In vitro and in vivo antibacterial activity of shrimp chitosan against some intestinal bacteria

ABSTRACT:   The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus , and six strains of Bifidobacterium were evaluated. In vitro , the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C .  perfringens . The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.     

甲壳低聚糖酶法制备工艺的优化

为制备具有较好水溶性和生理活性的甲壳低聚糖,本实验选用纤维素酶对壳聚糖进行降解,以还原糖浓度作为衡量降解效果的指标,通过单因素实验和正交实验优化酶解制备工艺,获得最佳酶解条件为:酶浓度1600U/g、温度55℃、反应时间7h、pH值5.4,在该条件下获得的酶解产物还原糖浓度为2.52mmol/L。得到的酶解液通过超滤分离,获得分子量小于10KDa的甲壳低聚糖,其产率达到69.51%。     

不同分子量壳聚糖对刺参(Apostichopus japonicus Selenka)生长和免疫功能的影响

以初始体重为(6.77±0.01)g的仿刺参(Apostichopus japonicus Selenka)为实验对象,在基础饲料中添加1％不同分子量的壳聚糖,分子量分别为35 kDa和400 kDa,进行为期56d的养殖实验,研究低分子壳聚糖(LMWC)和高分子壳聚糖(HMWC)对仿刺参生长和免疫相关酶活性的影响.结果显示,不同分子量的壳聚糖对仿刺参的生长均有促进作用,且LMWC能显著促进刺参的特定生长率(SGR)(P＜0.05).饲料中添加不同分子量壳聚糖,刺参体腔细胞中的总超氧化物歧化酶(T-SOD)活性比对照组均显著增高(P＜0.05).LMWC可显著增强刺参体腔细胞中酸性磷酸酶(ACP)活性(P＜0.05),并能提高碱性磷酸酶(AKP)和一氧化氮合酶(NOS)活性.HMWC对刺参体腔细胞中AKP和NOS活性均有显著增强作用(P＜0.05).研究表明,在刺参饲料中添加一定量的壳聚糖,对其生长和相关免疫酶活性有促进作用.     

Effects of dietary chitosan on growth,survival and stress tolerance of postlarval shrimp,Litopenaeus vannamei

The effect of chitosan, a polymer of glucosamine obtained by the deacetylation of chitin, on growth, survival and stress tolerance was studied in postlarval Litopenaeus vannamei. An experiment was performed with postlarval shrimp (mean initial wet weight 1.2 mg) fed five isoenergic and isonitrogenous diets containing five supplemented levels of chitosan (0, 0.5, 1, 2 and 4 g kg^?1 diet, respectively). The five compound diets (C0, C0.5, C1, C2 and C4) sustained shrimp growth throughout the experiment. Growth performance (final body weights; weight gain; SGR: specific growth rate) in shrimp fed diet C2 was significantly higher than that in shrimp fed diets C0, C0.5 and C1 (P < 0.05), diet C4 treatment provided intermediate growth result. The survival in shrimp fed diet C1 was significantly higher than that in shrimp fed C0 diet (P < 0.05), other diets treatments gave the intermediate survival results. No significant differences were found in growth and survival between diet C2 and C4 treatments. After 9 days of a stress tolerance test, survival in shrimp fed diets C1, C2 and C4 was significantly higher than that in shrimp fed diets C0 and C0.5. We concluded from this experiment that the incorporation of a moderate dietary chitosan was beneficial to the development of postlarval L. vannamei. Considering the effect of chitosan on both growth and survival of postlarval L. vannamei, second‐degree polynomial regression of SGR and survival indicated optimum supplement of dietary chitosan at 2.67 and 2.13 g kg^?1, respectively, so the level of chitosan supplemented in the diet should be between 2.13 and 2.67 g kg^?1.     

