Radical scavenging activity of hetero-chitooligosaccharides

Nine hetero-chitooligosaccharides (hetero-COSs) consisting of relatively high molecular weights (90-, 75-, and 50-HMWCOS), medium molecular weights (90-,75-, and 50-MMWCOS), and low molecular weights (90-, 75-, and 50-LMWCOS) were prepared from partially deacetylated hetero-chitosans (90%, 75%, and 50% deacetylated chitosan), and their antioxidant activity based on their scavenging potency on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was investigated. The results showed that the 90-MMWCOS, prepared from 90% deacetylated chitosan, showed the highest scavenging activity on the DPPH radical. Their scavenging pattern was dose- and time-dependent. Electron spin resonance (ESR) spectrum of DPPH treated with 90-MMWCOS decreased in a time-dependent manner and the reducing power of hetero-COSs increased in a dose-dependent manner. These results indicated that hetero-COSs have an antioxidant activity which is dependent on their degree of deacetylation and molecular weight.     

摄食壳聚糖对小鼠体内铅水平的影响

采用壳聚糖含量为1.05% 的饲料喂食小鼠90d,喂食的四种壳聚糖分别是高分子量壳聚糖(HCS)(重平均分子量Mw7.60 × 105,脱乙酰度(DDA) 85.5%、中分子量壳聚糖(MCS)(Mw3.27 × 104,DDA85.2%)、壳寡糖(COS)(Mw0.99 × 103,DDA 85.7%)和水溶性壳聚糖(WSC)(Mw3.91 × 104, DDA52.6%)。喂养期间,小鼠无非正常症状和死亡发生。实验结果表明,喂食HCS 对小鼠的肝、脾、心脏和肾脏中的铅含量几乎无影响,COS 和WSC使小鼠的肝、脾和心脏中的铅含量有所降低。但是,MCS 却明显增加了肝脏中铅含量。     

Preparation of chitosan oligomers and their antioxidant activity

Chitosan oligomers with different molecular weights were prepared by oxidative degradation method involving hydrogen peroxide (H₂O₂) and the combined degradation method using hydrogen peroxide and microwave radiation. Viscosity determination and end group analysis were applied to measure molecular weights of chitosan oligomers. Effects of concentration of H₂O₂ and degradation time on molecular weights of chitosan oligomers were studied. Both methods were effective to prepare chitosan oligomers from the initial chitosan (8.5×10⁵ Da). The degradation process of chitosan will be accelerated with the aid of microwave and degradation time may be reduced. The antioxidant activity of chitosan oligomers was evaluated as radical scavengers against superoxide anion and hydroxyl radical by application of flow injection chemiluminescence technology. Chitosan oligomers A, B, C and D (2300, 3270, 6120, and 15,250 Da) had different antioxidant activity. Among the four chitosan oligomers, oligomer D (15,250 Da) had the lowest scavenging ability against superoxide anion and hydroxyl radicals. For superoxide anion scavenging, the 50% inhibition concentrations (IC₅₀s) of other three oligomers A, B, and C were 5.54, 8.11, and 12.15 mg/mL, respectively. And for hydroxyl radical scavenging the values were 0.4, 0.76, and 1.54 mg/mL, respectively. At the concentration range examined, the maximal inhibiting efficacy of A, B, C, and D were 89, 75, 74, and 41% for superoxide anion, and 71, 65, 51, and 7% for hydroxyl radical. These results indicated that chitosan oligomers with lower molecular weight had better antioxidant activity.     

基于美拉德反应的低聚壳聚糖衍生物的生物活性研究

以低聚壳聚糖作为氨基供体分别与提供羰基的葡萄糖和麦芽糖进行美拉德反应(氨基与羰基的物质量比均为1:1),醇沉法提取低聚壳聚糖美拉德反应衍生物CG和CM。对两种衍生物进行红外表征和分子量测定,并研究其抗氧化性能和抑菌性能。结果显示:两种衍生物均保留着低聚壳聚糖的特征吸收峰;其对O2.-、.OH及DPPH的清除能力以及还原能力和对大肠杆菌、铜绿假单胞菌和副溶血性弧菌的抑菌活性均得到显著提高,同时CG的抗氧化性和抑菌活性明显优于CM。即与单糖进行美拉德反应制得的壳聚糖衍生物具有更好的生物活性。     

