克氏原螯虾蛋白肽及其美拉德反应产物抗氧化活性的研究

以碱性蛋白酶水解克氏原螯虾副产物得到的蛋白肽为原料,与葡萄糖发生美拉德反应(Maillard reaction,MR),在单因素实验的基础上,通过正交实验优化美拉德反应,并对虾蛋白肽和最优条件下获得的美拉德产物(Maillard reaction produces,MRPs)进行抗氧化性测定。实验结果表明:虾蛋白肽美拉德反应的最佳条件是反应时间为4h,蛋白肽与葡萄糖的质量比(肽糖比)为1∶3,pH为9,最适温度为100℃制备得到的美拉德反应产物还原力达0.929,DPPH自由基清除能力为87.75%。克氏原螯虾虾壳蛋白肽美拉德反应后抗氧化活性明显的增加,还原力、羟自由基清除能力,DPPH清除能力分别为反应前的8.367倍、1.829倍和2.027倍。     

裂壶藻蛋白肽美拉德反应产物的制备及其抗氧化特性

该研究以裂壶藻(Schizochytrium limacinum)渣为原料,采用酶解法制备得裂壶藻酶解物(S. limacinum hydrolysate, SLH),分析其美拉德反应条件,同时探究其不同超滤组分和葡聚糖凝胶柱层析组分美拉德反应产物(Maillard reaction products, MRPs)的抗氧化能力。结果表明,当还原糖为核糖、糖肽质量比为1∶1、反应pH为9、反应温度为100℃、反应时间为6 h时,SLH的MRPs还原力(5 mg·mL~(-1))为1.24,DPPH自由基清除率(12.5 mg·mL~(-1))为88.62%。将SLH经超滤后分别得到SLH-1 (5 kD)、SLH-2 (10 kD)和SLH-3 (50 kD)组,发现SLH-1组在美拉德反应前后的抗氧化能力最强。将SLH-1组经Sephadex G-25凝胶柱分离后分别得到SLH-1-Ⅰ、SLH-1-Ⅱ、SLH-1-Ⅲ和SLH-1-Ⅳ组,发现分子量较大的SLH-1-Ⅰ组经美拉德反应修饰后的抗氧化活性最好,其还原力(2 mg·mL~(-1))为0.741,DPPH自由基的清除率(5 mg·mL~(-1))为79.41%。此外,氨基酸组成分析表明美拉德反应可导致酪氨酸(Tyr)、赖氨酸(Lys)、组氨酸(His)、精氨酸(Arg)、色氨酸(Trp)等氨基酸含量明显降低,但对必需氨基酸总含量影响不大。     

淡水鱼类主要过敏原的模拟肠胃液消化

采用Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)和免疫印迹(Western-blot)方法,通过模拟肠胃液消化实验分析淡水鱼类肌肉中主要过敏原小清蛋白(parvalbumin,PV)的消化特性。Tricine-SDS-PAGE结果显示,纯化的鲫、鲢PV在模拟胃液消化实验中反应5～30min明显分解,而在模拟肠液消化实验中4h仍未见明显分解。鲫、鲢肌浆蛋白在模拟胃液消化实验中,肌浆蛋白中的PV比纯化PV的分解时间明显延长,消化30～60min仍未被完全分解,而其它肌浆蛋白消化10～30min就完全分解。采用抗PV多克隆抗体的Western-blot显示,该抗体能特异识别PV及其降解产物。研究结果表明,淡水鱼类PV相对于非过敏蛋白具有较高的消化稳定性,而胃蛋白酶能较好地分解该过敏原。     

