壳寡糖的制备、纯化及应用研究进展

壳寡糖(Chitooligosaccharide,COS)是几丁质或壳聚糖降解后获得的N-乙酰-D-葡萄糖胺和D-葡萄糖胺的同聚或异聚物,具有抗菌、抗氧化、抗肿瘤、免疫调节等多种生物活性,已被广泛应用于医药、食品、农业等领域。目前,酶法、物理法和化学法均已被报道用于制备COS。然而,单一法制备COS有一定的局限性,很难高效、绿色地获得具有特定聚合度和乙酰度的目标产物。因此,COS的制备已经从单一法转向至协同催化体系的探索。此外,借助超滤法、活性炭吸附法、色谱法等分离纯化技术也可以提高COS的纯度。本文对近年来COS的制备和纯化方法以及应用研究进展进行了综述,以期为高品质COS的生产以及应用领域的拓展提供理论基础。

烟酰化壳寡糖季铵盐的制备及抑菌活性研究

海洋生物体内蕴含着丰富的天然活性物质,许多都具有抑菌活性,据此研制新型海洋生物源抑菌药物,是海洋生物产业中的一个新领域,也是新型抑菌药物研发的重要途径。壳聚糖是来自海洋中虾蟹等甲壳类动物外壳的甲壳素的脱乙酰产物,壳寡糖是壳聚糖的降解产物,具有比壳聚糖更好的水溶性和生物活性,在食品和生物医药等领域具有更广阔的应用前景。为了提高壳寡糖的抑菌活性,本研究以壳寡糖和烟酸为原料、N, N-羰基二咪唑为催化剂合成了壳寡糖烟酸接枝衍生物,并与不同链长的溴代烷烃亲核反应得到其阳离子化衍生物。壳寡糖及其衍生物的化学结构通过红外光谱(FTIR)和核磁共振波谱(NMR)进行分析表征,壳寡糖及其衍生物对2种海洋致病细菌和3种植物致病真菌的体外抑菌能力分别采用肉汤稀释法和菌丝生长速率法进行研究和评估。结果表明,与壳寡糖相比,合成的终产物对致病菌的抑制能力得到了极大增强,其中含有己基的烟酰化壳寡糖季铵盐表现出最强的抑制海洋致病细菌活性(MIC=0.125mg/mL、MBC=0.25mg/mL),并且该化合物表现出较强的抗真菌活性,在1.0 mg/mL时,对灰葡萄孢菌和围小丛壳的抑制率分别为65.7%和51.5%。这些结果表明,含直链烷烃的烟酰化壳寡糖季铵盐具有较强的抑菌活性,在食品、生物医药等行业具有较大的应用潜力。

壳寡糖对羽衣甘蓝芽苗硫代葡萄糖苷及抗氧化活性的影响

为探究壳寡糖(COS)对羽衣甘蓝芽苗生长的影响,为芽苗生产上壳寡糖的合理施用提供参考,以京羽一号为试验材料,在种子发芽后施用不同质量浓度COS(25,50,75,100 mg/L)溶液,观察壳寡糖对羽衣甘蓝芽苗生长指标、硫代葡萄糖苷(简称硫苷)含量及抗氧化能力的影响。结果表明,施用75 mg/L COS处理的羽衣甘蓝芽苗总酚、总黄酮、总硫苷质量以及黑芥子酶活性和DPPH自由基清除率呈现上升趋势,且与对照差异显著,较对照分别增加25.99%,17.99%,29.68%,141.88%和2.51%;100 mg/L COS处理的羽衣甘蓝芽苗的产量及总酚、总黄酮、钠和硼含量及黑芥子酶活性也得到了相应提升,且与对照差异显著,较对照分别增加了15.21%,21.29%,22.47%,15.97%,32.63%,97.71%。经相关性分析,可以得出芽苗的产量与类胡萝卜素、总酚、总黄酮含量及DPPH自由基清除率均呈显著正相关关系。综上可得,经壳寡糖处理后可提高羽衣甘蓝芽苗的产量,提升芽苗的营养品质和抗氧化能力,增加芽苗的食用价值。

壳聚糖酶的结构特征及其应用

随着酶学研究和微生物学的发展,壳聚糖酶已从多种菌株筛选、分离和鉴定出来,通过基因工程和蛋白质工程的方法进行系统的酶学性质表征,可获得一系列高活性的壳聚糖酶。文章系统总结和归纳壳聚糖酶的研究进展状况,介绍壳聚糖酶的微生物来源、分类、酶学性质、三维结构和催化机制及其应用研究,指出其在科学研究上的瓶颈并提出展望。

