【作者】 王雅立；

【导师】 庞杰；

【作者基本信息】 福建农林大学 ， 食品科学， 2007， 硕士

【摘要】 随着科学技术的发展，人们在不断寻求具有新特性、新功能的智能高分子材料，尤其是具有响应特性的高分子水凝胶材料。魔芋葡甘聚糖（Konjac Glucomanan，简称KGM）是在魔芋植物中存在的一种水溶性极好的、高分子量的非离子型杂多糖，具有特殊的生物活性，广泛应用于食品、药物、化妆品、材料等领域，具有很大的经济效益和社会效益。魔芋葡甘聚糖具有分子量大、水合能力强、不带电荷等特性，这些特性决定了它具有优良的凝胶性能，即在一定的条件下（如加一定量胶凝剂调pH值等），魔芋溶胶液便会失去流动性，变为弹性固体或半固体状态；KGM凝胶性能还表现在可以与其它食品胶（卡拉胶、黄原胶等）复配成胶。但是在应用过程中发现KGM凝胶性能不稳定，结构是性能变化的原因，因此要研究KGM凝胶性能就要研究其结构。由于多糖本身结构的复杂性，对其结构的研究存在困难，而且多糖三维结构的研究都是通过间接手段进行的，不但底物要求复杂而且给出的结构不够直观。为了弥补这些不足，本文在实验和谱学研究的基础上利用分子模拟技术直观的给出分子的动态微观结构，对有关现实原型的各种观察实验事实做出科学解释，并研究分子与周围环境体系的作用过程。研究包括以下几个方面：本文以魔芋葡甘聚糖为试材，首先探讨了结构化学研究方法在凝胶稳定性研究中的应用，对魔芋葡甘聚糖的提纯进行了研究；接着探讨了结构生物学研究方法在凝胶稳定性研究中的应用，采用凝胶排阻色谱法对酶解后的魔芋葡甘聚糖的分子量测定进行了探讨，考察酶解时间和高聚物浓度等操作条件对所测分子量和分子量分布的影响；最后探讨理论研究方法在凝胶稳定性研究中的应用，运用分子模拟同仪器分析相结合的方法探讨了浓度、分子量、pH值、温度、氢键对不同分子量的魔芋葡甘聚糖凝胶稳定性的影响。主要研究内容与结果如下：1．结构化学研究方法在凝胶稳定性研究中的应用结构不同性能也不同，为了要研究KGM的结构，有必要对其进行提纯。魔芋精粉中SO₂的含量是其质量的重要指标。但由于加工工艺落后，含硫量高，严重影响了魔芋食品的稳定性，所以以SO₂脱除率为提纯指标。魔芋精粉3g在70℃、pH值为5的柠檬酸溶液中溶胀1h，可使魔芋精粉中的含硫量降至最低，SO₂脱除率可达90％，并且由于纯化过程中部分去除了原粉中含有蛋白质、淀粉等物质，纯化后的KGM较纯化前感官性状改善明显，纯化后的魔芋精粉洁白、无异味，且其水溶胶的稳定性比提纯前有所提高，抗菌能力得到加强，存放时间增长一倍。2．结构生物学研究方法在凝胶稳定性研究中的应用多糖的分子量对其结构、性能及应用方面具有很大的影响，分子量大小不同，其生理活性也不一样；将多糖降解到适宜的分子量，可得到高活性的多糖片段或寡糖。由于不同分子量大小的KGM具有不同的性质，因此有必要在它的分子量大小方面作选择。酶降解由于具有专一性，降解条件和过程易于控制，无副反应等优点，近年来常应用于多糖的降解。魔芋精粉3g中加入酶活力180U的纤维素酶，在50℃条件下，pH值为6.0，100 mL水中反应。由于KGM溶胶粘度会随着分子量的下降而显著下降。以粘度为参考指标，选择酶解时间为10min、20min、30min，制备不同分子量的魔芋葡甘聚糖。比较发现随着反应时间的增长其凝胶的稳定性也下降，容易析出水分，导致其抗菌性也下降。将不同分子量的魔芋葡甘聚糖的多糖样品分别溶于双蒸水中配成浓度为0.5％的溶液，离心，经0.22μm微孔滤膜过滤，利用凝胶排阻色谱法，用GPC软件计算各样品的分子量。浓度0.5％的KGM所得低聚糖的分子量主要分布在数均相对分子量在204922～265322之间，重均相对分子量在538737～697832之间。3．理论研究方法在凝胶稳定性研究中的应用运用分子模拟同谱学分析相结合的手段探讨了浓度、分子量、pH值、温度、氢键对不同分子量的魔芋葡甘聚糖凝胶稳定性的影响。分析表明：（1）采用两因素正交实验分别探讨了KGM的浓度、KGM的分子量二因子对KGM凝胶的稳定性的影响，通过实验发现：当KGM的浓度增大，分子量增大时，凝胶的粘度、凝胶强度和冻融稳定性都增大。通过分子动力学模拟发现，氢键作用是影响其稳定性的分子作用力，氢键作用的增强提高了其凝胶强度。（2）采用L₁₆（4⁵）正交实验分别探讨了KGM／κ-卡拉胶的浓度、KGM／κ-卡拉胶的比例、KGM的分子量对KGM凝胶稳定性的影响，通过实验发现：当增大KGM／κ-卡拉胶的浓度，KGM／κ-卡拉胶的配比为4：6时，分子量增大时凝胶的稳定性提高。通过分子动力学的模拟的稳定构象可以清楚的看出葡甘聚糖与卡拉胶共混时形成的分子间氢键数明显增多，氢键作用是影响其稳定性的分子作用力，氢键作用的增强提高了其凝胶强度。（3）采用L₁₆（4⁵）正交实验分别探讨了KGM的浓度、KGM的分子量、尿素对KGM凝胶的稳定性的影响，通过实验发现：当KGM的浓度增大，KGM的分子量增大，尿素的浓度减小，凝胶的稳定性提高。通过分子动力学模拟发现，尿素干扰了葡甘聚糖分子间作用即氢键作用，不但使葡甘聚糖与水性成的氢键数减少同时形成氢键的作用位点也发生了变化。（4）采用L₁₆（4⁵）正交实验分别探讨了KGM浓度、KGM分子量、pH值、温度四因子对KGM凝胶稳定性的影响，通过实验发现：当KGM浓度增大，KGM分子量增大，pH值为9，温度在90℃时，凝胶的稳定性最大。通过分子动力学模拟发现，KGM在碱性条件下由于化学作用乙酰基的脱除，超分子作用力分子间氢键作用的加强提高了凝胶强度。说明乙酰基是影响KGM凝胶稳定性的重要基团，氢键作用是影响其稳定性的分子作用力，乙酰基的脱落和氢键作用的增强提高了其凝胶强度。更多还原

【Abstract】 With the development of scientific technology, people are searching for intelligent materials of new properties and high functions, especially the macromolecule which has the respondent ability. Konjac Glucomannan （KGM） is the main content of the local resource Konjac as a kind of natural polymer. It is a water-soluble noninonic polysaccharide which now is successfully widely used in fields such as food, Pharmaceuticals, cosmetics, and