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%。     

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8d,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2～6d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.     

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.     

Evaluation of shrimp or tilapia protein hydrolysate at graded dosages in low fish meal diet for olive flounder (Paralichthys olivaceus)

A study was carried out to examine and optimize the inclusion levels of shrimp hydrolysate (SH) or tilapia hydrolysate (TH) in low fishmeal diets for olive flounder (Paralichthys olivaceus). A fishmeal (FM)‐based diet was considered as a high FM (HFM) diet, and a diet containing soy protein concentrate (SPC) as a FM replacer at 50% substitution level was regarded as a low FM (LFM) diet. Six other experimental diets were prepared by dietary supplementation of SH or TH to LFM diet at different inclusion levels of 15 g/kg, 30 or 45 g/kg in the expense of FM (designated as SH‐1.5, SH‐3.0, SH‐4.5, TH‐1.5, TH‐3.0 and TH‐4.5, respectively). After 10 weeks of a feeding trial, growth performance and feed utilization efficiency of fish were significantly higher in fish fed HFM, SH‐3.0, SH‐4.5, TH‐1.5 and TH‐3.0 diets compared to those of fish fed LFM diet. Intestine diameter, villus height and goblet cell counts of fish were significantly increased by dietary inclusion of SH or TH into LFM diet. Dry matter and protein digestibility of diets were significantly improved by SH or TH incorporation. Innate immunity of fish was significantly enhanced by dietary SH or TH supplementation into LFM diet. Disease resistance of fish was significantly increased against Edwardsiella tarda by dietary inclusion of SH and TH at the highest inclusion level (45 g/kg). The optimum inclusion level of SH or TH in a SPC‐based LFM diet could be ~30 g/kg and 15–30 g/kg, for olive flounder.     

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。     

卵形鲳鲹配合饲料中酶解鱼浆蛋白和陆生复合蛋白替代鱼粉的研究

为了评估卵形鲳鲹(Trachinotus ovatus)配合饲料中酶解鱼浆蛋白和陆生复合蛋白替代鱼粉的可行性,本研究设计了4种等蛋(42%)等脂(12%)配合饲料(D1~D4),其中,D1(对照组)含30%鱼粉,D2~D4(处理组)都含14%陆生复合蛋白且还分别含有16%、11%、6%鱼粉和0、5%、10%酶解鱼浆蛋白;各处理组都补充蛋氨酸和赖氨酸。将360尾初始体重为(7.28±0.10) g的卵形鲳鲹幼鱼随机分配到12个海上网箱中,每个网箱30尾鱼,每种饲料设3个网箱。将鱼以上述4种饲料饲养62 d后,测定其生长性能、体组成、血清生化指标、肠道消化酶活性与组织抗氧化指标。结果显示,各实验组鱼的增重率(WGR)、特定生长率(SGR)、蛋白质效率(PER)、饲料系数(FC)、胃蛋白酶(PEP)、脂肪酶(LPS)和淀粉酶(AMS)活性均无显著差异(P>0.05)。D3和D4组全鱼粗蛋白含量显著高于D1和D2组,D4组鱼肌肉脂肪含量显著低于D1~D3组(P<0.05);D2~D4组鱼血清谷草转氨酶(AST)及D2和D3组鱼谷丙转氨酶(ALT)活性都显著低于D1组(P<0.05),D1和D4组血清总蛋白(TP)和球蛋白(GLO)水平显著高于D2和D3组(P<0.05);D2~D4组肝脏过氧化氢酶(CAT)活性、总抗氧化能力(T-AOC)和肌肉超氧化物歧化酶(SOD)活性显著高于D1组(P<0.05),且肝脏丙二醛(MDA)含量低于D1组(P<0.05)。研究表明,含6%鱼粉的D4组鱼的生长性能与30%鱼粉D1组无差异,且全鱼蛋白含量及肝脏和肌肉抗氧化能力显著提高,说明14%陆生复合蛋白配合10%酶解鱼浆蛋白可有效替代卵形鲳鲹饲料中80%鱼粉,使饲料鱼粉使用量低至6%。本研究是首次探讨酶解鱼浆蛋白在卵形鲳鲹配合饲料中应用的可行性,结果可为研发高效低成本配合饲料提供参考依据。     

Effect of shrimp head protein hydrolysates on the state of water and denaturation of fish myofibrils during dehydration