低聚壳聚糖与葡萄糖和麦芽糖的美拉德反应及其衍生物的抗氧化性能

低聚壳聚糖(COS)作为氨基供体分别与提供羰基的葡萄糖和麦芽糖进行美拉德反应(氨基与羰基的物质量比均为3:1),考察了反应过程中吸光度和荧光值的变化。醇沉法提取低聚壳聚糖美拉德反应衍生物CG和CM。对两种衍生物进行红外表征和相对分子质量测定,并研究其对超氧阳离子O2.-、DPPH自由基的清除能力以及还原能力。结果显示:抗氧化能力强弱次序为CG>CM>COS,即美拉德反应后低聚壳聚糖衍生物抗氧化能力得到显著提高,且CG的抗氧化活性优于CM,表明与单糖进行美拉德反应制得的壳聚糖衍生物具有更好的抗氧化性。     

Sensory Characteristics and Antioxidant Activities of Maillard Reaction Products from Soy Protein Hydrolysates with Different Molecular Weight Distribution

Soy protein was hydrolyzed using two enzymes to obtain soy protein hydrolysate (SPH), the SPH was fractionated with ultrafiltration membranes to obtain peptide fractions below 1,000 Da (SP1) and 1,000–5,000 Da (SP2), and for the meantime, SPH was further completely hydrolyzed to get compound amino acids (CAA). Maillard reaction products (MRPs) were prepared from aqueous xylose–SPH/SP1/SP2/CAA model systems by heating at 120°C for 2.0 h. Compared with the original hydrolysates and other MRPs, the MRPs from SP2 exhibited a distinctly enhanced effect on flavor, including the caramel-like, soy sauce-like odors, umami and mouthful tastes and a greatly reduced bitterness in consomme′ soup. Antioxidant activities of SPH, SP1, SP2, CAA, and their MRPs were investigated through reducing power, DPPH radical-scavenging activity, and Fe²⁺ chelating activity. Before Maillard reaction, the antioxidant activities of peptide fractions with different molecular weights were quite different, and SP2 showed the highest activity; however, CAA exhibited very poor antioxidant activity. The antioxidant activities of SPH, SP1, SP2, and CAA were greatly enhanced by Maillard reaction, and the MRPs prepared from xylose–CAA model system exhibited a higher antioxidant activity than those from other model systems. Pyrazines, pyrroles, furans, and thiazoles were significantly correlated with reducing power and Fe²⁺ chelating activity by principal component analysis.     

Influence of marine oligosaccharides on the response of various biological systems to UV irradiation

Low molecular weight alginate-derived oligosaccharide (ADO) (373–571 Da) and chito-oligosaccharide (COS) (855–1671 Da) were purified from alginate and chitosan, and known as marine oligosaccharides with polyanionic and polycationic properties, respectively. We compared the effects of ADO and COS on cell regulation using several biological models (Candida albicans, Escherichia coli and Bacillus subtilis spore), cellular uptake determination, erythrocytes haemolysis inhibition and antioxidant capacity assay to investigate stress response under UV radiation. Our results further confirmed the anti-UVR potential of ADO and COS and their potential for commercial UVR protector application in the area of functional foods as food ingredients.     

降解壳聚糖对肉品保鲜效果的试验研究

目的:制备降解(低分子质量)壳聚糖并考察对肉品的保鲜效果。方法:采用H2O2均相氧化降解壳聚糖,以乌氏黏度法测其黏均分子质量;用牛津杯法测定降解壳聚糖对大肠杆菌、金葡菌、霉菌的抑菌圈直径;喷洒降解壳聚糖溶液于肉品表面,用表面涂抹法测定肉品在25℃放置过程中菌落总数的变化。结果:影响降解壳聚糖分子质量大小的因素顺序为:时间>温度>H2O2浓度>HAc浓度;在反应时间6h、温度75℃、H2O2体积分数6%和HAc体积分数4%时,可得黏均相对分子质量为2300的可溶性壳聚糖;降解壳聚糖对大肠杆菌、金葡菌的抑菌率高于未降解壳聚糖,而对霉菌的抑菌性变化不大;经质量分数6%、黏均分子质量2300壳聚糖溶液喷洒肉品表面后,鲜猪肉4d、熟猪肉6d可保持良好色泽、弹性、气味,且菌落总数符合GB2726-2005要求。结论:低分子质量壳聚糖可作为肉品防腐保鲜剂。     