Oxidative damages by cadmium and the protective effects of low‐molecular‐weight chitosan in the freshwater crab (Sinopotamon yangtsekiense Bott 1967)

Cadmium (Cd) is an environmental pollutant and has posed a potential threat for the growth and survival of freshwater crabs. Low‐molecular‐weight chitosan (LMWC) may promote growth in crab culture. The present study was designed to investigate the Cd‐induced oxidative damage and the protective role of LMWC against oxidation caused by Cd²⁺ in freshwater crab (Sinopotamon yangtsekiense Bott 1967). The results showed that Cd²⁺ significantly inhibited the activities of total antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase and peroxidase, while it increased malondialdehyde levels in the hepatopancreas and the gill. Moreover, Cd²⁺ at the concentration tested obviously increased the protein carbonyl contents and DNA–protein crosslinks coefficients in the hepatopancreas, gill, heart and muscle tissues of S. yangtsekiense in a dose‐dependent manner. In addition, Cd²⁺ induced a significant increase in the levels of nitric oxide and inducible nitric oxide synthase in the hepatopancreas, gill and muscle. The results also showed that LMWC plus Cd²⁺ significantly improved antioxidant markers. The observations suggested that the severe oxidative damage in multiple crab tissues was one of the important causes of the adverse influence of Cd²⁺ on S. yangtsekiense growth and indicated that LMWC could provide a protective effect against such an injury.     

Comparison of Lactic Acid Bacteria Fermentation with Acid Treatments for Chitosan Production from Shrimp Waste

The traditional procedure for chitosan production involves use of a strong acid (HCl) for demineralization of chitin. This study reports application of a mixed culture of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus lactis) fermentation in demineralization of chitin for chitosan production from shrimp waste. Chitosan produced from shrimp waste with lactic acid bacteria fermentation at 30°C for 72 h or 1 M lactic acid treatment at room temperature for 2 h followed by alkaline treatments (0.5 M NaOH at 25°C for 4 h followed by 12.5 M NaOH at 70°C for 10 h) contained very low protein (0.8–1.1%) and ash (<0.01%) contents with the same solubility (100%) as those of chitosan produced with hydrochloric acid treatment. Lactic acid bacteria fermentation and lactic acid treatment may be utilized in chitosan production from shrimp waste to reduce use of strong chemicals.     

Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus

ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus . Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. K _m and k _cat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N -acetylchitopentaose (GlcNAc₅). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N -acetylchitooligosaccharides (GlcNAc_n, n  = 2–6). Both chitinases hydrolyzed GlcNAc_n ( n  = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc₄ > GlcNAc₅ > GlcNAc₆ for the 38 kDa chitinase, and GlcNAc₆ > GlcNAc₅ > GlcNAc₄ for the 42 kDa chitinase. Both the chitinases released p -nitrophenol from p -nitrophenyl GlcNAc_n ( n  = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.     

The primary culture and subculture of lymphoid cells from shrimp,Penaeus chinensis on thermo‐sensitive CS/α, β‐GP hydrogel

A thermo‐sensitive hydrogel composed of chitosan and α, β‐glycerophosphate was successfully synthesized and used as basic substrate for primary culture and subculture of lymphoid cells from shrimp, Penaeus chinensis. When the ambient temperature increases from 4°C to 25°C, the chitosan/α, β‐glycerophosphate solution transforms into a semi‐solid three‐dimensional hydrogel with a rough surface. Primary lymphoid cells formed were round and grew homogenously over the surface of the hydrogel. The peroxidase activity indicated that cells seeded on the hydrogel suffered less stress than cells suspended in control culture flasks. The higher relative viability and alkaline phosphatase activity of primary cultured lymphoid cells indicated that the chitosan/α, β‐glycerophosphate hydrogel enhanced the growth and proliferation of lymphoid cells. Subsequent subculture of lymphoid cells starting from crumbled primary hydrogel culture was also successful.     

Preparation and characterization of water-soluble chitosan produced by Maillard reaction

ABSTRACT:   The objective of this research was to improve the solubility of chitosan by Maillard reaction with 1% chitosan and 2% reducing sugar (glucose or glucosamine) dissolved in 0.2 M acetic acid, which was adjusted to pH 6.0, and incubated at either 50°C or 70°C for 1–7 days. The physicochemical and rheological properties of the chitosan–saccharide derivatives were also investigated. Results indicated that the solubility of modified chitosan derivatives was significantly greater than that of native chitosan. The solubility of chitosan–glucosamine was higher than that of chitosan–glucose, and the chitosan–glucosamine derivative remained soluble at pH 10. The degree of deacetylation of the derivatives decreased with increasing reaction time. Rheological investigation revealed that the apparent viscosity of the water-soluble chitosan derivatives in aqueous solution depended upon system conditions such as pH, ionic strength, and solution temperature. The measured apparent viscosity decreased as all system conditions increased. As calculated by the Arrhenius equation, the activation energy ( E _a ) of the derivatives in aqueous solution generally decreased with increasing the extent of Maillard reaction with respect to the reducing sugars used.     