饲料中添加不同浓度的壳寡糖对黄河鲤幼鱼生长及抗氧化功能的影响

在水温(25.5±3.0)℃下,将体质量20 g左右的黄河鲤(Cyprinus carpio)饲养在循环水养殖可控实验系统0.4 m3水族中,每箱30尾,分别饲喂含0(基础饲料,对照组)、0.02%、0.04%、0.06%质量比壳寡糖(分子量小于3500 Da纯度为10%)的饲料。饲养56 d时,测定生长指标、血清血糖(GLU)、总蛋白质(TP)、白蛋白(ALB)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)和丙二醛(MDA)含量及溶菌酶、超氧化物歧化物酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,研究壳寡糖对黄河鲤生长及抗氧化功能的影响。结果表明,0.04%和0.06%组鲤的相对生长率分别比对照组提高4.43%和6.00%,但是差异不显著(P>0.05),而肝胰指数分别显著降低17.5%和12.5%(P<0.05)。各壳寡糖组鲤的血清溶菌酶活性比对照组均有提高,且随着壳寡糖添加量的增加上升。0.04%壳寡糖显著提高了血清SOD活性。不同浓度壳寡糖不同程度提高了血清和肝脏GSH-Px活性,但是差异不显著(P>0.05)。0.06%壳寡糖组鲤血清丙二醛含量显著降低(P<0.05)。总之,在高量植物性蛋白饲料中添加不同浓度的壳寡糖,尽管没有显著影响鲤的生长,但在一定程度上提高了鲤的免疫和抗氧化水平,即提高了鲤的机体健康水平。综合多种指标以及经济成本因素,建议饲料中壳寡糖的添加量为0.02%~0.04%。

纤维素纳米晶/壳寡糖复合物稳定的姜黄素皮克林乳液的制备及其研究

目的:本研究以纤维素纳米晶(CNC)和壳寡糖(COS)通过静电吸附形成的复合物作为稳定剂,并探索该复合物制备姜黄素皮克林乳剂的可能性。方法:采用高速剪切结合超声的制备方法制备CNC/COS-Cur,以平均粒径为主要指标筛选出最优处方工艺。结果:CNC/COS可以成功制备姜黄素皮克林乳剂。结果表明最优工艺为:离子强度为100mM/L;CNC用量为1.2wt%;CNC:COS=1:1.2。结论:纤维素纳米晶和壳寡糖复合物能用于制备皮克林乳剂,可以增大姜黄素的溶解度。

壳寡糖的生物学功能及其在生物医学领域中应用的研究进展

壳寡糖(COS)作为天然多糖壳聚糖(CS)的降解产物,既保留了CS的良好生物相容性、无毒和可生物降解等特点,同时由于糖链缩短使相对分子质量降低,其水溶性和生物活性均得到提高且更容易被生物体吸收利用,近年来受到越来越多的关注。目前国内外学者对CS生物学功能的研究及其在生物医学领域中应用的报道较多,但对COS功能及其应用的研究报道较少。现结合国内外最新研究成果,对COS的主要生物学功能(抗炎、抗肿瘤、抗菌和促组织再生等)及其可能的作用机制进行总结和分析,并阐述其在生物医学领域中应用的研究进展,以期为COS的深入研究及其在生物医学领域中更广泛的应用提供理论依据和参考。

壳寡糖的制备及应用的研究进展

甲壳质是自然界中分布广泛、产量很高的一种多糖,但是其内外氢键之间存在相互作用力,进而组成了有序的大分子结构,导致其在常用的溶剂中的溶解性受到抑制。它的脱乙酰基产物为壳聚糖,壳聚糖的水溶性仍然没有得到很好的改善。而壳寡糖是壳聚糖糖苷键经改性断裂后降解得到的寡糖产品,在改善难溶的物理特性的同时,也使其拥有了多种更高的生物活性,如抗氧化、抑菌、抗炎、抗肿瘤、促进作物生长等功能。文章综述了制备壳寡糖的几种方法并且比较了它们的优缺点,讨论了它们在食品等领域中的具体应用,最后尝试对不同相对分子质量、具有不同特性的壳寡糖在各个领域的应用进行了总结。