过氧化氢制备壳寡糖的工艺

为了简便高效的制备出壳寡糖,采用过氧化氢(质量分数1.0%)来降解稀醋酸溶液(质量分数1.0%)中含量为3.5%的壳聚糖,60℃下摇床反应4.5h后得到壳寡糖水溶液,检测溶液中还原性端基数并估算降解物平均聚合度来表征降解程度。降解液浓缩到25%,调pH7.0,用体积分数95%乙醇分级沉淀分离壳寡糖可以除掉部分单糖,总的回收率达93.0%。通过电喷雾电离质谱(ESI-MS)分析,3倍醇沉出的产物为2-4糖为主的壳寡糖产品。实验说明过氧化氢氧化降解壳聚糖可制取聚合度为2-4的壳寡糖。     

Enzymatic preparation of chitooligosaccharides by commercial lipase

The effect of a commercial lipase on chitosan degradation was investigated. When four chitosans with various degrees of deacetylation were used as substrates, the lipase showed higher optimal pH toward chitosan with higher DD (degree of deacetylation). The optimal temperature of the lipase was 55 °C for all chitosans. The enzyme exhibited higher activity to chitosans which were 82.8% and 73.2% deacetylated. Kinetics experiments show that chitosans with DD of 82.8% and 73.2% which resulted in lower K_m values had stronger affinity for the lipase. The chitosan hydrolysis carried out at 37 °C produced larger quantity of COS (chitooligosaccharides) than that at 55 °C when the reaction time was longer than 6 h, and COS yield of 24 h hydrolysis at 37 °C was 93.8%. Products analysis results demonstrate that the enzyme produced glucosamine and chitooligosaccharides with DP (degree of polymerization) of 2–6 and above, and it acted on chitosan in both exo- and endo-hydrolytic manner.     

壳寡糖-金属配合物对太平洋牡蛎体内中Cd脱除效果的研究

目的:以太平洋牡蛎为实验对象,研究壳寡糖(chitosan oligosaccharide,COS)相对分子质量及脱乙酰度对COS-金属配合物脱Cd效果的影响,并对其应用条件进行优化。方法:将染Cd牡蛎养殖在含不同COS-金属配合物的天然海水中,定期取样,采用原子吸收法测定Cd含量的变化。结果:在COS-Ca、COS-Mg、COS-Zn和COS-Ree4种配合物中,COS-Mg的脱Cd效果最好。COS的相对分子质量和脱乙酰度对COS-Mg的脱Cd效果均有显著影响,其中当COS相对分子质量为2000、脱乙酰度为90%时COS-Mg对太平洋牡蛎的Cd脱除效果最佳,可达到28.20%。对COS-Mg配合物的应用条件进行了优化,当配合物质量浓度为100mg/L海水、脱除时间48h时,Cd脱除率最高(34.68%)。结论:COS-Mg配合物可有效降低牡蛎体内的Cd残留,是一种非常有潜力的贝类重金属残留脱除剂。     

Synergistic effect of chitooligosaccharides and lysozyme for meat preservation

The objective of this study was to enhance the antibacterial spectrum of lysozyme with the use of chitooligosaccharides (COS) produced by radiation treatment of chitosan. Exposure of chitosan solution to γ radiation led to formation of oligosaccharides of different molecular weights. COS with molecular weight of 8.3 kDa were found to exhibit highest antioxidant potential in free radical scavenging assay but antibacterial activity decreased with decrease in molecular weight. Combination of COS and lysozyme was more effective against Gram-negative bacteria than when used alone. This clearly indicated the synergistic effect of the two antibacterial agents added together. When tested in meat model system, the combination treatment resulted in complete elimination of Escherichia coli, Pseudomonas fluorescens and Bacillus cereus and reduced the load of Staphylococcus aureus cells in packed inoculum and storage studies. The shelf life of minced meat containing COS-lysozyme mixture was extended up to 15 days at chilled temperatures.     

THE DEGRADATION OF CHlTOSAN WITH THE AID OF LIPASE FROM RHIZOPUS JAPONICUS FOR THE PRODUCTION OF SOLUBLE CHlTOSAN

Lipase from Rhizopus japonicus degraded chitosan resulting in soluble chitosan hydrolysates with molecular weight of about 30–50 kDa as shown by size exclusion chromatography. Optimal temperature for the hydrolysis of chitosan was 40C. The chitosan degradation products were fractionated stepwise according to their molecular weights by ultrafiltration with the filtration range of over 0.1 μm, 0. l μm to 30 kDa, 30 kDa to 10 kDa, 10 to 3 kDa, and 3 to 0.2 kDa. These fractions exhibited molecular weights of 50, 41, 41, 35, and 30 kDa, respectively. The molecular weights did not coincide with the pore size of filter membranes. Chitosan hydrolysate exhibited almost the same structural composition in IR spectra as chitosan flakes, except the peak of 1550 nm^?1 that appeared to be the COO residue shifted from sodium acetate buffer to amine residue of chitosan. All fractions showed high solubility at neutral pH. The chitosan hydrolysates exhibiting molecular weights between 30 and 41 kDa were considered to be most suitable as a food additive or functional agent as demonstrated by sensory evaluation.     