Cellulose digestion by common Japanese freshwater clam Corbicula japonica

ABSTRACT:   Cellulose digestion by Corbicula japonica was investigated according to the hypothesis that without any symbiotic aid, this organism can utilize cellulose as a carbon source. Enzymatic studies revealed the complete cellulase activity of this species, and molecular cloning resulted in the isolation of cDNA with an ORF encoding a 596-amino-acid protein that shares significant homology with abalone and termite cellulases with an amino acid identity of 52.2% and 50.5%, respectively. The isolated cellulase had a carbohydrate-binding module at the N-terminal region that was also reportedly present in abalone cellulase, and its mRNA were specifically expressed in the digestive gland. These findings strongly support the assumption that C. japonica has an endogenous cellulose, as well as abalones and termites. It is further believed that C. japonica plays an important roll in decomposing cellulose, and consequently contributes to the carbon-cycle in the aquatic environment, as termites do in terrestrial forests.     

用丰年虫卵壳制备甲壳素和壳聚糖

介绍了用丰年虫卵壳制备甲壳素及壳聚糖的方法及过程,通过傅立叶变换红外光谱仪的检测并与美国标准谱图库检索出的用蟹壳制备的壳聚糖光谱图对照,证实丰年虫卵壳制备的壳聚糖。     

Effect of dietary chitosan on growth performance,haematology, immune response,intestine morphology,intestine microbiota and disease resistance in gibel carp (Carassius auratus gibelio)

A 75‐day experiment was conducted with juvenile gibel carp (Carassius auratus gibelio) (4.80 ± 0.01 g) to evaluate effects of dietary chitosan on fish growth performance, haematology, intestine morphology and immune response. Six isonitrogenous (crude protein: 383 g kg^?1), isolipid (97.5 g kg^?1) and isocaloric (gross energy: 16.7 kJ g^?1) diets were formulated to contain 0, 1800, 4000, 7500, 10 000, 20 000 mg kg^?1 chitosan, respectively. The results showed that the growth was depressed when the fish fed with 10 000 mg kg^?1 chitosan. Serum cholesterol, triglyceride and low‐density lipoprotein decreased in 10 000 and 20 000 mg kg^?1 chitosan. On day 75, blood leucocyte phagocytic activity respiratory burst and alternative pathway of complement haemolytic activity were enhanced in 4000 mg kg^?1 chitosan. The number of goblet cell, intraepithelial lymphocyte of mid‐intestine and microvilli height of distal intestine increased at 4000 mg kg^?1 dietary chitosan. Dietary chitosan modulated intestine microbiota, depressed pathogen bacteria Aeromonas veronii‐like and improved Cellulomonas hominis‐like, Bacillus oceanisediminis‐like and two uncultured bacterium‐like species on day 75. Dietary 7500 and 10 000 mg kg^?1 chitosan enhanced the protection against Aeromonas hydrophila infection. In conclusion, oral administration of dietary 7500 mg kg^?1 chitosan for 75 days is recommended for the survival of gibel carp.     

鱼肉酶解物及壳聚糖对鲤鱼涂膜保鲜效果的研究

以感官评分、挥发性盐基氮、菌落总数、K值为指标,研究比较了鱼肉酶解物和壳聚糖为原料的涂膜液对鲤(Cyprinus carpio)(4℃)冷藏过程中品质变化的影响。结果显示:贮藏期间,对照组的感官评分显著低于各涂膜组(P<0.05)。贮藏前8 d,涂膜组能够显著抑制K值的升高(P<0.05)。鱼肉酶解物和壳聚糖涂膜组均能够显著抑制细菌的生长,而贮藏2～6 d内鱼肉酶解物涂膜鲤鱼的菌落总数显著低于壳聚糖涂膜组(P<0.05)。贮藏后期,鱼肉酶解物组能延缓TVB-N值的升高,壳聚糖涂膜组能够显著抑制TVB-N值的升高(P<0.05)。鱼肉酶解物可作为一种新的可食性涂膜材料,用于延长鲤鱼的贮藏期。     

