甲壳单糖、寡糖、低聚糖制备方法的优化与比较研究

该文通过大量实验,确定了制备单糖、寡糖及低聚糖的最佳生产工艺,并对几种制备方法做了比较研究.该文采用氧化降解和微波降解等不同技术对甲壳低聚糖的制备工艺进行了研究.H<,2>O<,2>作为氧化剂,在酸性条件下,采用4因素3水平进行正交实验,得到了制备分子量2000以下的寡糖的最佳条件.微波降解制备低聚糖目前尚未见报道,经过该实验证明此方法可降低能耗,减小污染,节省时间和原料,是一个具有产业化前景和市场潜力的好方法.通过以不同的壳聚糖为原料,探讨了在微波场作用下纯溶剂及盐效应对分子量变化的影响,最终产物的分子量均在1×10<'5>～10×10<'5>之间.根据IR光谱,壳聚糖降解前后主要峰的位置都无变化,只是随壳聚糖相对分子质量的降低各峰峰强有所变化,证明了壳聚糖氧化降解及微波降解制备寡糖、低聚糖是以开裂壳聚糖的β-1.4糖苷键来进行.该项研究对更好的开发利用甲壳质资源,促进中国海洋生物制品的发展具有十分重要的意义.

甲壳质、壳聚糖生产废水中虾青素、蛋白质的回收及壳聚糖在水处理中的应用

近年来，甲壳质生产发展迅速，但是在生产过程中所产生的废水引发了一系列的环境污染问题，对于该废水的处理，目前尚未有行之有效的方法，大多数生产厂家仍处于无处理排放状态。本文在处理甲壳质、壳聚糖生产废水的同时回收得到了具有多种生物学功能的虾青素以及可以作为饲料添加剂的粗蛋白，这对于我国甲壳质、壳聚糖产业的发展以及保护我国近海环境具有重要的意义；另一方面，壳聚糖因其具有优良的吸附性能、螯合性能、可再生性及生物降解性，在水处理工艺中已经显示出广阔的应用前景，本研究中将壳聚糖应用于甲壳质、壳聚糖生产废水以及海藻工业水处理工艺中，这样既保护了环境又达到了资源综合利用的目的，并且节约了能源和资源。 首先研究了从甲壳质、壳聚糖生产废水中回收提取虾青素以及所得的虾青素对DPPH自由基的清除作用。分别采用单元有机溶剂、二元有机溶剂作为萃取剂从废水中回收虾青素，并确定了有机溶剂萃取的最佳条件，实验结果表明，二元有机溶剂萃取的效果优于单元有机溶剂萃取的效果；将得到的虾青素经HPLC分析，游离虾青素的含量达30.02%；最后，对所得的虾青素进行了有机自由基DPPH的清除作用的测定，结果表明，所得的虾青素具有很强的清除DPPH自由基的能力，IC50可以达到0.84mg/ml。 将得到的虾青素进行了深加工，合成了虾青素-β-环糊精的包合物。结果表明，当虾青素与β-环糊精形成包合物时，虾青素与β-环糊精以1：4的比例进行包合，包合物的水溶性稍好于虾青素（虾青素不溶于水）；在水相中，包合物很容易形成超分子结构；包合物中虾青素对温度和光的稳定性明显增强。 其次，研究了甲壳质、壳聚糖生产废水中蛋白质的回收技术，通过实验确定了蛋白质回收的最佳条件；并对所得的蛋白质沉淀进行了氨基酸分析，结果表明，该沉淀中含有丰富的氨基酸，其总量可以达到20.56%，这些结果表明该沉淀可以开发为饲料用蛋白质添加剂。 在回收虾青素和蛋白质的同时，对甲壳质、壳聚糖生产废水处理新工艺的开发，并且确定了最佳实验条件。经过处理后的废水由青岛市环境保护监测站监测结果表明，处理后的废水达到了国家二级排放标准。 利用壳聚糖对海藻工业水进行了处理，结果表明，壳聚糖絮凝海带工业水中“糖胶”的效果明显优于工业生产中通常使用的方法，在此基础上确定了壳聚糖作为絮凝剂对海带工业水进行处理的最佳实验条件。在本研究中还测定了I-浓度的变化，从I-的浓度变化来看，应用本方法处理海带工业水不影响碘的提取。