Preparation of antibacterial chito‐oligosaccharide by altering the degree of deacetylation of β‐chitosan in a Trichoderma harzianum chitinase‐hydrolysing process

BACKGROUND: Chito‐oligosaccharide (COS) is generally known to possess many specific biological functions, especially antibacterial activity, depending on its size. To prepare a specific size range of COS, however, has proved difficult. The aim of this study was to establish a method for preparing a specific size range of antibacterially active COS by adjusting the degree of deacetylation (DD) of β‐chitosan in a Trichoderma harzianum chitinase‐hydrolysing process. RESULTS: The molecular weight spectrum, elucidated by viscosity‐average molecular weight, high‐performance liquid chromatography and thin layer chromatography, of COS in chitosan hydrolysate was significantly related to the DD of its original chitosan. Compared with the original form, COS produced at 90% DD showed superior activity against most Gram‐negative bacteria tested, with a minimum inhibition concentration (MIC) ranging from 55 ± 27 to 200 ± 122 µ g mL^?1. Conversely, most Gram‐positive strains tested were less sensitive to COS (MIC > 880 ± 438 µ g mL^?1) than to its original form. Among the Gram‐positive strains, Staphylococcus xylosus was the only exception in that it showed a high susceptibility to COS and had an MIC as low as 45 ± 11 µ g mL^?1. CONCLUSION: The results indicate that the production of a specific size range of COS product is possible by altering the DD of chitosan in the chitinase‐catalysed process. To produce various sizes of COS for versatile biological functions, as seen in this study to inhibit various types of bacteria, is made possible in this established process. Copyright © 2008 Society of Chemical Industry     

壳聚糖对亚硝酸盐清除作用的研究

研究了体外模拟胃液条件下壳聚糖对亚硝酸盐的清除作用；用分光光度法测定了壳聚糖对亚硝酸盐的清除率，考察了反应时间、壳聚糖用最、壳聚糖的脱乙酰度和分了量对清除亚硝酸盐效果的影响；结果表明：随反应时间的延长、壳聚糖用晕的增加，壳聚糖脱乙酰度的增高和分了量的降低，壳聚糖对亚硝酸盐的清除能力升高；分了量0．98万的壳聚糖37．5mg能完全清除亚硝酸盐。     

牛初乳中乳铁蛋白的分离纯化与免疫功能检测

牛初乳经离心、盐析后获得乳铁蛋白粗提物，通过CM-Sephadex C50离子交换色谱和Heparin-Sepharose CL-6B亲和色谱纯化获得纯度分别为76．98％和92．75％的乳铁蛋白，经SDS-PAGE检测乳铁蛋白存在分子质量分别为76000Da和78000Da的两种异构体，免疫电泳说明提纯样品与乳铁蛋白标准品具有相同的免疫原性。小白鼠喂养和免疫实验说明乳铁蛋白可提高机体免疫功能。     

Antioxidant activity of oligochitosan Maillard reaction products using oligochitosan as the amino or carbonyl groups donors

The purpose of this study was to evaluate the influence of oligochitosan (COS) as amino (–NH₂) or carbonyl group (–C=O) donors on the antioxidant activities of COS Maillard reaction (MR) products. Two kinds of COS MR products (COS MRPs) were prepared by using COS and glucose/α-alanine, respectively. Their structures were characterized by Fourier transform infrared spectroscopy (FT-IR), their molecular weights (Mws) were measured by gel permeation chromatography, and the –NH₂ contents of COS MRPs were determined. The antioxidant activities of COS MRPs were evaluated by measuring the scavenging activity of superoxide anion radical (O₂^?), 1,1-diphenyl-2-picrylhydrazyl radical, and the reducing power. The result showed that C=N absorbance can be observed in CG (MRPs of COS and glucose) and CA (MRPs of COS and α-alanine). The Mw of COS increased after MR. CG and CA showed dramatically enhanced antioxidant activity compared to COS, and the antioxidant activity of CG was better than that of CA. In other words, MR is an efficient way to enhance the antioxidant activity of COS. Moreover, it is a more efficient way to enhance the antioxidant activity of COS by using COS as amino group donor involved in MR. The results may provide some references to the modification and application of COS.     