materials. Properties of high molecular weight, good hydration capacity and without charge help with KGM excellent gel-forming ability. Factors like concentration, molecular weight, pH, temperature and H-bonds could strongly affect its gel strength. Under certain conditions, the temperature arising would enhance the gel strength. KGM can be deacetylated to form irreversible gel, under such conditions, it turns to be a solid or semi-solid gel. KGM also can be blended with other polymers such as carrageean, xanthan, and soybean protein to form gel. The synergistic interaction between them can improve properties of blend gel. Due to its structure, KGM gel is instable, so its structure should be studied. The study on polysaccharide is difficult because of its complex structure. Polysaccharide 3D structure was used to be studied by indirect methods, they need complex substrate but can not give visual structure image. In this text, molecular dynamics method was used to give visual molecule structure image, explain the experiment results and study the interaction between KGM and its reaction environment. Studies were as follows: firstly chemical study methods were used, the efficient way to purify the KGM was studied; secondly the biological study method was used, the purified KGM was enzymed, as well as effects of enzyme time and enzyme concentration on KGM molecular weight were studied, the molecular weight and distribution of enzymed KGM was determined by Size Exclusion Chromatography （SEC）;And finally the theory study method was used, the effects of concentration, molecular weight, pH, reaction temperature and H-bonds on KGM gel stability were also studied. The details are specified as follows:1. Chemical method on KGM gel stability studyDifferent structures lead to different properties. So structure study is a key to study KGM gel stability. In order to well study its structure, purification was used. The SO₂ percentage of KGM is an important index. High percentage of SO₂ could affect its stability, so it was used as a reference index in purification. In KGM purifying, best results were obtained in following conditions: KGM 3g, reaction temperature 70℃, reaction time 1h, and pH=5 Na₂HPO₄ solution. By this way, the SO₂ removing rate could reach 90%. It is purely white and has no strange smell for its protein and starch are partly removed. Purified KGM is shown to be superior to the raw KGM in freeze-thaw stability and fungistasis.2. Biological method on KGM gel stability studyThe molecular weight means much to the structure and properties of polysaccharide. Different molecular weight polysaccharide has different physiological properties, so it needs to study effects of different molecular weight on KGM properties. In order to prepare different molecular weight KGM, they were