ABSTRACT:   To utilize fisheries waste products as food materials with functional properties, shrimp head protein hydrolysates (SHPH) from three species of shrimp, that is, Northern pink shrimp ( Pandalus eous ), Endeavour shrimp ( Metapenaeus endeavouri ) and Black tiger shrimp ( Penaeus monodon ), were produced by enzymatic hydrolysis using endopeptidase derived from Bacillus subtilis and exopeptidase derived from Aspergillus oryzae at a level of 0.1% (w/w). SHPH were rich in protein (90–91%) and amino acids (71–84%) but little fat (0.01–0.02%). The average molecular weight of SHPH was 300–1400. The effect of 5% SHPH (dry basis) addition on the state of water and denaturation of lizard fish myofibrils (Mf) during the dehydration process was evaluated by the desorption isotherm and the Ca-ATPase activity, and compared with the effect of sodium glutamate (Na-Glu). SHPH decreased the water activity and the Ca-ATPase inactivation, and increased monolayer sorbed water and multilayer sorbed water of Mf, although these effects of SHPH were smaller than those of Na-Glu. These findings suggest that the SHPH suppressed dehydration-induced denaturation of myofibrillar protein by stabilizing the hydrated water surrounding myofibrils.     

Effects of including shrimp protein hydrolysate in practical diets on the growth and survival of redclaw crayfish hatchlings Cherax quadricarinatus (Von Martens, 1868)

An experiment was carried out to analyse the effects of substituting fish‐meal (FM) with soya bean meal and shrimp protein hydrolysate (SPH) on the percent survival, growth rate, feed efficiency and molt frequency of redclaw crayfish hatchlings, Cherax quadricarinatus. The experiment was performed in a closed recirculating‐water system over a 56‐day period. Five diets were tested: a control diet (SPH₀) comprising 100% FM without SPH, and four diets with 2.5%, 5.0%, 10.0% and 15.0% SPH substitution (diets SPH_2.5, SPH₅, SPH₁₀ and SPH₁₅ respectively). Analysis of water quality indicated that it was adequate to maintain a good growth and survival of hatchlings. The results showed that 100% survival was obtained for hatchlings fed with SPH₅, SPH₁₀ and SPH₁₅ diets. Higher mean values of final weight were obtained for the SPH₅ diet. The highest weight gain was observed in SPH_2.5 and SPH₅ diets. Hatchlings showed similar specific growth rates for all diets. The efficiency rate of the SPH₁₅ diet was lower compared to the other diets. Overall, individual feed consumption was higher in hatchlings fed diets of SPH₅ and SPH₁₅. It is recommended that 5.0% of SPH could be included in diets of hatchling redclaw crayfish to improve their growth efficiency.     

草鱼肠道一菌株产纤维素酶的分离纯化及性质研究

从草鱼肠道分离出1株枯草芽孢杆菌, 有较强的活性。该菌产生的纤维素酶粗酶液, 经盐析、透析, 并通过葡聚糖凝胶层析柱Sephadex G - 75分离纯化, 经SDS- PAGE后表明第2峰已纯化, 得到一种相对分子质量约为62. 43 kD纤维素酶。分离纯化后该酶的比活力提高了2. 924 倍, 回收率为6. 38%。酶学试验研究表明: 该酶的最适反应温度为55e , 最适pH 值为7. 0; L inew eaver- Burk法求得动力学参数, K m 和Vm ax分别为1. 02 @ 10- 3 g /mL、2. 727 @ 10- 2mg /( mL# m in)。     

Effect of protein hydrolysate from Antarctic krill meat on the state of water and denaturation by dehydration of lizard fish myofibrils

ABSTRACT: To utilize Antarctic krill as functional food, protein hydrolysates were prepared by enzymatic hydrolysis. Their effects on the state of water in myofibrils of lizard fish and dehydration-induced denaturation were compared with those from two species of shrimp, glucose, and sodium glutamate. Peptides are major components in hydrolysates, occupying approximately 85–93% of the total materials. The Antarctic krill protein hydrolysates stabilized the bonding of water molecules, leading to suppressed denaturation of myofibrils during the dehydration process. Similar effects were observed for shrimp protein hydrolysates. The effect of the hydrolysate was less than that by glucose and sodium glutamate.     

Suitable dietary chitosan improves the growth performance,survival and immune function of tiger shrimp,Penaeus monodon

Different levels of dietary chitosan on growth performance, survival and stress tolerance to air exposure was studied in tiger shrimp, Penaeus monodon. Shrimp (mean initial wet weight about 1.16 g) were fed with six different diets (C0, C0.05, C0.1, C0.2, C0.3 and C0.4) containing six level of chitosan (0%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% respectively) in triplicate for 60 days. Growth performance [final body wet weight (FBW); weight gain (WG); biomass gain (BG)] of shrimp fed chitosan‐containing diets were higher (P < 0.05) than that of shrimp fed the basal diet, shrimp fed C0.1 diet showed the highest value of growth performance. Survival of shrimp in C0.1 and C0.2 diet groups were higher (P < 0.05) than that of shrimp in C0, C0.05 and C0.4 diet groups but without statistical difference (P > 0.05) in shrimp fed C0.3 diet group. Whole body and muscle lipid contents decreased with increasing dietary chitosan levels. Plasma total cholesterol and triglyceride contents of shrimp fed C0 diet was significantly higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of shrimp fed C0 diet were higher than those of shrimp fed chitosan‐containing diets. Digestive gland malondialdehyde (MDA) and carbonyl protein contents of shrimp fed chitosan‐containing diets were lower (P < 0.05) than that of shrimp fed C0 diet. Total haemocyte count of shrimp fed C0 diet was lower (P < 0.05) than that of shrimp fed chitosan‐containing diets. On the contrary, the haemolymph clotting time of shrimp fed C0 diet was higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. In conclusion, all results suggested that dietary intake containing 0.1% and 0.2% chitosan enhanced the growth of shrimp, whereas a higher level than 0.3% and 0.4% decreased growth of shrimp. Second‐degree polynomial regression analysis of WG and BG indicated that the optimum supplement of dietary chitosan level should be 0.19–0.21%.     

