Low molecular weight chitosan prepared with the aid of cellulase,lysozyme and chitinase: Characterisation and antibacterial activity

A continuous set of low molecular weight chitosan (LMWC) products was successfully made for this study by coordinating three enzymes (chitinase, lysozyme and cellulase) and two different deacetylated chitosan substrates (80% and 92%). It was observed that the intrinsic viscosity molecular weight (M_V) and SEC-HPLC-determined M_W distribution of LMWC were directed by both the used enzyme and the degree of chitosan substrate acetylation. LMWC prepared using lysozyme and 92%-deacetylated chitosan had larger M_W and, therefore, possessed higher antibacterial activity, as compared to other combinations. LMWC enzyme-directed properties suggest chitinase is more predictable and flexible to produce the specified M_V of LMWC. LMWC’s solubility and antibacterial activity, determined as minimum inhibitory concentration (MIC), against Escherichia coli exhibited a negative linear relationship with log M_V. E. coli strains showed much higher susceptibility to LMWCs than Staphylococcus aureus strains did. Both species also showed intra-species sensitivity diversity toward LMWC.     

Antibacterial Activity and Physical Properties of Edible Chitosan Films Exposed to Low-pressure Plasma

The challenge for food industry is developing gentle processes concept, which will prevent food spoilage and leave a food natural, minimally processed, fresh-like and safe. A new technique of food preservation could be usage of combined methods of green process, such as cold gas plasma with bioactive substances, and protective coatings. The aim of this study was to determine the antibacterial activity of chitosan films incorporated with lysozyme exposed to helium plasma treatment as well as evaluate their physical properties. The edible films have been prepared basing on low molecular weight chitosan by casting from lactic acid solution with water solution of lysozyme in three various concentrations (0, 0.5 and 1 %). Dried films were then modified by exposition on cold helium plasma treatment for 0, 5 and 10 min. Obtained films were tested against growth of Listeria monocytogenes, Yersinia enterocolitica and Pseudomonas fluorescens. In order to characterize chitosan-based films, their mechanical properties, theromogravimetric analysis (TGA), dynamic mechanical thermal analysis (DMTA), contact angle measurement, water vapour permeability (WVP) and scanning electron microscopy (SEM) were evaluated. The films with 1 % lysozyme incorporation enhanced the inhibition efficiency of chitosan-based films against gram-positive (L. monocytogenes) and gram-negative (P. fluorescens) bacteria, where reduction zones were 42.5 and 69.8 mm, respectively. Besides that, hydrolytic changes of chitosan chain caused by lysozyme activity were confirmed by TGA and DMTA. Contact angles and WVP of tested films were not significantly affected by helium plasma exposition, nor lysozyme dosage. Hydrophilic natures of chitosan-based films were confirmed by both tests. Microscopy image of cross-section structure was smooth and continuous due to lysozyme addition in film composition. Application of chitosan films incorporated with lysozyme and low-pressure plasma treatment could be used as innovative preservation method in a wide range of food products.     

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.     

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     

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。     

Development of Shelf-stable Intermediate-moisture Meat Products Using Active Edible ChitosanCoating and Irradiation

Shelf‐stable intermediate‐moisture (IM) meat products were developed using a combination of hurdles such as reduced a_w, active edible coating of chitosan, and irradiation. Chitosan prepared from chitin had a viscosity of 16 c P, molecular weight of 17.54 kDa, and a degree of deacetylation (DD) of 74%. The nitrogen content of the chitosan was estimated to be 7.56%. The antioxidant activity of chitosan increased upon irradiation without significantly affecting its antimicrobial property. The effect of irradiated chitosan coating in terms of its antimicrobial and antioxidant properties in IM meat products immediately after irradiation and during storage was assessed. The a_w of meat products such as mutton sheek kababs and streaky bacon was first reduced to 0.85 ± 0.02. The products were then coated with chitosan and irradiated (4 kGy). No viable bacteria or fungi were detected in chitosan‐coated, irradiated products. In contrast, IM meat products that were not subjected to gamma radiation showed visible fungal growth within 2 wk. The chitosan‐coated products showed lower thiobarbituric acid‐reactive substances (TBARS) than the noncoated samples for up to 4 wk of storage at ambient temperature. The studies thus clearly indicated the potential use of chitosan coating for the preparation of safe and stable meat products.     

Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M_w) of 2-224 kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F_A = 0.16) were more active than the more acetylated chitosans (F_A = 0.48). No trends in antibacterial action related to increasing or decreasing M_w were observed although one of the chitosans (M_w 28.4 kDa, F_A = 0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.     