鸢乌贼肌原纤维蛋白糖基化产物功能特性研究

以葡聚糖、褐藻寡糖、葡萄糖、乳糖为糖基供体实现对鸢乌贼(Symplectoteuthis oualaniensis)肌原纤维蛋白糖基化改性,分析了不同糖的种类及改性时间对蛋白溶解性、结构、乳化性、起泡性等功能性质的影响。结果表明,肌原纤维蛋白与不同种类的糖质量比1∶1反应12 h后,SDS-PAGE分析显示糖基化产物分子量明显增大,小分子糖与肌原纤维蛋白反应更快;糖基化反应使肌原纤维蛋白的溶解性显著提高,表面疏水性降低;此外,肌原纤维蛋白二级结构明显改变,β-转角含量显著升高,β-折叠含量降低。糖基化反应未能改善肌原纤维蛋白的热稳定性。反应后的肌原纤维蛋白具有更好的起泡性及起泡稳定性,但乳化性和乳化稳定性降低。肌原纤维蛋白糖基化后在高离子浓度氯化钠(Na Cl)溶液中比在低离子浓度溶液中具有更高的乳化和起泡能力。同时,经糖基化改性的蛋白在低离子浓度时的溶解性和起泡能力也能得到明显改善。     

糖基化鸢乌贼肌原纤维蛋白体外消化产物抗氧化性研究

将鸢乌贼(Symplectoteuthis oualaniensis)肌原纤维蛋白与褐藻寡糖进行糖基化反应,并采用胃蛋白酶和胰酶对蛋白进行体外消化,分析糖基化改性对蛋白消化产物抗氧化活性和氨基酸组成的影响。结果显示,肌原纤维蛋白与褐藻寡糖在反应过程中自由氨基浓度显著降低,且反应前12 h降低更为明显,表明二者之间发生了明显的接枝反应。糖基化肌原纤维蛋白的DPPH自由基清除率及还原力在模拟消化前后均显著高于未接枝肌原纤维蛋白(P0.05),因此接枝物具有更高的抗氧化能力。消化后蛋白自由基清除能力及还原力均降低。SDSPAGE电泳图谱表明,肌原纤维蛋白与褐藻寡糖接枝反应促使高分子量蛋白接枝物的生成。酶解后糖基化肌原纤维蛋白的小分子蛋白、多肽的产生量明显增多,表明糖基化促进了蛋白水解反应。氨基酸分析显示,肌原纤维蛋白接枝物酶解液中总必需氨基酸含量减少而疏水性氨基酸含量增加。     

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.     

“红肉病”文蛤的组织病理学

利用组织学和组织化学方法,在光镜水平下研究了患“红肉病”文蛤(Meretrix meretrix Linnaeus)的组织病理学变化特征。结果显示,患病文蛤的病理学变化主要表现为组织结构紊乱,上皮膨大、脱落,鳃、外套膜、消化盲囊等组织发现异常结构及寄生物,如嗜碱性的包涵体、嗜酸性颗粒及寄生性原生动物等。另外,组织化学研究结果显示,病蛤在糖含量、磷酸酶活性等方面也有明显变化,表现为消化盲囊、肠等部位吸收细胞内糖含量增加。消化盲囊、消化管各处酸性磷酸酶(ACP)活性减弱,碱性磷酸酶活性(AKP)增强。鳃组织ACP活性增强,AKP活性减弱等。     

不同热加工方式对刀额新对虾过敏原活性的影响

选取煮(100 ℃)、蒸(100 ℃)、高压(121 ℃,0.1 MPa)这3种常见热加工方法,分别处理刀额新对虾不同的时间,通过检测总可溶性蛋白、主要过敏原含量和免疫活性的变化,分析3种热加工方式对虾类过敏原免疫活性的影响,并通过质地剖面分析,研究虾肉经过处理后组织结构的变化。经过3种热处理后,分子量为36 ku的主要过敏原蛋白仍然存在,免疫印迹的结果显示其免疫活性有不同程度的下降,高压对免疫活性的降低程度最大,30 min时免疫活性降低了97%;在25~35 ku区域出现一条新的IgE结合蛋白。质构分析结果显示,经过高压热处理后,虾肉的硬度和咀嚼度都有很大程度的降低,口感变差。结果表明:热高压处理在降低虾过敏活性的同时,对其口感也有一定的影响,有必要通过优化高温高压处理工艺,在保持虾口感及营养的基础上,降低其过敏活性。     