The use of squid protein hydrolysate as a protein source in microdiets for gilthead seabream Sparus aurata larvae

In the present study, the use of predigested proteins as an ingredient of microdiets offered to gilthead seabream larvae was tested. The protein source (freeze-dried squid powder) was hydrolysed with protease (trypsin and pancreatin). Different levels of raw squid protein and hydrolysate (100% protein, 50% protein/50% hydrolysate, 100% hydrolysate) were added to the microdiets to produce a dietary protein level of 65%. For comparison, cofeeding of Artemia nauplii and microdiet as well as microdiet supplemented with pancreatin were also offered to the larvae. The final average dry weights of 32-day-old larvae were 1.65 ± 0.04 mg, 1.38 ± 0.06 mg and 1.13 ± 0.1 mg, respectively, for larvae cofed 0%, 50% and 100% hydrolysate microdiets and Artemia nauplii. Survival of larvae was not affected by protein source. The survival of larvae cofed Artemia nauplii and microdiet was significantly higher than that of larvae fed exclusively on microdiet (68% and 80%, respectively). These results suggest that the use of hydrolysate (at 50% greater) as a protein source in diets for seabream larvae is not to be recommended.     

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.     

三相流化床生物反应器同时产酶同时降解壳聚糖的研究

采用多孔聚酯泡沫块固定里氏木霉大三相流化床固定化反应器中同时产酶同时降解壳聚糖，结果表明，通过控制降解时间可以得到不同平均聚合度的降解物，在28℃，pH4.8，通气量2.5vvm条件下，重复利用菌丝降解2%（w/v)浓度壳聚糖，每批产生的壳聚糖酶活力平均达到150mU.mL^-1以上，壳聚糖平均降解率为72%以上，利用此固定化反应器，在45天内连续进行15批同时产酶降解试验，结果发现壳聚糖酶活和壳聚糖降解率能保持稳定。     

Assessment of the Antibacterial Effect of Chitosan Coated with Heracleum persicum Oil on Rainbow Trout Quality

Today, due to the rapid spoilage of fish, the use of natural preservatives is a priority over those of synthetic varieties. Also, the natural antimicrobial effects of essential oils can help to increase shelf life. There are very few studies concerning the use of essential oils in this regard. In the present study, the antibacterial effect of chitosan coated with Heracleum persicum oil was investigated on the quality of rainbow trout. The control and the coated fish samples were analyzed periodically by generalized estimating equation (GEE) for total viable count (TVC), psychrophilic bacteria, total volatile base nitrogen (TVB-N), polyunsaturated fatty acids (PUFA), texture, taste, odor, and overall acceptance parameters. The results showed that by increasing the amount of chitosan, TVC, psychrophilic bacteria, and TVB-N decreased while PUFA, texture, taste, odor, and overall acceptance parameters increased.     

An evaluation of replacing fish meal with cottonseed meal protein hydrolysate in diet for juvenile blunt snout bream (Megalobrama amblycephala): Growth,antioxidant, innate immunity and disease resistance

This study evaluated effects of fishmeal replacement by cottonseed meal protein hydrolysate (CPH) on growth, antioxidant and immunity of Megalobrama amblycephala and its resistance to Aeromonas hydrophila. Fish (average weight: 38.66 ± 0.08 g) were divided into five groups and fed with five isonitrogenous (320 g/kg crude protein), isolipidic (70 g/kg crude fat) and isocaloric (17.8 MJ/kg gross energy) diets replacing fishmeal with 0%, 1%, 3%, 5% and 7% CPH, respectively. The control diet contained 60 g/kg fishmeal. Final weight and weight gain of fish fed 5% and 7% CPH were significantly lower than that of fish fed control diet (p < 0.05). 3% CPH significantly increased total protein, globulin, acid phosphatase, C3 and C4 contents of plasma and total‐superoxide dismutase and catalase activities of liver (p < 0.05), as well as upregulated the antimicrobial peptides 1 and 2 (Leap‐1 and Leap‐2) expression levels in liver and spleen (p < 0.05). After challenge, the lowest mortality was observed in fish fed 3% CPH, and it was significantly lower than that in fish fed the 7% CPH (p < 0.05). In conclusion, dietary fishmeal replacement by 3% CPH could increase antioxidative capacity, as well as enhance immunity of fish.     

