加工处理对鲢小清蛋白免疫活性的影响

采用鱼丸加工、烘烤、油炸、高压4种方式对鲢进行加工,并比较在这4种鲢制品中小清蛋白的含量、免疫活性以及体外模拟胃液消化稳定性的变化情况.通过Tricine-SDS-PAGE和Western-blotting分析表明,鱼丸加工、烘烤、油炸、高压等方式处理可降低鲢制品中小清蛋白的含量,但在一定程度上提高了小清蛋白的胃液消化稳定性;高压几乎可完全破坏小清蛋白的IgG结合能力,是较为理想的降低食物过敏原免疫活性的加工方式之一.     

鲢鱼骨骼肌过敏原小清蛋白的分离纯化及多克隆抗体制备与应用

小清蛋白是鱼类的主要过敏原,对该蛋白的研究不仅有利于过敏原检测方法的建立也可为低致敏性水产品的开发提供理论依据。通过组织捣碎、冷冻离心、热处理、Superdex75凝胶过滤等方法从鲢白色肉中纯化得到过敏原小清蛋白。Tricine-SDS-PAGE显示,在非还原条件下,该蛋白呈分子量分别为12ku、14ku、24ku的3个条带。而在还原条件下,仅有分子量为12ku的条带。Western-blotting分析表明,分子量为12ku、14ku和24ku的这3个条带都与小鼠抗蛙小清蛋白单克隆抗体(PARV-19)发生特异性反应,提示它们均为小清蛋白的不同形态。用纯化的小清蛋白制备多克隆抗体,经Protein A Sepharose亲和层析纯化得到高纯度的免疫球蛋白G(IgG)。Dot-blot检测发现,抗体稀释至1/51200时仍能与纯化的小清蛋白有显色反应。用制备的多克隆抗体进行Western-blotting分析,能特异地检测4种鱼(鲤、鲢、鲫、黄鳍鲷)中的小清蛋白。     

甘露糖—小清蛋白美拉德反应产物的免疫活性

为探讨以甘露糖为还原糖进行美拉德反应对小清蛋白免疫活性的影响,通过将鲢重组小清蛋白(r PV)和甘露糖混合物在干热条件下(100°C)反应100 min制得糖化的r PV(Mr PV),采用Tricine-SDS-PAGE和斑点杂交(Dot-blotting)分析糖化前后r PV的聚合特性、免疫活性及消化稳定性的变化;采用圆二色谱仪和扫描电子显微镜分析糖化对r PV的二级结构及微观结构的影响。结果显示,r PV糖化后形成大分子量的片状聚集体;甘露糖糖化修饰的r PV免疫活性及消化稳定性均显著下降;美拉德反应导致r PV二级结构中的α-螺旋和无规则卷曲的含量有一定程度的减少,而β-折叠的含量显著增加。研究表明,以甘露糖为还原糖进行美拉德反应可有效降低r PV的免疫活性及消化稳定性,这可能与糖化后r PV聚集体的形成及二级结构的改变有关。     

日本鳗鲡胶原蛋白和小清蛋白的过敏原性

以日本鳗鲡皮与肌肉组织为研究对象,采用碱溶、酸溶、盐析、冻干等方法纯化得到胶原蛋白,采用加热、饱和硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析等方法纯化得到两种亚型的小清蛋白(PV-Ⅰ和PV-Ⅱ),纯化的目标蛋白经动物特异性抗体的免疫印迹实验确证。酶联免疫吸附测定和免疫印迹分析结果显示,纯化的胶原蛋白和小清蛋白分别与鱼类过敏患者阳性血清发生特异性反应,且二者之间无免疫交叉反应。体外模拟胃液消化实验和SDS-PAGE分析结果显示,胶原蛋白和小清蛋白均具有较高的消化稳定性。结果提示,日本鳗胶原蛋白和小清蛋白具有较高的消化稳定性和免疫原性,二者可引发不同患者的IgE介导特异性超敏反应。     