不同脱乙酰度蚕蛹壳聚糖结构分析及其清除自由基能力的比较研究

首次采用无水乙醇浸泡辅助脱乙酰基与间歇式碱处理相结合的方法,通过控制反应时间制备不同脱乙酰度的蚕蛹壳聚糖。同时对其脱乙酰度、表观黏度和分子量等理化指标进行测定,用红外光谱对其基本结构进行表征,并分别采用邻二氮菲-Fe2＋氧化法、核黄素-光-氮蓝四唑法和DPPH法对其清除羟自由基（·OH）、超氧阴离子自由基（O2-·）和DPPH自由基能力进行了测定。结果表明：当反应时间为5、7、9h时,蚕蛹壳聚糖的脱乙酰度分别为：78.12%、86.45%、95.96%;蚕蛹甲壳素和不同脱乙酰度壳聚糖的红外光谱图的不同说明其结构上的差异,可为进一步研究其性质提供理论参考;蚕蛹壳聚糖对羟自由基和超氧阴离子自由基的清除率均较低,但均高于虾蟹壳来源的壳聚糖;对DPPH·的清除率较高。三种壳聚糖对三种自由基清除率的大小顺序均为：脱乙酰度为95.96%的壳聚糖〉脱乙酰度为86.45%的壳聚糖〉脱乙酰度为78.12%的壳聚糖。     

不同脱乙酰度蚕蛹壳聚糖抑菌性能的比较

研究了3种不同脱乙酰度蚕蛹壳聚糖对9种供试菌的抑菌效果。结果表明:3种壳聚糖对蜡状芽孢杆菌、鼠伤寒杆菌、枯草芽孢杆菌、白色念珠球菌、绿脓假单胞菌、大肠杆菌和金黄色葡萄球菌有明显的抑制作用,对巨大芽孢杆菌和嗜热脂肪芽孢杆菌的抑制作用不明显。随着壳聚糖溶液浓度的增大,其抑菌能力也增强。3种壳聚糖的抑菌活性为:脱乙酰度为95.96%的壳聚糖>脱乙酰度为86.45%的壳聚糖>脱乙酰度为78.12%的壳聚糖。壳聚糖的抑菌活性呈现随pH降低而增加的趋势,当pH值在pH 4.0～5.0,壳聚糖对所有供试菌均能完全抑制。高脱乙酰度蚕蛹壳聚糖作为热加工食品的防腐剂,可稳定保持其抑菌防腐性能。     

Synthesis,characterization and functional properties of galactosylated derivatives of chitosan through amide formation

Low molecular weight chitosan (LMWC) and chitooligosaccharide (COS) derivatives were obtained by the introduction of lactobionic acid (LA) through amide formation, obtaining different complexes COS-LA and LMWC-LA (1–5), with a degree of substitution (DS) between 3 and 16%. The synthesis of these derivatives was monitored by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR), High-Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and proton Nuclear Magnetic Resonance (¹H NMR) analyses. Different functional properties, solubility, water binding capacity (WBC) and fat binding capacity (FBC), as well as the antioxidant activity (DPPH radical scavenging activity) of these derivatives were evaluated. Solubility, WBC and FBC increased in all of the chitosan derivatives respect to those of the native LMWC or COS. The most substituted derivative (LMWC-LA1, DS 15%) presented the highest value of solubility (14.4 mg/mL) while the highest levels of WBC and FBC were obtained for the derivative with a DS of 3% (LMWC-LA5; 4730% and 7100%, respectively). COS and COS-LA presented the best DPPH scavenging abilities, as shown by their low values of EC₅₀ (1.29 and 3.45 mg/mL, respectively). An inverse relationship between the DS of chitosan derivatives and antioxidant activity was observed. LMWC-LA5 (3% DS) was the derivative with the highest DPPH activity, being higher than LMWC in all the concentrations assayed (10.2–14.3% and 6.9–13.7%, respectively). Due to their enhanced functional properties, these chitosan derivatives could be considered as very promising for their future use as additives in the food industry (i.e. to bind fat and cholesterol or avoid hardening of foods).