Effect of dietary chitosan on haematology,innate immunity and disease resistance of Asian seabass Lates calcarifer (Bloch)

This study was performed to evaluate the effect of dietary chitosan on haematology, innate immunity and protection against Vibrio anguillarum in Asian seabass, Lates calcarifer. A basal diet supplemented with 0, 5, 10 and 20 g chitosan kg^?1 diet was fed to the four different groups for 60 days. The haematological (total erythrocyte count, total leucocyte count, total serum protein, albumin, globulin and albumin‐globulin ratio) and innate immune parameters (phagocytic ratio, respiratory burst, serum lysozyme and serum bactericidal activities) were monitored at fortnight interval to assess the effect of chitosan feeding in Asian seabass. All the studied haematological and innate immune parameters were increased significantly (P ≤ 0.05) in chitosan‐fed groups in comparison with control. However, the group fed diet containing 10 g chitosan Kg^?1 feed showed highest haematological and innate immune parameters on 45^th day in comparison with other groups. Moreover, the fish fed the diet containing 10 g chitosan Kg^?1 feed had significantly higher post‐challenge survival (75.56 ± 4.44%) on the 30th day following V. anguillarum challenge. Therefore, this study suggests that chitosan at 10 g kg^?1 diet could be used as prophylactic in Asian seabass culture to enhance the protection against any possible infection by V. anguillarum.     

Chitosan as a wall material for a microencapsulated delivery system for Macrobrachium rosenbergii (de Man) larvae

Chitosan has been widely accepted as a wall material for preparing microcapsules of various purposes in human medicine. The possibility of using chitosan as a wall material for microencapsulating nutrients and drugs for aquaculture purposes, specifically to Macrobrachium rosenbergii larvae was evaluated in this study. Two types of chitosan-coated microcapsules were prepared using either acetone (MEC-A) or NaOH (MEC-N) as the cross-linking agents. They were compared with a microbound diet relative to total leaching of nutrients and free amino acids (FAA). Among the microcapsules, MEC-N showed the lowest level of total leaching of nutrients (23.3%) during 5 h of immersion in seawater and released 65% FAA after 60 min. During laboratory trials, 75% larvae had accepted the MEC-N capsule. The results of the study suggest that chitosan can be used as a wall material for preparing microcapsules to deliver drugs and nutrients to M. rosenbergii larvae.     

枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质

构建了枯草芽孢杆菌CH_2菌株(Bacillus subtilis CH_2)壳聚糖酶基因的真核表达载体p PIC9K-CH2-Cns,在毕赤酵母GS115(Pichia pastoris)中进行重组表达,获得了有生物学活性的重组壳聚糖酶,对重组壳聚糖酶的酶学特性及酶解产物进行了分析。结果显示,重组壳聚糖酶的分子量为29 k D,粗酶的比酶活达到133.60 U/mg,纯化后重组蛋白的比酶活为338.08 U/mg;该酶的最适作用温度为50℃,最适pH为4.5,酶动力学常数Vmax=24.39(μmol/mg)·min~(-1),Km=5.48 mg/m L;Fe2+、K+等对其酶活力有一定的激活作用,其中Mn~(2+)能使酶活力提升2.4倍,Ag~+、Mg~(2+)、Hg~(2+)、EDTA、EGTA和SDS等存在时则对其酶活力有强烈抑制作用。利用重组壳聚糖酶酶解壳聚糖,酶解产物主要为聚合度2~10的壳寡糖,且分布均匀。以上结果表明,枯草芽孢杆菌CH_2菌株壳聚糖酶基因在毕赤酵母GS115中成功表达,获得了一种内切型的高酶活力重组壳聚糖酶,该重组酶具有反应条件温和、酶解产物均一等特点,可被应用于海洋甲壳质加工应用中。     

