| The main objective of this research was to screen the stains of producing metabolism enzymes that can remove three kind of bitterness components including naringin, limonin and nomilin in pomelo, the purified enzymes which can remove bitterness for productingpomelo juice without bitterness and further processed product would be obtained, it is necessary conditions for screening the stains to establish the effective detection method and extract bitterness, Determination of bitterness is researched at first and a HPLC method for determination of naringin, limonin and nomilin was established. And then processing parameters for extracting bitterness components of pomelo as inducers for screening stains that can produce enzymes which can remove bitterness would be optimized subsequently. The study showed that:(1)The HPLC condition determinating the main natural bitterness components including naringin, limonin and nomilin in Guaixi pomilo by using Symmetry C18 column was methyl alcohol: acetonitrile: PBS(containing 0.01mol/L postassium dihydrogan phospate pH=3.5)= 18:3:14 as mobile phase with a flow rate of 1.0ml/min at the detection wavelength of 210nm and the temperature of the column was 35℃.(2)The Calibration curve was linear in the range of 1.80～115ug/ml for naringin(R~2=0.999, n=6), 4.30～137.5ug/ml for limonin(R~2=0.9968, n=6)and 5.82～186.25ug/ml for nomilin(R~2=0.9993, n=6) respectively. The addition recoveries were 97.80% (RSD=1.86), 95.32%(RSD=2.04) and 98.54%(RSD=3.17) respectively. And this method is accurate, sensitive and convenient.(3)Different concentration of the bitterness components was determinated in different parts of Guaixi pomilo, the distribution of the concentration of naringin was Pericarp＞White cortex＞Oil cyst layer＞Pulp; the distribution of the concentration of limonin was Pericarp＞Oil cyst layer＞Pulp＞White cortex, the distribution of the concentration of nomilin was Pericarp＞Pulp, the concentration ofnomilin in white cortex and oil cyst layer are under The detection limit. The highest concentration of the three bitterness components were determinated in pericarp, the concentration were 976.13 (μg/g raw material), 860.46 (μg/g raw material), 351.83(μg/graw material).(4) The optimized extraction process for the main bitterness components including naringin, limonin and nomilin in ripe Guanxi pomelo was: the powder of pericarp was defatted by ligarinc in soxhlet's extractor, afier, then, mixed with 20-fold (volume/mass) acetone at 40℃and pHT.0 and extracted for 15h, the concentration of total bitterness was up to 3467.71μg/g(raw material), and the concentration of naringin 2425.97μg/g(raw material), limonin 716.63μg/g(raw material), and nomilin 325.111μg/g(raw material).(5)The result of the screening of the stains of producing metabolism enzymes that can remove bitterness components was: the cultivation of the strains in selective culture medium that use the three bitterness components concentratation as carbon source, obtain 324 natural strains with enrichment, isolating, and elementary screening. Then the survivable strains were fermentated in tube and analyzed of the content of the bitterness by HPLC, so obtain 106 strains that be able to hydrolyze the three bitterness and 128 strains that only be able to hydrolyze naringin and 21 strains that be able to hydrolyze limonin and nomilin. A strain that have higher activities to hydrolyze bitterness components was obtained, the laboratory number was 4-D5, its activies of naringinase was 121μg/ml. min and limoninase was 5.6μg/ml·min. It is identificated as AsporgiUus, belongs to Subdivision: Deuteromycotina, Class: Hyphomycetes Order: Moniliales, Family: Moniliaceae, Genus: Aspcrgillus.
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