甲壳质衍生物共混改性水性聚氨酯

采用聚酯218、聚醚310、IPDI、DMBA为主要原料合成水性聚氨酯(WPU),将一系列质量浓度梯度的壳聚糖(CS)或壳寡糖(COS)与WPU乳液共混。结果表明:由于CS相对分子质量过大,即使调整CS溶液、WPU乳液的质量浓度及pH,两者均无法稳定共混。但采用聚合度较低的COS与WPU共混可获得稳定乳液,经探究可知COS溶液的临界质量浓度为0.4 mg/mL,与WPU乳液共混的临界质量比为2∶8。测试结果表明共混改性后混合乳液的粒径、抗菌性、耐水性有所提升,对乳液稳定性及胶膜的热稳定性几乎无影响。

壳寡糖对大黄鱼肌肉口感及营养价值的影响

为探究壳寡糖对舟山大黄鱼肌肉口感及营养成分的影响,选取舟山市大黄鱼养殖示范点网箱大黄鱼鱼苗为研究对象,分别投喂壳寡糖添加量为0‰、0.3‰、0.6‰的饲料160 d。结果表明,在饲料中添加适量壳寡糖可提高大黄鱼肌肉的硬度、咀嚼性和弹性,改善了大黄鱼肌肉口感,提高了大黄鱼肌肉中粗蛋白质含量,降低了粗脂肪含量;在一定程度上改善氨基酸、脂肪酸含量结构,提高了大黄鱼肌肉营养价值及品质,且壳寡糖在大黄鱼饲料中的推荐添加量约为0.3‰。

“海之星”有机营养型氨基酸水溶肥对郁金香观赏期延长的研究

郁金香(Tulipa L.)花色丰富,耐低温,常用于早春的花境及花海打造,当前主要通过改善土壤及温度条件来调控花期。针对郁金香开花所需要的有效积温、有效光照、最佳激素配比及其与其他影响因子的交互效应,尤其是对影响郁金香花芽分化及花器官发育的基础研究很少。基于此,通过试验规律地喷施1000倍“海之星”有机营养型氨基酸水溶肥能够有效延长郁金香的观赏期7~9 d,同时能不同程度地提前郁金香观赏期,提升郁金香花期观赏状态,为提前和延长其他花卉观赏期提供参考。

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

AIM: To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats.METHODS: In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS: Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides (100 mg/L) had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance.CONCLUSION: Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.

Anti-proliferation and radiosensitization effects of chitooligosaccharides on human lung cancer line Hep G2

Objective: To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides(COS) on human lung cancer cell line Hep G2. Methods: CCK-8 assay was employed to obtain the inhibition ratio of COS on Hep G2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY+COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY+COS group after 24 h treatment. Results: COS inhibited the proliferation of Hep G2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY+COS group. The cell viabilities of RAY+COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy(P<0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G2/M phase and apoptosis rate, and lower percentage at S phase in RAY+COS group versus the other two groups(P<0.01). Conclusions: COS can inhibit the proliferation of Hep G2 cells, and enhance the radiosensitization of Hep G2 cells, induce apoptosis and G2/M phase arrest.

Comparative study of the chitooligosaccharides effect on the proliferation inhibition and radiosensitization of three types of human gastric cancer cell line

Objective:To observe the chitooligosaccharides(COS) effect on the proliferation inhibition and radiosensitivity of three types of human gastric cancer cell line.Mothods:CCK-8 assay was employed to obtain the inhibition ratio of COS on BGC823 cells,MKN45 cells and SGC7901 cells at 48 h after treatment and the proliferation-inhibition curve was drawn with the inhibition ratio of COS on three types of cells.The clonogenic assay was used to detect the cell viability of 0,1,2,4,6 and 8 Gy(6 dose grades) in RAY group and RAY+COS group after X-ray,and the cell survival curve was used to analyze the sensitization enhancement ratio of COS.Flow cytometry was employed to detect cell cycle and apoptosis rate in control group,RAY group and RAY+COS group after 48 h treatment.Results:COS inhibited the proliferation of three types of cells.The inhibition rate was positively correlated with the concentration of COS,and the susceptibility of MKN45 cells,SGC7901 cells and BGC823 cells to COS decreased in turn.The cell viability decreased gradually with the increasing radiation dose in RAY group and RAY+COS group(P<0.01).The cell viabilities of RAY+COS group were lower than those of RAY group at all the dose grades under X-ray exposure(P<0.01),and the sensitization enhancement ratios of COS on BGC823 cells,MKN45 cells and SGC7901 cells were 1.06,1.28 and 1.15 respectively.In controlled trials,apoptosis rate and percentage in the G_2/M phase of three types of cells in RAY+COS group were higher than those in control group and RAY group,and percentage in the S phase and the G_0/G_1 phase in RAY+COS group were lower than those in the other two groups(P<0.01).Conclusions:COS can inhibit the proliferation of three types of human gastric cancer cells and enhance the radiosensitivity by inducing apoptosis and G_2/M phase arrest.

