| Mannatide is a novel immune enhancer developed by Chinese scientists.Former studies have shown that Mannatide could inhibit the growth of tumor,enhance the immune system and stimulate hematopoietic function of patients.As the result, the clinical demand of Mannatide is growing fastly. Currently,Mannatide is extracted from the fermentation products of α-hemolytic Streptococcus strains 33, however, the production of Mannatide is rarely low.Therefore, it is necessary to seek other methods to produce Mannatide.China enjoys the largest plantation and production of konjac around the world. At present, the major application of amorphophallus is glucomannan. In contrast, most of Konjac powder has been abandoned. Researches have shown that konjac powder is abundant in mannan, protein and glycoprotein, which made konjac powder a good source for obtaining mannatide.Konjac powder was selected as the raw materials to prepare konjac mannatide in this research. Firstly, the konjac glycoprotein was extracted and konjac glycopeptide was prepared by enzymatic hydrolysis. Then, the glycopeptide impurities were removed by the way of Sevage. Lastly, konjac mannatide was purified by chromatography of DEAE-52, Sephadex G-100 and Con A-Sepharose4 B. Qualitative analysis, the content and the physical and chemical properties of the konjac mannatide was studied. Experiment results can be concluded as follows:1. The extraction process of konjac glycoprotein by alkaline dissolution and acid precipitation methods was studied. The protein extraction yield was taken as an index. And through the single factor and orthogonal experiment, the extraction process of konjac glycoprotein was optimized. The result showed that the isoelectric point of konjac glycoprotein was 3.8 p H value, the optimum condition was: solid-to-solvent ratio 1:35, temperature 50 ℃, time 50 min, p Hvalue 10.0. At the optimum condition, the protein extraction yield could reach34.72%.2. The preparation process of konjac glycopeptide using enzymatic hydrolysis was studied. The TCA-SN and the hydrolysis degree were taken as indexes for the preparation of konjac glycopeptide. Alkaline protease was choosed to hydrolyze konjac glycoprotein. And through the single factor and orthogonal experiments, the preparation process of konjac glycopeptide was optimized. The results showed that the optimum conditions of enzymolysis were:p H value 8.5, temperature 50 ℃, enzyme concentration 3500 U/g, time 150 min.Under the optimum conditions, the TCA-SN was 11.98%, the degree of hydrolysis was 9.19%.3. The removal of glycopeptide impurities using the way of Sevage was studied. Peptide removal rate was taken as an index. According to the single factor and orthogonal experiment, the removal of glycopeptide impurities was optimized. The result showed that the optimum conditions were: sevage reagent ratio 1:2, ratio of trichloromethane and n-butyl alcohol 4:1, time 40 min. Under the optimum conditions, the peptide removal rate was 24.27%.4. The extraction of konjac mannatide using chromatography of DEAE-52,Sephadex G-100 and Con A-Sepharose4 B was studied.The result of DEAE-52 chromatography was collecting eluent between number 27 and 44. The result of Sephadex G-100 chromatography was collecting eluent between number 6 and13,number 21 and 35. Konjac mannatide was lastly extracted by using Con A-Sepharose4 B chromatography.5. Qualitative analysis of konjac mannatide was studied by high performance liquid chromatography(HPLC). The result indicated that the aminoacids of konjac mannatide was the same composition as standard product of mannatide. The monosaccharide composition of konjac mannatide was also mannose and glucose. The content of konjac mannatide was 71.5%.6. Konjac mannatide had strong moisture absorption.But its foamability and emulsibility was poor.And the molecular weight was between 27 KD and 35 KD. |
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