壳聚糖对鲢鱼糜凝胶特性的影响

将壳聚糖添加到鲢鱼糜制品中,测定鱼糜制品的凝胶强度、全质构(TPA)、失水率和色泽,研究壳聚糖的脱乙酰度(DD)、分子量(MW)以及添加量对鲢鱼糜凝胶特性的影响,采用电镜扫描观察凝胶的微观结构,结果表明,壳聚糖DD对鱼糜制品凝胶特性影响较大,DD为64%时,凝胶强度提高了约34%,失水率减少了29.1%;壳聚糖MW对鱼糜制品的凝胶强度影响小;随着壳聚糖添加量的增加,鱼糜凝胶强度、TPA都有明显的增加,失水率减少(P<0.05),添加1.0%壳聚糖的鱼糜凝胶强度与添加4.0%淀粉的鱼糜凝胶强度相当;微观结构可看出壳聚糖与鱼糜形成网络结构。结果说明壳聚糖是鱼糜制品良好的品质改良剂。     

Conditions for the induction of some selective enzymes from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and their hydrolysis ability against mackerel and gracilar (asparagus, <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>)

ABSTRACT:   This study was conducted to optimize the cultivation conditions for Bacillus subtilis to produce proteases, amylases and cellulase, and to further investigate the hydrolysis ability against mackerel and asparagus. The extracellular enzymes from B. subtilis after 2 and 4 days incubation in a modified medium, containing 1% skim milk, 1% soya meal, 0.25% starch, 0.25% K₂HPO₄, 0.5% NaCl and 0.05% MgCl₂ were collected for the hydrolysis of asparagus and minced mackerel, respectively. Except for the α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging ability of the hydrolyzed asparagus, the trolox equivalent antioxidation capacity and DPPH scavenging ability of both samples increased significantly ( P  < 0.05) after 1 h hydrolysis and further increased during elongated hydrolysis at 50°C. Sodium dodecylsulfate–polyacrylamide gel electrophoresis indicated severe degradation of muscle proteins during hydrolysis. Changes in reducing sugar, soluble proteins and peptides before/after hydrolysis suggested the extracellular enzymes from B. subtilis could effectively hydrolyze the mackerel or asparagus, and subsequently improve their antioxidation ability.     

Effects of dietary cellulase addition on growth performance,nutrient digestibility and digestive enzyme activities of juvenile crucian carp Carassius auratus

This study was conducted to determine the effects of dietary cellulase addition on improving the nutritive value of Chlorella for juvenile crucian carp Carassius auratus (initial body weight: 2.99 ± 0.02 g, mean ± SEM). Five isonitrogenous and isoenergetic experimental diets were formulated to contain 0.0 (control), 0.5, 1.0, 1.5 and 2.0 g kg^?1 cellulase, respectively. Each experimental diet was randomly assigned to triplicate groups with 25 juvenile fish per fibreglass tank for 8 weeks. The results showed that weight gain, specific growth rate, feed intake and the trypsin activity in the anterior intestine increased with increasing dietary cellulase to 1.5 g kg^?1 and then declined with further addition. However, the mRNA expression levels of Mrf4 and Myf5, the apparent digestibility coefficients for dry matter, protein, energy and the majority of amino acids, and the activity of lipase in the anterior intestine were highest in fish fed the 1.0 g kg^?1 cellulase diet, and then tended to decline with further cellulase supplementation. In conclusion, the optimal dietary cellulase supplementation level was 1.0–1.5 g kg^?1, which can improve growth performance, digestive activities and nutrient digestibility in crucian carp.     

三种免疫添加剂对草鱼非特异性免疫功能的影响

在饲料中添加不同剂量的Vc、酵母多糖和壳聚糖,连续投喂42d,并在试验的第14、28、35和42天取样检测,研究其对草鱼血清溶菌酶活性、血清超氧化物歧化酶活性（SOD）活性、血清碱性磷酸酶（AKP）活性、补体C3、含量丙二醛（MDA）含量和血液白细胞吞噬活性共6种指标的影响。结果显示：实验组与对照组相比,饲料中添加Vc可以提高血清溶菌酶活性、SOD活性、AKP活性、补体C3含量和血液白细胞吞噬活性高,并降低MDA含量。添加酵母多糖可以提高血清溶菌酶活性、AKP活性、补体C3含量和血液白细胞吞噬活性,并降低MDA含量,但不能提高SOD活性。添加壳聚糖可以提高血清溶菌酶活性、SOD活性、AKP活性、补体C3含量和血液白细胞吞噬活性,但不能降低MDA含量。饲料中添加Vc、酵母多糖和壳聚糖均对草鱼的非特异性免疫功能具有一定的促进作用。