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%。     

甲壳低聚糖的特性研究

甲壳低聚糖(COS)具有优越的功能性质和生理活性,是国际上近年来发展的一种新型的低聚糖,有关甲壳低聚糖的一些特性的研究国内外未见报道。本文对这种低聚糖的带电性、热性质、吸湿保湿性、抑制淀粉老化等特性进行了研究。结果表明:(1)0.5%COS溶液当pH<9.7时,带正电荷。它的第一等当点是pH5.1,第二等当点是pH9.7;(2)DSC分析表明:COS的热分解温度范围为211～219℃,低于壳聚糖,COS的热稳定较壳聚糖差;(3)COS有优良的吸湿保湿性;(4)COS对淀粉老化具有显著的抑制作用。     

马氏珠母贝蛋白的分离及分子量分布研究

试验结果表明,马氏珠母贝全脏器各分离蛋白组分占总蛋白含量分别为:非蛋白氮1.48%,肌浆蛋白33.20%,肌原纤维蛋白13.14%,碱溶性蛋白21.54%,基质蛋白30.65%;贝肌肉各分离蛋白组分占总蛋白含量分别为:非蛋白氮0.83%,肌浆蛋白26.56%,肌原纤维蛋白18.48%,碱溶性蛋白30.34%,基质蛋白23.79%;马氏珠母贝蛋白氨基酸组成均衡,呈味氨基酸及支链氨基酸含量高,但在各蛋白组分中的含量存在一定差异性.利用聚丙烯酰胺凝胶电泳(SDS-PAGE)测定的分子量分布结果显示,2种原料蛋白的肌浆蛋白分子量分布范围较宽,集中在200～19 kDa,肌原纤维蛋白分子量分布集中在200～45 kDa,碱溶性蛋白集中分布在41～3 kDa.     

琼胶低聚糖清除自由基的活性

采用体外鲁米诺发光和DPPH体系对琼胶低聚糖清除氧自由基活性进行了研究。实验结果表明,琼胶低聚糖ML对水溶性的·OH和O2·-有很好的清除作用,IC50分别为0.05mg·mL-1、0.7mg·mL-1,效果与自由基清除剂肌肽相近。琼胶低聚糖ML对醇溶性的DPPH自由基有一定的清除活性(IC50为6.4mg·mL-1),但是清除效果远不如Vc和丙丁酚。对不同的聚合度、含硫量和摩尔浓度的琼胶低聚糖的清除O2·-的活性进行比较,实验发现含硫量高、分子量大的低聚糖在清除自由基过程中发挥主要作用。琼胶低聚糖ML对LPS+D-GalN急性肝损伤小鼠的实验表明,一定浓度的ML灌胃可以有效抑制肝损伤小鼠血清GPT活性和MDA浓度的升高,保护抗氧化酶SOD和GSH Px的活力,对肝损伤有很好的保护作用。     

固定化木瓜蛋白酶水解甲壳胺的研究

木瓜蛋白酶经戊二醛固定于自制的甲壳质上制成固定化木瓜蛋白酶，其水解甲壳胺的最佳条件是：45℃，pH4.0的1％的甲壳胺溶液（醋酸缓冲液配置），酶／底物比1：3．5（质量比）。震荡反应24h后，用超滤器分离不同分子量的甲壳胺低聚糖产物，结果产物中分子量小于10000的甲壳胺低聚糖得率约为45．55％，分子量小于5000的约为34．0％，优于游离木瓜蛋白酶水解甲壳胺的效果。此固定化酶可反复使用至少10次以上，总反应时间达192h，提高了酶的使用效率和酶解效果。     

蛋白质水解物的超过滤分馏

为浓缩富集低分子量抗氧化肽（2kDa）鱼肉蛋白水解物（绿青鳕，Pollachius virens），测试了不同材质和截留分子量的五种有机管式纳滤膜。为限制浓差极化效应，研究了在低污染状况下膜的材质和截留分子量（MWCO）对渗透通量、水解吸附程度、膜的清洁和多肽保留时间的影响。水渗透通量显现出对MWCO和膜的材质都敏感。高疏水性材质的聚醚砜膜（PES）表现出比改进的聚醚砜（m—PES）膜和聚砜（PS）膜更低的透水通量。     

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.     

