Screening And Identifying Of Strains Degrading Limonin And Study On Characteristics

Limonin is the main bitter substance in citrus fruits.It is a kind of triterpenoid compounds that lead to the strong"post-bitter"of citrus fruit juice in later processing,which greatly affects the taste of citrus fruit juice.Reducing effectively the content of bitter substances in citrus foods,improving the taste properties of foods,and developing enzymes that can degrade limonin efficiently under acidic conditions,which has important significance and good application prospect in the processing of related citrus foods.The purpose of this paper is to search for the microbial enzyme that can efficiently degrade limonin in acidic environment,and screen the microbial strain with the ability of degrading limonin efficiently by using acid vinegar mash as the source of isolation,and study the growth characteristics and the action characteristics of the corresponding limonin degrading enzyme.The main research contents and results are as follows:1.Screening and Identification of strains which degrade limonin in vinegar fermented grains.Eight limonin-degrading strains using limonin as the sole carbon source were isolated from fresh fermented grains of vinegar,and their corresponding limonin degradation rates were all above70%in the cultured system.Among them,strains 6,7,8 exhibited higher degradation rates than others of 95.14%,98.21%,94.31%respectively.Basedonmorphologicalobservationsandmolecularbiology identifications,strains 6,7,8 were identified as Raoultella ornithinolytica.2.Localization of enzyme degrading limonin.Comparing the limonin degradation ability of extracellular protein,intracellular protein,and bacterial fluid in three strains 6,7,8,the results indicated that enzymes having limonin degradation ability of three strains were located as extracellular enzymes,there was almost no limonin degradation in intracellular protein of three strains,and extracellular enzymes of strain 6had the strongest limonin degradation ability with the limonin degradation rate of 45.57%.3.Study on degradation characteristics of strain with high limonin-degrading efficiency.The growth cycle of limonin-degrading strain was determined after 12 h,the growth trend of strain 6 with limonin as the sole carbon source was consistent with the change of the degradation rate of limonin in culture medium,which was supposed to be a consttutive enzyme.The effects of the age of strain,the amount of limonin added,the inoculation amount and the culture time on the degradation of limonin were investigated.On the basis of single factor,the optimum degradation conditions of strain limonin-degrading were determined by response surface analysis:the age of strain was 10 h,the dosage of limonin was 4 mg/L,and the culture time was 12 hunder this condition,the degradation rate of strain 6 was 91.28%.4.Study on preliminary purification and characterizations of limonin-degrading enzyme.The extracellular limonin degrading enzyme of strain 6 was purified by Sephadex G-100 gel filtration chromatography and DEAE-cellulose column chromatography with ammonium sulfate precipitation.The molecular weight of protease determined by SDS-PAGE molecular weight is about 27 kDa,and the electrophoretic purity was achieved.After purification,the specific activity of protease is increased by 9.54 times.The optimum temperature of limonin-degrading enzyme was 35?,and it had good thermal stability at 35?,and the activity of enzyme had been decreased slowly when it was higher than35?.Metal ion Fe³⁺could increase the activity of limonin-degrading enzyme,while metal ions Ca²⁺and Zn²⁺could inhibit the activity of enzyme obviously.