海洋藻类功能性蛋白质的纯化、鉴定及其应用的研究

使用膨化柱和离子交换或羟基磷灰石柱层析相结合的方法，分别从多管藻、坛紫菜及钝顶螺旋藻中分离纯化了R-藻红蛋白溶液和C-藻蓝蛋白。光谱检测及电泳分析结果证明完全符合经典的藻胆蛋白纯度标准。彭化床最突出的优点是克服了常规分离方法堵塞色谱柱的难题，纯化速度快、产量高、不需要常规色谱方法所要求的填料的平衡及粗提液的预处理，仅需一步操作就可以得到满足一般食品添加剂纯度要求的藻胆蛋白，极大地简化了后续的纯化程序，减少了分离纯化的步骤和时间，而其产率及纯度均高于常规的藻胆蛋白分离方法。这同时也降低了藻胆蛋白分离纯化的成本。 本文通过戊二醛或环氧氯丙烷交联的方法，合成了四种壳聚糖-氨基酸共聚小球。并选取吸附性好的戊二醛交联孔球和戊二醛交联微球系统测定了其对R-藻红蛋白和C-藻蓝蛋白的吸附和缓释性能。 纯化了藓羽藻中与其细胞器团聚密切相关的一种凝集素并进行了部分性质的鉴定。N端前15个氨基酸序列及LC-ESI-MS质谱分析结果证明此凝集素属于一种新的蛋白质族。实验证明，凝血活性与细胞器团聚活性并不完全依赖于此凝集素分子相同的结构域。 通过异双功能试剂SPDP处理藓羽藻凝集素使之衍生化，DTT处理R-PE在其分子内引入外源巯基，然后将活化的R-藻红蛋白与凝集素进行交联反应。交联产物经凝胶过滤纯化并检测，但电泳及荧光显微镜检测结果并不能证明交联探针的成功制备。

含硫、磷壳聚糖新衍生物的制备、结构与抑菌活性关系研究

由于化学农药和抗生素兽药残留问题严重影响着环境和人类健康，生物农药和兽药的开发迫在眉睫。壳聚糖无毒、无污染、可生物降解，已经成为这一方向研究热点。但是，目前对壳聚糖抑菌活性的研究大都集中于分子量和脱乙酰度对其活性的影响，而对壳聚糖衍生物的研究相对较少。本文制备了壳聚糖磺酰胺类、酰基异硫氰酸酯类和α-氨基烷基膦酸酯类共计40余种新型壳聚糖含硫、磷衍生物，研究了它们对6种真菌（棉花枯萎病菌、番茄早疫病菌、葡萄炭疽病菌、姜叶斑点病菌、黄瓜灰霉病菌、芦笋茎枯病）和4种细菌（大肠杆菌、绿脓杆菌、金黄色葡萄球菌、八叠球菌）的抑菌活性，结果发现，所有衍生物的抑菌活性均强于壳聚糖原料，其中4种新型衍生物对于真菌具有较强抑制活性，其抑制率大于或接近阳性对照武夷菌素；10余种衍生物对于细菌有较强的杀菌、抑菌活性，如4-HBSA(l)CMCS、4-HBSA(l)CSS等对四种细菌具有广谱性，效果接近甚至强于阳性对照诺氟沙星。 对影响因素、构效关系和机理进行了深入探讨。衍生物的分子量、浓度、溶解性、α-位C原子空间位阻、吸电子空间诱导效应、烷基链的增长、等因素变化均会对抑菌活性产生重大影响。