哈维氏弧菌TS-628菌株胞外产物(ECP)蛋白酶活性的研究

采用复性电泳技术,研究环境pH、温度及培养时间对哈维氏弧菌TS-628菌株胞外产物(ECP)蛋白酶活性的影响。结果表明,胞外产物蛋白酶最适反应pH在8.5左右,酸对ECP蛋白酶活性的抑制明显强于碱对它的抑制作用;最适反应温度在20～50℃之间,20℃以下或50℃以上蛋白酶活性则受到明显抑制,表明蛋白酶不仅对高温敏感,对低温也很敏感;培养12h的胞外产物蛋白酶活性最强。最适温度和pH条件下,主要出现3种蛋白酶,其中分子量为94kDa和26kDa的两种蛋白显示了很强的蛋白酶活性,而且能在比较极端的温度和pH值条件下保持活性,而分子量为35kDa蛋白酶只在最适反应条件下出现,并且该蛋白酶的最适反应条件与弧菌病暴发时的环境条件极为相似。由此可推测分子量94kDa和分子量为26kDa的两种蛋白可能是弧菌维持生存所必需,而分子量为35kDa的蛋白酶虽然只出现在特定条件下,却可能在致病过程中起重要作用。     

赤点石斑鱼诺达病毒的纯化及其衣壳蛋白的Western-blot分析

采用差速离心,蔗糖(10%～40%,W/V)密度梯度离心和氯化铯(30%～40%,W/W)等密度梯度离心法,对赤点石斑鱼诺达病毒大亚湾株(RGCN)进行了纯化,测定计算其浮密度为1.3102～1.3243g·cm-3。通过Westernblot法检测到的病毒的结构蛋白的分子量为37和31kDa,而以31kDa的蛋白为主。     

具有结合胆酸盐作用卵形鲳鲹蛋白酶解物的制备和分子量分布研究

以卵形鲳鲹(Trachinotus ovatus)鱼肉为原材料,以酶解产物与胆酸盐的体外结合率为指标,从5种常用食品用蛋白酶中筛选最优蛋白酶。进而采用正交法优化酶解条件,以提高酶解产物的胆酸盐结合率,并采用GPC法分析产物中蛋白肽的分子量分布情况。结果表明,胰蛋白酶酶解产物(100 mg·m L~(-1))与胆酸钠、甘氨胆酸钠和牛磺胆酸钠等3种胆酸盐的体外结合能力均好于其他4种蛋白酶,结合率分别达到42.1%、33.5%和30.1%,分别相当于降血脂药物考来烯胺散(20 mg·m L~(-1))的78.3%、74.4%和76.8%;经过正交试验优化的制备卵形鲳鲹蛋白肽的工艺条件为酶解时间2 h,加酶量2 000 U·g~(-1),料液比1∶4(g·m L~(-1)、p H 8、37℃)。该条件下酶解产物(20 mg·m L~(-1))对甘氨胆酸钠体外结合率达到同浓度阳性对照物(考来烯胺散)的48.3%,酶解产物中相对分子量3 k D以下的肽类组分占总蛋白水解物的77.30%。     

Lipid nutrition of juvenile Litopenaeus vannamei: I. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction

Two 6-week experiments were conducted in recirculating systems to determine dietary requirements of Litopenaeus vannamei for phospholipids (PL) and cholesterol, and their potential interaction. A 3×4 factorial design consisting of three cholesterol levels (0%, 0.2%, and 0.5% of diet) and four PL levels (0%, 1.5%, 3.0%, and 5.0% of diet) provided by de-oiled soybean lecithin (SL) was applied. Overall survival was 97.7% with no significant difference among dietary treatments. There was a highly significant interaction between PL and cholesterol on shrimp growth (P=0.0001). Dietary cholesterol at 0.2% or 0.5% gave similar shrimp growth at any given level of PL.

In a second factorial experiment, the same four levels of PL were evaluated in conjunction with smaller intervals between dietary cholesterol levels, 0%, 0.05%, 0.1%, 0.2%, and 0.4%, in an attempt to define dietary requirements and interaction better. Overall survival was 99.7%. A highly significant interaction between dietary PL and cholesterol on growth of shrimp was detected again (P=0.0001). The cholesterol requirement was estimated to be 0.35% of diet in the absence of supplemental PL. At 1.5% and 3% PL, dietary cholesterol requirements were reduced to 0.14% and 0.13% of diet, respectively. When PL were provided at 5% of diet, 0.05% dietary cholesterol was needed for optimal growth. Shrimp growth was significantly enhanced as level of PL increased, but as the level of dietary cholesterol level increased, the growth-promoting effect of PL was diminished. Lipid composition of hepatopancreas (mid-gut gland) and muscle tissue of shrimp fed the different diets was also analyzed. Diets supplemented with PL resulted in higher total lipid in hepatopancreas and lower total lipid in muscle of shrimp than those of shrimp fed diets without supplemental PL. Interaction between dietary PL and cholesterol also affected total lipid and triglycerides (TG) in the hepatopancreas, as well as cholesterol concentration in the muscle.     