盐度升高对中华绒螯蟹几种非特异性免疫因子的影响

采用逐步增盐法, 结合生物酶学测定, 研究了从淡水升高盐度到 7、14、21、28和35对二秋龄雌、雄中华绒螯蟹血清总蛋白、氧合血蓝蛋白含量及碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、酚氧化酶(PO)活力的影响。结果显示, 随着盐度的升高, 中华绒螯蟹的血清总蛋白、氧合血蓝蛋白含量逐渐降低; AKP活性先略升高, 而后逐渐降低; 与雄蟹的PO活力逐渐降低相比, 雌蟹的PO活力先降低, 当盐度高于21后维持稳定水平; SOD活性先略降低, 盐度高于21被激活。除SOD外, 雌蟹血清各测定指标均显著高于雄蟹(P<0.05)。研究结果表明, 盐度接近或超过28将显著影响中华绒螯蟹3种免疫酶的活性及总蛋白和氧合血蓝蛋白的含量, 进而影响其免疫防御能力。     

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。     

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。     

The digestibility of the proteins of broad bean (Vicia faba) and soya bean (Glycine max) under in vitro conditions simulating the alimentary tracts of rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio)

The in vitro digestibility of the proteins of broad bean and soya bean was compared by using a physiologically oriented method developed in our laboratory. The physical and chemical conditions of the alimentary tracts of rainbow trout and carp were simulated in vitro as closely as possible. Generally protein digestibility of broad bean is poorer than that of soya bean. Differences in digestibility are more pronounced after digestion under the simulated conditions of the carp gut than under those of the trout gut. The deficiency in the sulfuric amino acids cysteine + methionine is more marked in broad bean than in soya bean. Furthermore, in broad bean, threonine and lysine (for carp) and phenylalanine (for trout) are near or slightly below the limits of dietary requirement.     

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。     

Apparent digestibility of Asian carp‐ and common carp‐derived fish meals in feeds for hybrid striped bass Morone saxatilis ♀ × M. chrysops ♂ and rainbow trout Oncorhynchus mykiss

Apparent digestibility coefficients (ADCs) of nutrients (crude protein, amino acids, crude lipid, fatty acids, and minerals) were determined for fish meals derived from menhaden, Asian carp (combination of silver and bighead carps), and common carp in feeds for hybrid striped bass and rainbow trout. Extruded test diets were formulated to contain a 70 : 30 mixture of reference diet and test ingredient with yttrium oxide (1 g kg^?1) serving as the inert marker. Diets were randomly assigned to triplicate tanks and fish were fed once per day at 2% body weight. Fecal samples were collected by manual stripping. The ADCs were calculated according to standard procedures. The composition and digestibility of Asian carp and common carp meals was broadly similar to menhaden meal. Protein digestibility ranged from 86.5% (Asian carp meal) to 93.1% (common carp meal). Lipid was highly digestible with ADCs >100% for all ingredients. Although the Asian carp meal was less digestible than the other two fish meals, it was still a highly digestible ingredient. Our data suggest that fish meals derived from Asian or common carp would be valuable feedstuffs in diets for hybrid striped bass, rainbow trout, and possibly other cultured fishes.     

An in vitro method to determine phosphorus digestibility of rainbow trout Oncorhynchus mykiss (Walbaum) feed ingredients