Studies on the Antimicrobial Activity of Chitooligosaccharides from Housefly Larvae, Musca domestica vicina Macquart(Diptera: Muscidae)

The inhibition effect of chitooligosaccharides from housefly larvae on pathogens ofcrops seeds, fruits or vegetables was studied and the main factors influencing theantimicrobial activity of chitooligosaccharides were also investigated. It was foundthat chitooligosaccharides from housefly larvae had wide spectrum fungistasis. It canstrongly inhibit the development of 31 kinds of plant pathogenic fungi such as corticiumrolfsii Saccardo. The results indicated that the degree of deacelylation (DD) or theaverage molecular weight(MW)of chitooligosaccharides were related to the antimicrobialactivity. The antimicrobial activity increased with the rising of DD or the declining ofMW. Pot culture results showed that chitooligosaccharide could enhance the rate ofgermination and emergence of the seeds of maize, wheat and cotton. Chitooligosaccharidehad certain effect on corn southern leaf blight caused by Helminthosporium maydis.

Adsorption characteristics of chitooligosaccharides onto activated charcoal in aqueous solutions

To investigate the adsorption characteristics of chitooligosaccharides in solution onto activated charcoal,we studied the optimal adsorption conditions and the adsorption mechanisms of the chitooligosaccharides onto activated charcoal,which will greatly promote the application of activated charcoal in the chitooligosaccharides separation and purification.We studied the effects of particle size of activated charcoal,pH of solution,contact time,temperature,and initial concentration of chitooligosaccharides on the adsorption behavior in batch mode experiments.Activated charcoal in fine particle size showed a high uptake of chitooligosaccharides.Weak alkaline solution(pH 8–9)was the most favorable to the adsorption.The adsorption equilibrium after 60 min was established,which followed a pseudo-second-order kinetic model.The adsorption capacity(Q_(max))reached 0.195 g/g(chitooligosaccharides/activated charcoal)at 298 K.The adsorption was temperature-insensitive,and the adsorption isotherms could be best described by the Langmuir equation.Chitooligosaccharides adsorbed on activated charcoal could be desorbed in 50%ethanol solution in combination with an acidic condition(pH 2),reaching desorption efficiency of 96.0%.These findings are of great significance for the production and purification of amino oligosaccharides including chitooligosaccharides using activated charcoal.

Improving the protective effects of a FGF for peripheral nerve injury repair using sulfated chitooligosaccharides

Injury to the peripheral nerves can result in temporary or life-long neuronal dysfunction and subsequent economic or social disability. Acidic fibroblast growth factor(a FGF) promotes the growth and survival of neurons and is a possible treatment for peripheral nerve injury. Yet,the actual therapeutic utility of a FGF is limited by its short half-life and instability in vivo. In the present study,we prepared sulfated chitooligosaccharides(SCOS),which have heparinlike properties,to improve the bioactivity of a FGF. We investigated the protective effects of SCOS with or without a FGF on RSC96 cells exposed to Na2 S2 O4 hypoxia/reoxygenation injury. Cell viability was measured by MTT assay and cytotoxicity induced by Na2 S2 O4 was assessed by lactate dehydrogenase(LDH) release into the culture medium. Pretreatment with a FGF and SCOS dramatically decreased LDH release after injury compared to pretreatment with a FGF or SCOS alone. We subsequently prepared an a FGF/SCOS thermo-sensitive hydrogel with poloxamer and examined its effects in vivo. Paw withdrawal thresholds and thermal withdrawal latencies were measured in rats with sciatic nerve injury. Local injection of the a FGF/SCOS hydrogels(a FGF: 40,80 μg/kg) increased the efficiency of sciatic nerve repair compared to a FGF(80 μg/kg) hydrogel alone. Especially a FGF/SCOS thermo-sensitive hydrogel decreased paw withdrawal thresholds from 117.75 ± 8.38(g,4 d) to 65.74 ± 3.39(g,10 d),but a FGF alone group were 140.58 ± 27.54(g,4 d) to 89.12 ± 5.60(g,10 d)(a FGF dose was 80 μg/kg,P < 0.05,n = 8). The thermal withdrawal latencies decreased from 11.61 ± 2.26(s,4 d) to 2.37 ±0.67(s,10 d). However,a FGF alone group were from 17.69 ± 1.47(s,4 d) to4.65 ± 1.73(s,10 d)(P < 0.05,n = 8). Furthermore,the a FGF/SCOS hydrogels also exhibited good biocompatibility in mice. In summary,SCOS improved the protective effects of a FGF in RSC96 cells injured with Na2S2O4 and increased the efficiency of nerve repair and recovery of function in rats with sciatic nerve injury. These findings pave an avenue for the development of novel prophylactic and therapeutic strategies for peripheral nerve injury.