豚链球菌和停乳链球菌类M蛋白及其抗原性分析

分别用热酸抽提法、碱抽提法和胃蛋白酶消化法分离豚链球菌NUF849的类M蛋白，通过SDS-PAGE和Western-blotting印迹法分析比较三种方法所提类M蛋白的组成和抗原性差异。结果表明，热酸抽提法分离制备的类M蛋白得率和纯度都较其它两种方法高，蛋白的抗原性保持较好。采用热酸提取法提取5株豚链球菌NUF633、NUF693、 NUF701、 NUF812、 NUF849和2株停乳链球菌SD、L2的类M蛋白，其SDS-PAGE图谱明显不同，种间差异明显, 豚链球菌主要类M蛋白的分子量分别为60、48、37、30、27、24和15kDa，停乳链球菌主要类M蛋白的分子量分别为68、48、42、37、29、26、23、21、19、17、15、14和11kDa。Western-blotting结果显示，兔抗豚链球菌血清可与豚链球菌的两条类M蛋白带结合，分子量分别为60和37kDa；与停乳链球菌的两条类M蛋白带结合，分子量为68和37 kDa。两种链球菌主要类M蛋白组成及其抗原性差异明显。     

Identification of a resistant protein from scallop shell extract and its bile acid-binding activity

In this study, we showed that feeding rats the organic extract of scallop shells (scallop shell extract) caused a decrease in the weights of white adipose tissues in rats fed a high-fat diet. In addition, the cholesterol concentration in the serum of rats that received a diet containing scallop shell extract was significantly lower than that in the serum of rats on the control diet. Feeding this scallop shell extract to rats increased the fecal weight as well as the fecal excretion of bile acids. The amino acid composition of the feces from rats fed the scallop shell extract was different from that of feces from rats fed the control diet, and treatment of the extract with pepsin and pancreatin identified a protein with a molecular weight of 90 kDa (90-kDa protein) as one of the indigestible proteins. Interestingly the 90-kDa protein was found to be identical to a free radical-scavenging protein we previously identified and showed the ability to bind bile acids. These results suggest that indigestible proteins (resistant proteins) in the scallop shell extract, including the 90-kDa protein, inhibit the absorption of bile acid by binding to it and cause increased excretion of fecal bile acid, which subsequently may decrease the serum cholesterol level.     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

饲料脂肪水平对锦鲤幼鱼生长和血液生化指标的影响

本实验以初始体重为(5.85±0.19)g红白锦鲤(Cyprinus carpio L)幼鱼为实验对象,研究在螺旋藻添加量为12%时,饲料脂肪水平对锦鲤生长、饲料表观消化率、肠道消化酶和血液生化指标的影响。实验设5.36%、7.74%、10.52%、12.85%、15.45%5个脂肪梯度,每组设3个重复,每个重复10尾鱼,表观饱食投喂60 d。结果显示:脂肪水平为5.36%～10.52%,锦鲤增重率(BWG)、特定生长率(SGR)随脂肪水平的升高而升高,均在脂肪水平为10.52%时,达到最大值。实验鱼血清谷丙转氨酶(ALT)随脂肪添加比例呈现先下降再升高的趋势,脂肪水平为12.85%后,血清ALT显著升高(P<0.05),脂肪水平为15.45%时,血清GLU浓度显著升高(P<0.05),而甘油三酯(TG)、总胆固醇(CHOL)随脂肪水平的升高各实验组间无显著性差异(P>0.05)。结果表明,螺旋藻添加量一定时,锦鲤适宜脂肪添加水平为10.52%。     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

Antioxidant Properties and Arsenic Speciation of Ultrafiltration and Nanofiltration Derived Abalone Viscera Hydrolysate Fraction

A fraction with molecular weight of 0.2–1.0 kDa was prepared from abalone viscera hydrolysate by ultrafiltration and nanofiltration. The contents of protein, carbohydrate, and fat in abalone viscera were 45.75%, 18.19%, and 13.60%, respectively. The protein in abalone viscera hydrolysate faction (AVHF) reached up to 75.73%, while carbohydrate and fat fell to 1.80% and 0.20%, respectively. The leucine (Leu) content was the highest in the AVHF. On the other hand, the hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and reducing power increased with increased concentration of AVHF. The inhibiting ability of AVHF on lipid peroxidation was higher than that of ascorbic acid. The total content of K, Na, and P in AVHF was 9.78 g/100 g, and total arsenic content reached 16.23 mg/kg. The proportion of arsenobetaine and arsenocholine accounted for more than 96%, while the proportion of arsenate was 0.47%, and arsenite was not detectable. It is concluded that the AVHF from abalone viscera is a safe natural antioxidant.     

