliminating the impurity protein and 60%ammonium sulfate was used for precipitating naringinase.By SDS-PAGE,it determined P-61 strains' crude enzyme liquid which after 5-fold ultrafitration and concentration.The crude enzyme's molecular weight was found to be about 100KDa and its activity and stability depended on the environment.At the condition of pH4.0 and at temperature 50℃,enzyme had the highest activity.The solution's ion species had some effection on the enzyme activity of aspergillus niger P-61.Ca²⁺(5mol/L) and had intensive activation for this crude enzyme.In addition,Zn²⁺,K⁺,EDTA had very strong inhibition for this crude enzyme.