made in following conditions: KGM 3g, reaction temperature 50℃, pH=6, 100mL water, reaction time were 10min, 20min and 30min. Using viscosity as a reference index. The results show that KGM gel stability and fungistasis decreasing as the molecular weight decreasing. The aboved KGM samples were dissolved to prepare 0.5% solution, centrifuged and ultra filtered. Their molecular weights were analyzed by SEC and GPC software. The results show that the Number Average Molecular Weight （Mn） of 0.5% KGM is between 204922～265322 while its Weight Average Molecular Weight （Mw） is between 538737～697832.3. Theory method on KGM gel stability studyEffects of factors, such as concentration, molecular weight, pH, temperature and hydrogen-bonds on KGM gel stability were studied by using molecular simulation and modern instruments. The results are specified as follows:（1） Effects of KGM concentration and its molecular weight on KGM gel stability were studied by two factors orthogonal experiments. The results show that the gel viscosity, gel strength, and freeze-thaw stability are all enhanced as the molecular weight increase. By molecular dynamics method, it shows that the increase of intra-molecular hydrogen bonds help with gel strength.（2） Effects of KGM/κ-carrageen concentration, KGM/κ-carrageen mixed ratio and KGM molecular weight on KGM gel stability were studied by three factors orthogonal experiments. The results showed that by increasing the KGM/κ-carrageen concentration and the KGM molecular weight while the KGM/κ-carrageen mixed ratio is 4:6, the gel stability could be enhanced. By molecular dynamics method, it shows that, compared with single gel, hydrogen-bonded function of blend gel is enhanced, the two polymers are compatible.（3） Effects of KGM concentration, KGM molecular weight and urea on KGM gel stability were studied by three factors orthogonal experiments. The results show that by increasing the KGM concentration and KGM molecular weight while decreasing the urea concentration, the gel stability could be enhanced. By molecular dynamics method, it shows that urea not only affect the formation of intra-molecular hydrogen bonds, but also change the position of hydrogen bonds and it destroys the KGM hydrogen bond network. That is because removing acetyl groups help with enhancing the intra-molecular hydrogen bond function.（4） Effects of KGM concentration, KGM molecular weight, pH, and reaction temperature on KGM gel stability were studied by four factors orthogonal experiments. The results show that by increasing the KGM concentration and KGM molecular weight when the reaction temperature is 90℃and pH=9, the gel stability is the best. By molecular dynamics method, it shows that, under alkaline conditions, the increase of intra-molecular hydrogen bond interaction improves gel strength.更多还原