Isolation And Purification Of Garlic 1-FFT And Characterization Of Garlic Oil-Cyclodextrin Complex

To investigate the purification and characterization of fructan synthetase in garlic,fructan: fructan 1-fructosyltransferase?1-FFT?was extracted from garlic bulbs.Ammonium sulfate precipitation and successive column chromatography using affinity chromatography,gel filtration chromatography and ion exchange chromatography were applied for protein purification,then the characterization of 1-FFT was investigated.Furthermore,3 types of garlic oil-cyclodextrin complex?GO-CD?were prepared using garlic oil and ?-,?-,and ?-cyclodextrin.The concentrations of volatile organic compounds?VOC?were determined via selected ion flow tube-mass spectrometry?SIFT/MS?to evalutate the encapsulation ability of ?-,?-,and ?-CD to VOC in garlic oil.The results show as follows:?1?Method for measurement of enzyme activity was established: 1-FFT was measured in an incubation mixture of enzyme?100 ?L?and 0.1 M 1-kestose?100 ?L?at 25°C.Reaction was terminated by addition of ethyl alcohol absolute?1 mL?and the production of 1,1-kestotetraose was measured via HPLC-ELSD.Besides,conditions of HPLC-ELSD gradient elution have been modified,which shortened the analysis time of single sample from 45 min to 15 min,saving 30 min.?2?1-FFT was partially purified using different types of column chromatography.1-FFT could not combine with Con A Sepharose under acidic,alkaline and neutrallty conditions,which indicated that 1-FFT possessed low extent of glycosylation,or was not glycoprotein.?3?1-FFT mainly existed in the 40%-50% ammonium sulfate fraction.The optimum pH of 1-FFT was 6.0,1-FFT was stable in a range of p H 5.5-6.5 for 1 h.The optimum temperature was 25°C,1-FFT was stable at 15-25°C for 1 h.1-FFT was activated by low concentration of Mn²⁺,Mg²⁺ and Fe3+,inhibited by Cu²⁺,Zn²⁺ and Ag+.Enzymatic activity increased with the ascending concentration of substrate,and Km was 0.6977 M,Vmax was 116.23 ?g/h.?4?Inclusion ratio of garlic oil encapsulated by ?-cyclodextrin was 13.8%,which was 1.7% higher than ?-cyclodextrin and 1.2% higher than ?-cyclodextrin,indicating that ?-cyclodextrin was optimum wall material among 3 types of cyclodextrin.Inclusion ratios of monopolysulfides encapsulated by different types of cyclodextrin increased with the ascending of the molecular mass of guest molecule.?5?Inclusion ratios of diallyl mono-polysulfides encapsulated by 3 types of cyclodextrin were higher than methyl allyl mono-polysulfides: inclusion ratio of diallyl mono-polysulfides encapsulated by ?-cyclodextrin was 2.6%-3.4% higher than methyl allyl mono-polysulfides;inclusion ratio of diallyl mono-polysulfides encapsulated by ?-cyclodextrin was 1.8%-5.6% higher than methyl allyl mono-polysulfides;inclusion ratio of diallyl mono-polysulfides encapsulated by ?-cyclodextrin was 5.2%-7.9% higher than methyl allyl mono-polysulfides.