鲫和鳜主要过敏原小清蛋白的基因克隆及序列分析

小清蛋白(parvalbumin, PV)是鱼类的主要过敏原, 为分析淡水鱼PV的序列及结构特征,采用RT-PCR方法, 从鲫和鳜肌肉中分别克隆得到2种小清蛋白的基因序列。生物信息学分析结果显示, 4种基因的序列长度均为330 bp, 编码109个氨基酸残基, 推导分子量在11.6 ku左右, 等电点为4.45~4.69。氨基酸序列分析表明, 克隆得到的这4种PV序列均含有丙氨酸-14、亮氨酸-16、半胱氨酸-19、苯丙氨酸-67、谷氨酰胺-69以及苏氨酸-79等β型PV特征性残基序列, 表明克隆的目的基因均为β型PV。鲫的2种PV序列相似性为80.73 %, 鳜的2种PV的序列相似性为83.49 %。对克隆得到的PV序列进行空间结构分析显示, 这4种PV序列均含有3个螺旋-松弛-螺旋结构, 即EF-手型结构, 其中靠近C端功能域的两个手型结构为Ca^2﹢结合位点。     

加工方式对刀额新对虾主要过敏原免疫原性的影响

选取冻干、烘干、虾丸化以及油炸4种加工方式处理刀额新对虾,通过检测可溶性蛋白、主要过敏原含量和免疫活性的变化以及体外模拟胃液消化实验,分析加工方式对虾类过敏原免疫活性及抗酶解能力的影响。结果表明经过不同的加工处理后,分子量为35 ku左右的主要过敏原蛋白仍然存在,免疫印迹显示其免疫活性有不同程度的下降,其中虾丸化处理对免疫活性的降低程度最大,达87%,冻干处理的影响最小,仅达6.8%;而主要过敏原蛋白抗酶解能力变化不明显。结论:不同的加工方式对虾过敏原活性的降低有比较大的差异,但对于抗酶解能力影响不大。     

TG酶催化过程中鲢肌原纤维蛋白凝胶性能的变化

为探究转谷氨酰胺酶（TG酶）催化过程中,鲢肌原纤维蛋白凝胶性能的变化,进一步提升鲢鱼糜制品特性和营养价值。对不同催化时间（0、15、30、60 min）后鲢肌原纤维蛋白凝胶的交联度、凝胶强度、白度、持水性、微观结构及消化特性变化进行了分析。实验结果表明,TG酶诱导蛋白胶凝后,蛋白凝胶交联度显著提高。在TG酶催化过程中,随交联时间的延长,白度值增加、持水性提高。通过扫描电镜分析发现凝胶网络孔径变小,结构变得更加致密。在催化时间30 min下,交联度、白度值及持水力显著提高,弛豫时间T22显著减小。TG酶催化后的蛋白凝胶在消化初始阶段水解程度较低,因为交联度提升,蛋白网络结构越来越致密,降低了胃蛋白酶对其水解位点的可及性。蛋白消化90 min后,网络遭到破坏,导致其水解速率明显提高。以上结果说明,在适当的交联时间下,有助于提升蛋白凝胶的凝胶特性。该研究为TG酶对鱼肉制品品质及消化特性的改善提供了实验依据。     

鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立

鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。     

南极磷虾主要过敏原的分离纯化及性质研究

为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-...     

淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定

为揭示淇河鲫(Carassius auratus var.Qihe)白细胞介素-8(interleukin-8,IL-8)的功能,本研究构建了原核表达系统,并制备了兔抗鲫IL-8多克隆抗体。首先采用RT-PCR法扩增淇河鲫IL-8基因编码序列中不含信号肽的基因片段,克隆到pET-32a(+)载体后转化入Rosetta菌株构建原核表达系统。经IPTG诱导表达出相对分子质量约27.8 kD的目标融合蛋白。将纯化的融合蛋白免疫家兔制备多克隆抗体,效价达1︰10~5。经免疫亲和层析纯化的抗体能特异性识别重组和天然的淇河鲫IL-8蛋白。比较不同组织的免疫组化和荧光定量PCR结果,IL-8蛋白量和mRNA的表达量在不同组织间变化趋势一致,在肌肉、脾和头肾均检测到较高的表达量,肠道的表达量较低。本研究为进一步探讨淇河鲫IL-8的生物学功能奠定了基础。     

Digestive dynamics during chyme formation of Octopus maya (Mollusca,Cephalopoda)

In the present study, digestive dynamics, measured through the changes in the in situ pH along the digestive tract, was evaluated along with the glycogen concentration in the digestive gland (DG) and the proteolytic enzyme activity of the gastric juice (GJ) (both acid and alkaline proteases) to obtain useful information that will allow for the understanding of the DG's function and the role that it plays as an extracellular digestive enzyme source and for energy storage, providing new information on the digestive physiology of Octopus maya. The results showed that pH along the digestive tract changed according to the postprandial time following the food transit. At the beginning, the pH on crop (St) was 5, changing to 6 when the food arrived. Similar changes were observed on the caecum (Ce) and DG. Glycogen from DG is used as a source of energy during digestion and recovered 8 h after feeding. Maximum activities of digestive gland GJ enzymes were observed 6 h after feeding, indicating that chyme enzymes are still active when they arrive at the DG's lumen. The presence of food in the digestive tract modifies the pH, which, in combination with GJ, favours the activity of the released enzymes of O. maya.     