水溶性壳聚糖衍生物的取代基团及氨基正电性对抑菌活性的影响

壳聚糖是一种天然的聚阳离子多糖，可以从甲壳类动物、菌类、昆虫等自然资源中提取得到。壳聚糖具有良好的生物相容性，天然无毒，可生物降解，从而在生物工程、制药、化妆品、纺织品、农业等领域引起了广泛关注。 随着对壳聚糖以及壳聚糖衍生物研究的深入，壳聚糖的抑菌活性由于农业杀菌剂的环境问题和抗药性问题的出现而得到重视。但是，对于壳聚糖抑菌活性的研究大都集中于壳聚糖的分子量和脱乙酰度对其活性的影响，而相应的壳聚糖衍生物很少。本文合成了羧甲基壳聚糖希夫碱，N-取代羧甲基壳聚糖，脲取代羧甲基壳聚糖，壳聚糖季铵盐以及羧甲基壳聚糖季铵盐，并对它们的抑菌活性进行了研究，同时探讨了导入基团与抑菌活性间的构效关系。 羧甲基壳聚糖希夫碱对苹果腐烂病菌、番茄早疫病菌和棉花枯萎病菌的抑制活性研究结果表明2-（2-羟基-5-硝基苯亚胺基）羧甲基壳聚糖和2-（2-羟基-5-氯苯亚胺基）羧甲基壳聚糖的抑菌活性高于壳聚糖、羧甲基壳聚糖和2-（2-羟基苯亚胺基）羧甲基壳聚糖，原因可能是2-羟基-5-硝基苯亚胺基和2-羟基-5-氯苯亚胺基两个活性基团的引入。对番茄早疫病菌和苹果轮纹病菌的抑制效果表明，N-取代羧甲基壳聚糖的抑菌活性低于壳聚糖和羧甲基壳聚糖，脲取代羧甲基壳聚糖的抑菌活性高于羧甲基壳聚糖。 测定了壳聚糖季铵盐对灰葡萄孢以及炭疽病菌的抑制活性，结果表明壳聚糖季铵盐的抑制活性高于壳聚糖。因为壳聚糖分子的正电荷可以和菌体细胞壁的负离子相互结合，从而导致菌体死亡，而壳聚糖季铵盐分子中明显的正电性可以促进这种结合能力，从而更进一步的增强抑菌活性。由于相同的原因，抑菌活性随着正电荷的增强而增强，与此同时，高分子量壳聚糖季铵盐由于其较大的体积而具有比低分子量壳聚糖季铵盐更高的抑菌活性。 为了进一步验证氨基的正电性与抑菌活性的关系，合成了羧甲基壳聚糖季铵盐并对这类衍生物对灰葡萄孢和炭疽病菌的抑制活性进行了测定，结果表明，羧甲基壳聚糖季铵盐的抑菌活性高于羧甲基壳聚糖，且部分羧甲基壳聚糖季铵盐的抑菌活性高于壳聚糖。500μg/mL时，N-(2-羟基-5-硝基苯亚甲基）-N,N-二甲基羧甲基壳聚糖和N-(2-羟基-5-氯苯亚甲基）-N,N-二甲基羧甲基壳聚糖对苹果轮纹病菌的抑制率都达到100%。壳聚糖季铵盐中正电荷在抑菌活性中的作用再一次被证实。

The preparation and antioxidant activity of glucosamine sulfate

Glucosamine sulfate was prepared from glucosamine hydrochloride that was produced by acidic hydrolysis of chitin by ion-exchange method. Optical rotation and elemental analysis characterized the degree of its purity. In addition, the antioxidant potency of chitosan derivative-glucosamine sulfate was investigated in various established in vitro systems, such as superoxide (O (2) (-) )/hydroxyl (center dot OH) radicals scavenging, reducing power, iron ion chelating. The following results are obtained: first, glucosamine sulfate had pronounced scavenging effect on superoxide radical. For example the O (2) (-) scavenging activity of glucosamine sulfate was 92.11% at 0.8 mg/mL. Second, the center dot OH scavenging activity of glucosamine sulfate was also strong, and was about 50% at 3.2 mg/mL. Third, the reducing power of glucosamine sulfate was more pronounced. The reducing power of glucosamine sulfate was 0.643 at 0.75 mg/mL. However, its potency for ferrous ion chelating was weak. Furthermore, except for ferrous ion chelating potency, the scavenging rate of radical and reducing power of glucosamine sulfate were concentration-dependent and increased with their increasing concentrations, but its ferrous ion chelating potency decreased with the increasing concentration. The multiple antioxidant activities of glucosamine sulfate were evidents of reducing power and superoxide/hydroxyl radicals scavenging ability. These in vitro results suggest the possibility that glucosamine sulfate could be used effectively as an ingredient in health or functional food, to alleviate oxidative stress.