Formation of Glycated Products and Quality Attributes of Shrimp Patties Affected by Different Cooking Conditions

ABSTRACT

The objective of this study was to firstly investigate the impact of the cooking conditions, including boiled, baked, and microwaved treatments, in the presence of 3% powdered sugar and 1% NaCl on the changes of quality attributes and the formation of glycated products in shrimp patties. The levels of carboxymethyllysine (CML), furosine, and fluorescent intensity in boiled shrimp patties were much lower than that in baked shrimp patties and microwaved shrimp patties. Addition of powdered sugar alone in baked shrimp patties and microwaved shrimp patties increased the level of furosine and fluorescent intensity. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a notably decreased intensity in the band with MW of 17–22 kDa in boiled shrimp patties and microwaved shrimp patties, and a stronger intensity of band, with MW of 75–100 kDa, in baked shrimp patties. Principal components analysis (PCA) further confirmed that the changes of quality attributes and glycated products in shrimp patties caused by cooking methods were greater than that caused by the addition of powdered sugar or NaCl.     

Serum lipoproteins in haddock, Melanogrammus aeglefinus L.

The lipoprotein and lipid composition of the serum in postabsorptive haddock was characterized. A low level of very low density lipoproteins (VLDL; <50 mg dL^?1) was observed in serum. High density lipoprotein (HDL) was the predominant lipoprotein class followed by low density lipoproteins (LDL). Of the lipoprotein classes, the highest proportions of triacylglycerol (28%) and phospholipid (64%) were observed in the lipid of VLDL and HDL, respectively. Serum lipid (48%), phospholipid (50%) cholesterol ester (65%) and HDL (51%) contained a high level of polyunsaturated FA, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Serum triacylglycerol (38%) and VLDL (36%) contained a lower proportion of polyunsaturated fatty acid. The phospholipid, triacylglycerol, cholesterol ester and cholesterol contents of the whole serum were 739, 214, 201 and 141 mg dL^?1 respectively. On the basis of total acyl carbon number, the 38C (36%) and 36C (24%) molecules were the predominant phospholipids. The 50C (11%), 52C (17%), 54C (21%), 56C (21%) and 58C (15%) molecules were the major triacylglycerols. High molecular weight apo B‐like proteins were observed in the VLDL, LDL and intermediate density lipoprotein (IDL) fractions. An apo AI‐like protein was predominant in the HDL fraction. The prevalence of enlarged fatty livers in cultured gadoid fish (i.e. haddock and cod) is a major constraint to their commercialization and has been associated with the level of dietary lipid. Information on lipid (triacylglycerol) transport out of the liver through the serum as lipoproteins (VLDL) has implications for our understanding of fatty liver development in cultured gadoid fish. The low level of VLDL triacylglycerol (<12 mg dL^?1) circulating in the serum of postabsorptive haddock suggests either a low level of lipid transport out of the liver as triacylglycerol or relatively rapid turn over of VLDL.     

牡蛎酶解液的抗氧化活性

检测了牡蛎(Crassotera gigas)木瓜蛋白酶和中性蛋白酶酶解液的抗氧化活性。由Sephadex G-15葡聚糖凝胶柱层析分析牡蛎酶解液,得到具抗氧化活性组分的分子量分布。分子量为1191 D和826 D左右的木瓜蛋白酶酶解活性肽组分的自由羟基清除活性最强,当其质量浓度分别为0.184 mg/mL和0.673 mg/mL时,体外自由羟基清除活性分别为53.6%和66.5%;分子量分别为1074 D和735 D左右的中性蛋白酶酶解活性肽的自由羟基清除活性最强,当其质量浓度分别为0.166 mg/mL和0.830 mg/mL时,体外自由羟基清除活性分别57.6%和70.5%。HPLC分析结果表明,木瓜蛋白酶与中性蛋白酶酶解的活性肽组分分别由几种抗氧化肽组成。抗氧化活性最强的木瓜蛋白酶与中性蛋白酶酶解的肽组分在质量浓度为2.5 mg/mL时,活性分别可达83.6%和80.8%。用牡蛎原浆与各酶解液灌胃昆明小白鼠。研究结果表明,用木瓜蛋白酶与中性蛋白酶酶解原液及经超滤纯化的酶解液对小白鼠实施灌胃,其肝脏组织的SOD活力都显著提高(P<0.05);灌胃经超滤纯化的木瓜蛋白酶和中性蛋白酶酶解液,小白鼠肝脏组织GSH-PX活力、GSH含量显著提高(P<0.05),MDA含量显著降低(P<0.05)。