壳聚糖抗菌性能的研究

本实验研究了客观因素对壳聚糖抗菌性能的影响,以确定壳聚糖的最佳抗菌条件。以金黄色葡萄球菌为实验菌,接种到含一定量壳聚糖的液体培养基中,通过测定光密度值研究壳聚糖的浓度、溶解度、分子量、培养基pH值对其抗菌活性的影响。抑菌实验结果表明:脱乙酰度、分子量相同的壳聚糖,对金黄色葡萄球菌的抑制能力随浓度的降低而减弱;壳聚糖溶于浓度为2%的醋酸溶液中,培养基pH控制在6.0最有利于发挥壳聚糖的抗菌活性;通过微波辐射法制得的低分子量壳聚糖水溶性很好,但对金黄色葡萄球菌的抑制能力有所下降。     

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。     

Effect of Molecular Weight,Acid, and Plasticizer on the Physicochemical and Antibacterial Properties of β‐Chitosan Based Films

Abstract: Effects of chitosan molecular weight (1815 and 366 kDa), type of acid (1% acetic, formic, and propionic acid, or 0.5% lactic acid) and plasticizer (0, 25% glycerol or sorbital w/w chitosan) on the mechanical, water barrier, and antibacterial properties of β‐chitosan films were investigated. Tensile strength (TS) of high molecular weight (Hw) films was 53% higher than that of low molecular weight (Lw) ones, acetate, and propionate films had the highest TS (43 and 40 MPa) among tested acids, and plasticizer‐reduced film TS 34%. Film elongation at break (EL) was higher in Hw films than in Lw ones, in which formate and acetate films were the highest (9% and 8%, respectively), and plasticizer increased the film EL 128%. Molecular weight of chitosan did not influence water vapor permeability (WVP) of the films. Acetate and propionate films had lower WVP than other acid types of films, and plasticizer increased film WVP about 35%. No difference was found between glycerol and sorbitol films in terms of film mechanical and water barrier properties. Lw β‐chitosan films showed significant antibacterial activity against E. coli and L. innocua. This study demonstrated that β‐chitosan films are compatible to α‐chitosan films in physicochemical properties and antibacterial activity, yet with simple sample preparation. Practical Application: β‐chitosan based edible films at molecular weight of about 300 kDa showed great antibacterial activity against Gram‐positive and Gram‐negative bacteria. The films have similar mechanical and water barrier properties to α‐chitosan based films at the similar molecular weight, but simple sample preparation procedures and more attractive color. The release of active chitosan fragment from the film matrix acts as an antibacterial agent, making β‐chitosan films suitable as intelligent food wraps or coatings for a wide range of food products to control moisture loss and prevent surface bacterial growth.     

Synthesis and antibacterial properties of new monomethyl fumaric acid-modified chitosan oligosaccharide derivatives

Inventions into chitosan oligosaccharide (COS) derivatives over recent decades have hugely broadened their application in food safety. Herein, monomethyl fumaric acid (MFA) was grafted on the OH position of COS to synthesise new COS-O-MFA derivatives and evaluated their in vitro antibacterial properties. The degrees of substitution (DS) of the derivatives yielded almost 15% with the range of 0.093 to 0.260. The in vitro antibacterial properties of the new COS-O-MFA1-3 derivatives against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were significantly enhanced concerning COS. Based on the finding, the introduction of MFA and increasing DS have a great impact on the enhancement of antibacterial properties of the COS-O-MFA1-3 derivatives. This work proves that COS-O-MFA1-3 derivatives can be potential novel antibacterial agents and also open up a new avenue to improve their antibacterial attributes.     

2,4-二氯苯氧乙酸壳寡糖酯的制备及抗菌活性研究

以壳寡糖、2,4-二氯苯氧乙酸为原料,经壳寡糖氨基保护、2,4-二氯苯氧乙酸的羧基酰氯化、酰氯化后的2,4-二氯苯氧乙酸与壳寡糖羟基反应、脱除壳寡糖氨基保护最终得到2,4-二氯苯氧乙酸壳寡糖酯(Dcpo-O-COS)。通过傅里叶红外、紫外可见吸收、1 H核磁共振对2,4-二氯苯氧乙酸壳寡糖酯进行了结构表征,证明成功合成终产物。经X射线衍射仪、热分析仪、抗菌性研究测定,终产物的热稳定性、对大肠杆菌及金黄色葡萄球菌的抗菌性较原料壳寡糖有所提高。     

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。     

Preparation of water-soluble chitosan from shrimp shell and its antibacterial activity

Crude chitosan was prepared from shrimp shell by HCl, NaOH and ethanol solution successively. Hydrogen peroxide was used to degrade the crude chitosan into water-soluble chitosan. A mathematical model between degradation conditions (H₂O₂ level, time and temperature) and the recovery of water-soluble chitosan was constructed using response surface methodology. Each factor showed a significant effect on the recovery. The model was confirmed to have a good fitness by analysis of variance. The optimal conditions to obtain the highest recovery of water-soluble chitosan were 5.5% of H₂O₂ level, 3.5 h of time and 42.8 °C of temperature. The predicted recovery was 93.5%. Through testing the number of colony, both crude and water-soluble chitosan showed good inhibition activities against B. subtilis. By determination of inhibition zone diameter, water-soluble chitosan showed significantly (P < 0.05) higher inhibition capabilities against E. coli, B. subtilis and S. aureus than crude chitosan.Industrial relevanceChitin is the second abundant polymer next to cellulose over the world. Its deacetylated product, chitosan, is an important ingredient in medicine and food. However, the low solubility in water limits the application of chitosan. In this work, the chitosan was degraded by H₂O₂ to produce water-soluble chitosan. Response surface methodology was taken to construct a model between degradation conditions and the recovery of water-soluble chitosan. By determination of the antibacterial activity, water-soluble chitosan showed better antibacterial activity than crude chitosan without degradation treatment. The results indicated the high potential of water-soluble chitosan as an antibacterial agent. This work was helpful for applying this product in industry.     