Preparation of high-molecular weight and high-sulfate content chitosans and their potential antioxidant activity in vitro

The influence of molecular weight and substitution degree of sulfated polysaccharides on their biological activity is considered in majority of works involving the anticoagulant or antiviral properties of these substances. Therefore, the present paper describes the effect of preparation conditions of sulfated chitosans on their molecular weight and sulfur content, such as different reaction time, acid solvent and temperature. Foregoing literature expounded the action of dichloroacetic acid (DCAA) as acid solvent in homogeneous reaction. However, DCAA is expensive and noxious, therefore, in the present paper cheap and non-noxious formic acid (88%) was in place of DCAA. Furthermore, during reaction formic acid was not dehydrated. Under formic acid we obtained the satisfying results that was higher yield and equivalent sulfur contents compared to DCAA. IR and C-13 NMR spectrums proved the structure of the resultant obtained under formic acid or DCAA to be same. Now, it has not been reported for formic acid as acid solvent in homogeneous reaction of chitosan sulfatation. In this present paper, we also determined antioxidant activity of high-molecular weight and high-sulfate-content chitosans (HCTS). The results showed that HCTS could scavenge superoxide and hydroxyl radical. Its IC50 is 0.012 and 3.269 mg/mL, respectively. It had obviously reducing power and slight chelating activity. The data obtained in in vitro models clearly establish the antioxidant potency of HCTS. It is a potential antioxidant in vitro. (C) 2005 Elsevier Ltd. All rights reserved.

Preparation of low-molecular-weight and high-sulfate-content chitosans under microwave radiation and their potential antioxidant activity in vitro

In the present paper microwave radiation has been used to introduce N-sulfo and O-sulfo groups into chitosan with a thigh degree of substitution and low-molecular weight. The sulfation of chitosan was performed in microwave ovens. It was found that microwave heating is a convenient way to obtain a wide range of products of different degrees of substitution and molecular weight only by changing reaction time or/and radiation power. Moreover, microwave radiation accelerated the degradation of sulfated chitosan, and the molecular weight of sulfated chitosan was considerably lower than that obtained by traditional heating. There are no differences in the chemical structure of sulfated chitosan obtained by microwave and by conventional technology. FTIR and C-13 NMR spectral analyses demonstrated that a significantly shorter time is required to obtain a satisfactory degree of substitution and molecular weight by microwave radiation than by conventional technology. In this present paper, we also determined antioxidant activity of low-molecular-weight and high-sulfate-content chitosans (LCTS). The results showed LCTS could scavenge superoxide and hydroxyl radical. Its IC50 is 0.025 and 1.32mg/mL, respectively. It is a potential antioxidant in vitro. (C) 2004 Published by Elsevier Ltd.

Recovery of protein from discharged wastewater during the production of chitin

Studies were carried out to optimize the conditions for the recovery of protein. The results showed that pH of 6.00 for wastewater, the dosage of 1% chitosan solution in 1% acetic acid aqueous solution of 2.0 ml for 50 ml wastewater and 1% FeCl3 aqueous solution of 2 ml for 50 ml wastewater, the flocculation time of 4.0 h were the optimal conditions for the recovery of protein. The obtained protein sediment contained abundant amino acids, especially isoleucine, methione and lysine that are absent in other protein resource. (c) 2007 Elsevier Ltd. All rights reserved.

A new cellular model for in vitro biocompatibility evaluation of microcapsule materials

Fibrosis caused by the host response to long-term transplanted microcapsules and the limitation of traditional L929 cell model for biocompatibility testing inspire the development of an assay of biocompatibility based on macrophage behavior. In this paper, the human monocytic cell line THP-1 was utilized for biocompatibility evaluation of microcapsule materials. The cell viability and secretion of nitric oxide (NO) and cytokines served as index of biocompatibility were assayed. It was found that the evaluated microcapsule materials had no effect on the stimulation of NO and cytokines secretion, which meant that these materials were biocompatible. Furthermore, it suggests the THP-1 cell a convenient in vitro experimental model that might be useful for long-term predictions of material biocompatibility.

Insight into permeability of protein through microcapsule membranes

The objective of this paper is to analyze the characteristics of protein permeability in alainate-polylysine-alginate (APA) and alginate-chitosan-alginate (ACA) microcapsules by mathematical models based on the balance of chemical potential. The comparison between calculated results and experimental data shows that the models can describe the process of protein diffusion from microcapsule and protein release into microcapsule successfully. The influences of membrane composition on the permeability of ACA microcapsule have been investigated and analyzed. The effect of resistance on the mass transfer is further analyzed theoretically with the aid of mathematical modeling. (c) 2005 Elsevier B.V. All rights reserved.