日本蟳消化系统组织结构研究

应用石蜡切片和苏木精—伊红染色技术对日本蟳消化系统组织结构进行研究,以期为日本蟳的人工养殖模式建立和饲料开发提供基础资料。研究结果表明,日本蟳消化系统包括消化道和消化腺两部分。消化道由口、食道、胃、中肠、后肠及肛门组成,消化道管壁由内向外为黏膜层、黏膜下层、肌层和外膜。食道黏膜下层中含有丰富的食道腺,可分泌黏液帮助食物的吞咽;贲门胃黏膜上皮覆盖的几丁质层有刚毛或骨板等构成的胃磨可磨碎食物;幽门胃中几丁质层特化形成密集排列的刚毛,起到过滤的作用;中肠黏膜上皮无几丁质层覆盖,形成纹状缘,可参与食物的吸收。消化腺主要是肝胰腺,肝胰腺为复管状腺,由许多肝小管组成。肝小管黏膜上皮由单层柱状上皮细胞组成,上皮细胞有4种类型:吸收细胞、分泌细胞、纤维细胞和胚细胞。     

栉江珧消化系统组织学与组织化学的研究

运用组织学和组织化学方法对栉江珧消化系统的结构和功能进行了研究.栉江珧消化管的粘膜上皮由纤毛柱状细胞和杯状细胞组成,唇瓣、中肠和上行肠的上皮层还有嗜酸性粒分泌细胞;消化盲囊的腺上皮由分泌细胞和消化(吸收)细胞组成.皮下结缔组织中富含血腔隙,结缔组织和血腔隙中有"颗粒细胞".食道、胃、中肠、晶杆囊及消化盲囊能分泌消化酶,对食物进行消化和吸收;结缔组织、"颗粒细胞"也参与了食物的消化吸收.唇瓣、食道、晶杆囊和中肠的上皮细胞及结缔组织中的"颗粒细胞"内含铁,结缔组织中含钙.     

Response of gastrointestinal melatonin,antioxidants, and digestive enzymes to altered feeding conditions in carp (<Emphasis Type="Italic">Catla catla</Emphasis>)

The purpose of present study was to ascertain whether the response of gastrointestinal (gut) melatonin to altered feeding conditions was related to the levels of different antioxidants and digestive enzymes in the same gut tissues of a sub-tropical carp (Catla catla). Accordingly, the fish were subjected to food deprivation for 4 or 8 days and separately to re-feeding for 4 or 8 or 12 days after deprivation of food for 8 days, and their gut tissue homogenates were used to measure the levels of melatonin, both enzymatic [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST)] and non-enzymatic [reduced glutathione (GSH)] antioxidants, as well as different digestive enzymes (α-amylase, cellulase, protease, and lipase). Notably, the gut levels of melatonin, SOD, CAT, GPx, and GST underwent gradual increase with the progress of food deprivation, but a sudden fall after restoration of food supply for 4 days and a rise thereafter. Conversely, the activity of all the digestive enzymes significantly decreased after deprivation of food, but started increasing when food supply was reinforced. Gut melatonin concentrations by showing a positive correlation with the titers of different antioxidants (in both food-deprived and re-fed fish groups) and a negative (in food-deprived fish) or a positive (in re-fed fish) correlation with the activity of each digestive enzyme underlined possible physiological interplay between them. Collectively, our findings lend support to the hypothesis that gut melatonin response to altered feeding conditions in carp might be associated with the oxidative status as well as the digestive functions of the gastrointestinal tissues itself.     

Histology of the developing digestive system and the effect of food deprivation in larval green sturgeon (Acipenser medirostris)

The histological development of the digestive tract in hatchery-reared green sturgeon (Acipenser medirostris) larvae and the effects of food deprivation on the digestive system organization were studied from hatching until 31 days post-hatching (dph). At hatching, the larval digestive system consisted of two rudiments: a large endodermal yolk sac and a primordial hind-gut. During the endogenous feeding phase, the wall of the yolk sac differentiated into the stomach (glandular and non-glandular regions) and the anterior and intermediate intestine, while the hind-gut primordium differentiated into the spiral valve and rectum. At the onset of exogenous feeding (15 dph at 16 °C), the organization and cytoarchitecture of the digestive system in green sturgeon larvae was generally similar to those of juveniles and adults. Larvae deprived of food exhibited a progressive deterioration, with subtle pathological changes observed after 5-d starvation: shrinkage of digestive epithelia, tissue degeneration, and necrosis were observed at 10–15 d of starvation (30 dph). No changes were observed in the mucous secretion of different regions of the digestive tract of food-deprived larvae. The histological analysis of the larval digestive system may be used to evaluate the nutritional condition of larval green sturgeon in their nursery habitats in spawning rivers, which are affected by dams and flow diversions.     