1H NMR‐based metabolomics studies on the effect of size‐fractionated fish protein hydrolysate,fish meal and plant protein in diet for juvenile turbot (Scophthalmus maximus L.)

This study was designed to evaluate changes in the metabolic profile of liver and muscle of turbot (Scophthalmus maximus L.) fed fishmeal‐based diet, diets containing size‐fractionated fish protein hydrolysate and plant protein‐based diet using ¹H NMR‐based metabolomics approach combined with the growth. Fish protein hydrolysate (FPH) was obtained by enzymatic treatment, permeate fraction was obtained as UF by ultrafiltered step, and retentate fraction was retained as RF. FM diet contained fish meal used as a single protein source. Four other diets (PP, UF, FPH and RF) contained 180 g kg^?1 diet fish meal. 54, 55 and 55 g kg^?1 dry diet UF, FPH and RF were supplemented to UF, FPH and RF diets. All diets were formulated to be isolipidic and isonitrogenous fed to five triplicate groups of turbot (16.05 ± 0.03 g) for 68 days. O‐PLS‐DA in FM versus UF, FM versus FPH, FM versus RF and FM versus PP resulted in a reliable model for muscle and liver tissue, while O‐PLS‐DA in UF versus FPH and UF versus RF only showed metabolites changes in liver tissue. Results indicated that metabolite changes among the different treatments were consistent with the growth tendency.     

中国明对虾serpin型丝氨酸蛋白酶抑制剂基因的重组表达及活性分析

Serpin家族成员作为主要的丝氨酸蛋白酶抑制剂,通过调节一系列丝氨酸蛋白酶和半胱氨酸蛋白酶的活性来调节蛋白酶级联反应,进而参与了包括血液凝结、补体激活、纤维蛋白溶解、炎症反应、肿瘤抑制和激素转运等大量的基本生物过程,在调节机体免疫及其它重要生理功能方面发挥着重要作用。我们在前期研究中,从中国明对虾血细胞中克隆得到一个serpin型丝氨酸蛋白酶抑制剂基因（Fc-serpin, GenBank注册号：DQ318857）,初步研究显示它在转录水平参与了病原菌和白斑杆状病毒（WSSV）引发的对虾先天免疫应答反应;本研究利用原核重组表达系统,成功获得了重组的对虾serpin型蛋白酶抑制剂（rFc-serpin）,纯化复性后得率为0.3g/L;活性分析显示,重组目的蛋白（rFc-serpin）对多种病原菌的生长均有一定抑制作用,研究结果进一步证实它参与病原微生物引发的对虾防御应答过程,为探讨甲壳动物先天免疫防御应答机制提供了有价值的参考。     

Evaluation of Agave lechuguilla by‐product crude extract as a feed additive for juvenile shrimp Litopenaeus vannamei

Agave lechuguilla is a succulent plant species, mainly distributed in the northeast of Mexico and south of the United States of America. The main use for this plant is the fibre´s extraction (known as Tampico fibres), resulting in 15% of fibres and 85% of a by‐product waste named guishe. The lechuguilla collectors, normally incinerate the guishe, thus causing environmental contamination. Interestingly, recent studies showed that guishe contains molecules with nutritional properties, such as saponins, flavonoids and sugars. Therefore, in this work, we evaluated the effect of the crude extract of guishe as a feed additive in whiteleg shrimp diets. According to that, MS‐HPLC analysis of the extract showed the presence of saponins such as diosgenin, smilagenin, hecogenin, manogenin, tigogenin hexose, yucagenin, chlorogenin, diosgenin diglucoside and the flavonol, quercetin. After chemical analysis, the crude extract was included into an experimental diet in four levels; 0% (L0%), 0.1% (L0.1%), 0.3% (L0.3%) and 0.6% (L0.6%). Dietary incorporation of the extract was evaluated by zootechnical performance, haemolymph biochemistry, histomorphology and digestive enzyme activity of shrimps. After 5‐week feeding, the L0.3% diet showed significantly higher growth and better feed utilization among treatments. A significant increase in tubule epithelium height and tubule coverage area from hepatopancreas in shrimp under L0.3% diet compared with the control diet suggest an improvement of the health and nutritional status of the shrimp. Inclusion of L0.3% and L0.6% of the crude extract resulted in a reduction in amylase activity, without effect in glucose levels in the haemolymph. Thus, we suggest that lechuguilla guishe crude extract contains nutritional molecules that may be used as a feed additive to promote shrimp productivity.