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 α-,β-,and γ-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 α-,β-,and γ-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 μL）and 0.1 M 1-kestose（100 μL）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 μg/h.（4）Inclusion ratio of garlic oil encapsulated by γ-cyclodextrin was 13.8%,which was 1.7% higher than α-cyclodextrin and 1.2% higher than β-cyclodextrin,indicating that γ-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 α-cyclodextrin was 2.6%-3.4% higher than methyl allyl mono-polysulfides;inclusion ratio of diallyl mono-polysulfides encapsulated by β-cyclodextrin was 1.8%-5.6% higher than methyl allyl mono-polysulfides;inclusion ratio of diallyl mono-polysulfides encapsulated by γ-cyclodextrin was 5.2%-7.9% higher than methyl allyl mono-polysulfides.