| Truffle and foie gras, caviar are called the world’s top three foods with rich nutrition and excellent healthcare function. It’s the precious edible and medicinal fungi with high commercial and edible value. Polysaccharide as one of the main effective components in truffle, currently about the truffle polysaccharide research more concentrated on the optimization of extraction process and the antioxidant activities, and the less about the analysis of its structure and research of immunological activity. So far, for the research on carboxymethylated derivative of truffle polysaccharide, the academic field has not been reported. In this study, the Tuber indicum as raw material, truffle polysaccharide of traditional hot water extraction process was optimized, crude polysaccharide was separated and purified, the structure was characterized, truffle polysaccharide carboxymethyl modified and activity were studied. The main research results are as follows:1. The study of extraction process of polysaccharide from truffle was through the water extraction and alcohol sink method, and the optimization of extraction technique was carried out by the orthogonal experiment. The results showed that liquid-solid ratio was the most important factor for the yield of polysaccharide, and the extraction times, the next was the temperature and the least important factor was extraction time. The optimal extraction technology conditions were that the liquid-solid ratio was 1:40, the temperature was 90 °C, the time was 2 h, and the extraction time was 3 times. The yield was 13.6%.2. Truffle polysaccharide was obtained through the water extraction in the optimium conditions, with deproteinized and ethanol subsided. Truffle polysaccharide was purified by DEAE Sepharose Fast Flow ion-exchange column chromatography to gain two components, which named TP1 and TP2. Then the effect of TP1 and TP2 on the proliferation, phagocytic ability and secretion of cytoline IL-6 to mouse macrophages(RAW264.7 cells) had been detected. The results showed that TP1 and TP2 all had no proliferation to RAW264.7 cells and they all can significantly enhance the the phagocytic ability and secretion of IL-6 in RAW264.7 cells. However, the effect of TP1 was better than TP2, so TP1 was chosen for subsequent experiments. Through the Sephadex G-100 glucan gel column chromatography, we got a single component from TP1 called TP1-1. Then the content of polysaccharide in TP1-1 had been detected, that was 91.51%.3. Through the gel permeation chromatography, the number average molecular weight mass of TP1-1 was measured, that was 757910 Da. HPLC analysis of TP1-1 proved that the monosaccharide composition of TP1-1 contained D-glucose and D-mannose with the percentage was 88.94% and 1.19%, respectively. There was no proteins and nucleic acids in TP1-1 from the the results of ultraviolet spectrum. The infrared spectra indicated that TP1-1 contained the stretching vibration of O-H at 3357 cm-1, the stretching vibration of 2922 cm-1, the stretching vibration of a group of carboxylic at 1368 cm-1, which were characteristic absorption peak of polysaccharides. The absorption peak on 1081 cm-1 and 1024 cm-1 was the stretching vibration of pyranoid ring and the absorption peak on 850 cm-1 made its glucosidic bond was α-type. The absorption peak on 761 cm-1 and 709 cm-1 indicated that it has mannose. Periodate oxidant showed that TP1-1 had 1→6 or 1→ type, 1→4 or 1→2, 1→3 glycosidic bond with the percentage of 12.4%, 13.0% and 74.6%, respectively. NMR analysis proved that TP1-1 linked by α-configuration and that was a pyranoid ring. C2, C3, C4 and C5 were not replaced and there were replaced C6 and not replaced C6, which indicated that may be there is other α-branch structure. Congo red test showed that TP1-1 existed standed helical and I2-KI experiments proved TP1-1 had chain and branch.4. TP1-1 reacted with chloroacetate acid to obtain the carboxymethyl polysaccharide of Truffle(CMTP1-1) with the degree of substitution was 0.875. The effect of TP1-1 and CMTP1-1 on the proliferation, phagocytic ability and secretion of cytoline IL-6 to mouse macrophages(RAW264.7 cells), and the antioxidant activity was studied. The results showed that TP1-1 and CMTP1-1 all had no proliferation to RAW264.7 cells and the all could significantly elevate the phagocytic ability and secretion of cytoline IL-6 of RAW264.7 cells. Meanwhile, the activity of CMTP1-1 was significantly better than TP1-1 on the phagocytic ability and secretion of cytoline IL-6 of RAW264.7 cells. To detecte the antioxidant activity of TP1-1 and CMTP1-1, three different kinds of systems(reducing power, hydroxyl radical OH?, DPPH?) were used. The results showed that CMTP1-1 had stronger antioxidant activity than TP1-1 in the same concentration in three systems. So the phagocytic ability and antioxidant activity were elevated after TP1-1 had been modified by carboxymethylation. |
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