Application of chitosan-incorporated LDPE film to sliced fresh red meats for shelf life extension

Chitosan lactate was impregnated as an antimicrobial additive into low density polyethylene (LDPE) with different concentrations. The antimicrobial effectiveness was tested with three pathogenic bacteria, specifically Listeria monocytogenes, Escherichia coli and Salmonella enteritidis. Also, these chitosan incorporated films were applied on red meat surfaces to determine the effectiveness of chitosan on color shelf life extension and microbial growth inhibition. Chitosan was exposed to 0.1% peptone water containing the three pathogens in separate tests and inhibited microbial growth a higher levels with increasing concentration of chitosan in the film matrix. Oxygen permeability was not affected by the incorporation of chitosan, while the water vapor permeability increased with the addition of chitosan. Film elongation decreased with the addition of chitosan. When chitosan incorporated films were applied on fresh red meat, microorganisms on the meat surface were not inhibited but significant extension of red color shelf life were observed in refrigerated, sliced red meats.     

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。     

Physicochemical characterisation of β-chitosan from Sepioteuthis lessoniana gladius

β-Chitin and its chitosan from the gladius of Sepioteuthis lessoniana have been isolated, purified, characterised and compared with the commercial chitosan. Ash, moisture, mineral, metal and elemental content were analyzed using standard techniques. The optical activity of chitin was found to be levorotatory. The degree of deacetylation was calculated by potentiometric titration and ¹H NMR. Viscosity average molecular weight of β-chitosan was calculated by viscometry and size average molecular weight by GPC. The structure of β-chitosan was elucidated with FT-IR and NMR. Thermal nature, crystalline structure and morphology of β-chitosan were characterised through DSC, XRD and SEM, respectively. The water and fat binding capacity of β-chitosan presently studied was significantly higher than that of the commercial chitosan. The result of the present study adds that S. lessoniana gladius is also an additional source of β-chitin and chitosan of higher yield, lower molecular weight and higher degree of deacetylation.     

Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells

In this study, chitosan was extracted from Metapenaeus stebbingi shells. In order to determine physicochemical characteristics of the extracted chitosan, the yield, moisture and ash contents, degree of deacetylation, molecular weight, water and fat binding capacities, apparent viscosity and colour properties were measured using a variety of techniques including Fourier transform infrared spectroscopy, scanning electron microscopy and X-ray diffraction. In addition, the physicochemical characteristics of the chitosan extracted from M. stebbingi shells were compared to commercial chitosan. The degree of deacetylation was calculated by the titration method and elemental analysis. The molecular weight was determined by viscosimetric methods. The results of the study indicate that shrimp shells are a rich source of chitosan as 17.48% of the shell’s dry weight is consisted of this material. Extracted chitosan exhibited a lower molecular weight, higher degree of deacetylation, higher viscosity and higher water and fat binding capacities compared to the commercial chitosan.     

Antimicrobial activity and physical properties of chitosan–tapioca starch based edible films and coatings

Antimicrobial activity of edible coating solutions based on chitosan and blends of chitosan–tapioca starch with or without potassium sorbate (KS) addition was studied. The agar well diffusion assay showed an antagonist effect on the efficiency of chitosan against Lactobacillus spp. when KS and/or tapioca starch were present. A salmon slice coating assay showed that the chitosan solution was the best coating since aerobic mesophilic and psychrophilic cell counts were reduced, pH and weight loss remained acceptable throughout refrigerated storage, extending global quality to 6-days. Chitosan–tapioca starch based films reduced Zygosaccharomyces bailii external spoilage in a semisolid product but were not effective against Lactobacillus spp. The results suggest that antibacterial action depended on the application technique, due to the fact that chitosan is more available in a coating solution than in a film matrix. Interactions between chitosan–starch and/or KS could affect film physical properties and the antimicrobial activity of chitosan. The addition of chitosan reduced water vapor permeability and solubility of starch films.     

不同分子量壳聚糖对大肠杆菌抑制作用规律及其机理探讨

考察了不同分子量的壳聚糖对大肠杆菌的抗菌、抑菌作用的影响规律,并利用壳聚糖的席夫碱反应对其氨基加以保护,初步提出了壳聚糖对大肠杆菌的抗菌机理。研究结果表明,壳聚糖分子量越小,对大肠杆菌的抗菌作用越明显;壳聚糖对大肠杆菌的抑菌作用与其氨基的质子化有关。