Preparation,characterization,and antifungal evaluation of a new type of aminourea chitooligosaccharide derivatives

Phytopathogenic fungi cause heavy negative impact on the agricultural economy,but most existing fungicides are toxic and pose a threat to both human health and environments.A green and efficient fungicide is urgently needed.Chitooligosaccharides(COSs),the degradation products of natural polysaccharide chitosan,are nontoxic and biodegradable antifungal substances.In this study,a novel type of aminourea chitooligosaccharide derivatives(AUCOS)was synthesized by successively grafting a hydrazine group and an amine-carbonyl group onto a chitooligosaccharide backbone to enhance the antifungal capability of COSs.The structures of the target compounds were identified by FTIR,1H NMR,and 13C NMR,and the degree of substitution of each product was calculated from the results of the elemental analysis.The antifungal activities of the prepared chitooligosaccharide derivatives against Fusarium solani,Verticillium albo-atrum and Phytophthora capsici were tested in vitro.The AUCOSs had better inhibitory efficiencies against the three plant pathogen fungi than that of chitooligosaccharide,of which aminourea chitooligosaccharide 2(AUCOS2)was the most promising antifungal compound,whose highest inhibition rates were 60.12%,82.95%,and 85.23%against F.solani,V.albo-atrum and P.capsici,respectively.The synthesized derivatives have good application prospects in crop protection and deserve further research.

Conjugation of Chitooligosaccharide-5-fluorouracil with Bovine Serum Albumin

The interaction between chitooligosaccharide-5-fluorouracil （COS-5FU） and bovine serum albumin （BSA） was studied by fluorescence spectroscopy. It was found that an energy transfer between COS-5FU and BSA had been occurred. The binding constants were calculated, k298K=1.175×10^4 L·mol^-1. Based on the mechanism of energy transfer of dipole-dipole interaction between the donor and acceptor, the distance between BSA and COS-SFU was determined.

Control Efficacy of Streptomyces sp. SY-L12 and Chitooligosaccharide on Kiwifruit Bacterial Canker, Pseudomonas syringae pv. actinidiae

[ Objective] The paper was to develop a priority scheme for prevention and control of kiwifruit bacterial canker with biological agents. [ Method ] The inhibitory effect of Streptomyces sp. SY-L12 on the growth of Pseudomonas syringae pv. actinidiae were assessed using liquid co-culture and inhibition zone meth- ods. The control efficiency of Streptomyces sp. fermentation broth against kiwifruit bacterial canker was studied via potted trial. [ Result ] As Streptomyces sp. SY- L12 was incubated for 24 h, the growth inhibition rate of its fermentation broth on P. syr/ngae pv. actinidiae was 94.0%. The control efficiency of fermentation broth against P. syringae pv. actinidiae was the best of 85.4% in potted trial when Streptomyces sp. SY-L12 grew at 28~C, 150 rpm for 8 -9 d, better than that of control agent copper hydroxide. The control efficiency was improved as the agent was mixed with 1 mg/L chitooligosaccharide. [ Conclusion] Streptomyces sp. SY-L12 could effectively inhibit the growth of P. syr/ngae pv. actinidiae, and can be developed as biological agent against kiwifruit bacterial canker.