河南华溪蟹消化系统的形态结构观察和组织学研究

为研究河南华溪蟹消化系统的形态结构及组织学特征,采用石蜡切片、H.E.染色和光镜等技术,对河南华溪蟹成蟹消化系统各组织器官的结构进行了观察。结果显示,河南华溪蟹的消化系统主要由消化道和消化腺组成。消化道主要包括食道、胃、中肠和后肠,消化腺为肝胰腺。胃包括贲门胃和幽门胃:贲门胃内有钙化的几丁质齿、隆嵴、短刺及刚毛等组成的胃磨,可磨碎食物;幽门胃内侧可见特殊形态的梳状结构,可过滤食物。在光学显微镜下,食道、中肠和后肠均由黏膜层、肌层和结缔组织层构成,其各段内表面均有由上皮细胞与基膜内凸形成的多级皱褶和嵴,有由环形、纵形和辐射状排列的肌层。仅中肠表面有典型微绒毛结构,无角质膜。食道与后肠上皮表面均有较发达的纤毛层。肝胰腺主要由基膜和4种上皮细胞构成,即吸收细胞(R细胞)、泡状细胞(B细胞)、纤维细胞(F细胞)和胚细胞(E细胞)。     

The effect of dietary exogenous digestive enzymes on ingestion,assimilation, growth and survival of gilthead seabream (Sparus aurata,Sparidae, Linnaeus) larvae

The success of microdiets commonly used in the cultivation of marine fish larvae is limited to serving as partial replacements for live food. This limited success is thought to be associated with a reduced digestive ability due to an incompletely developed digestive system. The enhanced growth obtained from live food has been partially attributed to the digestive enzyme activity of the food organism. The present study was designed to test the effect of an exogenous digestive enzyme incorporated. into a microdiet on the growth of Sparus aurata. Larval gilthead seabream, 20–32 days old, were fed ¹⁴C labelled microdiets containing a commercial pancreatic enzyme at different concentrations (0, 0.1 and 0.05g / 100 g dry diet). Rates of ingestion and assimilation were measured and their relationship to dry weight was determined. Our results show that the success of the microdiet as a food for larval gilthead seabream was limited by the larva's low ingestion rate which only approached its maintenance requirement. In addition, the presence of digestive enzyme in the microdiet enhanced its assimilability by 30%. Larval growth over ten days was 0, 100 and 200% on microdiet free of added enzymes, one with added enzymes and a live food regime, respectively. It is our opinion that successful development of microdiets for Sparus aurata must be based on diets improved both in digestibility and attraction to the larvae. Further studies are now underway to determine the nutritional requirements of gilthead seabream larvae using the experimental method developed in the present study. This research was carried out in partial fulfillment of the requirements for the M.Sc. degree.     

Enzyme‐producing bacteria isolated from fish gut: a review

Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes. Each of these factors is affected by a multitude of secondary factors. The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition. It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme‐producing microbiota. In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors. However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length. The present review will critically evaluate the results to establish whether or not intestinal microbiota do contribute to fish nutrition.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

斜带石斑鱼消化系统胚后发育的组织学研究

利用形态学和连续组织切片技术，对出膜后1-60d的斜带石斑鱼各期仔鱼、稚鱼和幼鱼的消化系统进行了光镜观察，描述了其消化器官发育过程和组织学特性。研究表明，实验水温为22.0-27.8℃时，孵化后第4天，上下颌形成，卵黄囊被吸收，消化系统明显分化成食道、胃、肠、直肠以及肝脏、胆囊和胰脏等，鱼体由内源性营养转向外源性摄食营养，表明其消化系统的形态变化与食性的变化相适应。此后随着鱼体的生长，其消化系统从功能和结构上逐步完善成熟。胰脏在出膜后第4天出现，是和肝脏相互分开的一个独立的器官，但是发育到第35-60天，位于肠道后部的胰脏组织内出现许多大空泡。