An in vitro method was developed to assess the digestibility of phosphorus in 12 plant and animal feed ingredients for rainbow trout Oncorhynchus mykiss (Walbaum). The method simulates the gastrointestinal tract of the rainbow trout with regard to pH and gastrointestinal enzymes. Phosphorus solubility was measured after acid digestion (pH 3) with and without gastric enzymes, after alkaline digestion (pH 9) with and without intestinal enzymes, and after a two-step process involving acid and alkaline digestion. Commercially available digestive enzymes from mammals were compared with digestive enzymes from rainbow trout. Correlating in vitro digestibility with in vivo digestibility showed that acid digestion with both commercial enzymes ( r ²=0.98, P  < 0.05) and trout enzymes ( r ²=0.94, P  < 0.05) predicted the in vivo digestibility of animal feed ingredients. Alkaline digestion with both enzyme systems (commercial r ²=0.79; trout r ²=0.74, P  < 0.05) or without ( r ²=0.82, P  < 0.05) enzymes predicted the in vivo digestibility of ingredients from animal byproducts but not those from plant products. The in vitro digestibility with two enzyme steps (acid and alkaline) predicted in vivo digestibility of plant and animal ingredients ( r ²=0.79 for commercial enzymes and r ²=0.74 for trout enzymes) better than did one-step acid or alkaline digestion.     

Effects of feeding time,water temperature,feeding frequency and dietary composition on apparent nutrient digestibility in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio

A series of experiments was conducted to evaluate the effects of feeding time (daytime vs nighttime feeding), dietary fat content (8 and 20%), feeding frequency (trout 1–3 times/day, carp 2–7 times/day), water temperature (trout 18 and 11°C, carp 25 and 17°C), on the apparent nutrient digestibility in rainbow trout and common carp. The feeding time had little effect on the macronutrient digestibility in both species. In trout, starch digestibility decreased with the decrease of water temperature and with increase of feeding frequency, but in carp, increase of the feeding frequency markedly decreased the macronutrient digestibility and phosphrrus absorption of the high fat diet. Fat digestibility of the beef tallow diet decreased relative to the pollock oil diet in carp, without affecting the phosphorus absorption. Inclusion of raw starch, the digestibility of which was lower than that of gelatinized starch, increased the phosphorus absorption in carp. These results suggest that reduction of water temperature and increase of feeding frequency notably decreased starch digestibility in trout while in carp, the effects of feeding frequency and water temperature on macronutrient digestibility and phosphorus absorption are notable for a high fat diet.     

鲤、鲢、鳙、草鱼消化道消化酶种类和活性的比较研究

采用聚丙烯酰胺凝胶电泳方法研究了鲤、鲢、鳙、草鱼亲鱼消化道消化酶的种类；用比色法及滴定法分别测其消化酶相对活性。结果表明：鲤、鲢、鳙、草鱼4种鱼消化道的蛋白酶、淀粉酶活性与食性有明显的相关性。其中，蛋白酶活性依次为鲤〉鲢〉鳙〉草鱼；淀粉酶活性依次为草鱼〉鲤〉鳙〉鲢。脂肪酶活性与食性的关系不明显。四种鱼消化酶的表达与消化道的组织结构特征相一致，消化道前2／5部位（G1、G2）的消化酶活性最高，是无胃鱼消化道的主要消化场所。消化道中央部位（G3）的消化酶活性次之，能将未消化完全的食糜进一步消化，而消化道后段（G4、G5）消化酶活性最低。可见，消化酶活性及消化功能呈递减形式，食物在消化道中随着消化、吸收，逐渐排出体外。本研究为鲤、鲢、草鱼、鳙4种鱼的营养生理学、高效饲料配比的研制及种质资源的科学管理提供理论依据。     

彭泽鲫的分子遗传分析及其与方正银鲫A系的比较

Genetic homogeneity between Pengze crucian carp and strain A of silver crucian carp was studied by using transferrin,isozyme and RAPD markers.The studied individuals of Pengze crucian carp showed transferring patterns were the same of silver crucian carp A strain while distinct from those of other crucian carp populations.As far as isozyme is concerned,the MDH,LDH and EST are all of the same with only slight differences in SOD between them.The RPAD patterns clearly indicated high homogeneity among 16 individuals (6 sampled from individuals of two years old and the others aged one) from crucian carp of Pengze and 5 individuals from strain A of silver crucain carp.Nearly indentical banding patterns were observed among all individuals.Average genetic distance within all the individuals is only 0.011,suggesting crucian carp of Pengze might possess indentical genetic background with strain A of silver crucian carp.