施氏鲟卵黄蛋白的分离纯化及其性质

采用组织匀浆、饱和硫酸铵分步沉淀和Sephadex G-100葡聚糖凝胶层析的方法提取和纯化施氏鲟卵黄蛋白,并对其性质进行了研究。结果表明:采用50%和70%饱和度的硫酸铵分步沉淀与Sephadex G-100凝胶层析相结合的方法,可以获得一种卵黄蛋白纯品。Native-PAGE和SDS-PAGE分析表明,该纯化蛋白纯度为100%,由一种同源亚基组成,其亚基的相对分子质量约为30 kD。油红O、免疫印迹(Western-blot)和罗丹明B染色均为阳性,表明该蛋白为施氏鲟卵中的一种脂磷蛋白。     

牙鲆皮肤黏液免疫球蛋白的纯化及部分特性分析

采用饱和硫酸铵分步盐析结合Sephacryl S-300凝胶过滤层析及DEAE Sepharose离子层析法,对牙鲆皮肤黏液中的免疫球蛋白(Ig)进行了分离纯化,并通过SDS-PAGE及Western-blotting技术对纯化蛋白的部分特性进行了分析。结果表明,30%、50%饱和硫酸铵分步盐析可以去除牙鲆黏液中除Ig外的很多杂蛋白,得到粗提黏液Ig;再经Sephacryl S-300纯化,Ig纯度较高,其中含有72和26 ku的条带;DEAE Sepharose层析法可进一步纯化Sephacryl S-300柱层析的产物,所提取黏液Ig经SDS-PAGE检测,只含有72和26 ku两个条带,初步认为是牙鲆黏液Ig的重链和轻链。Western-blotting结果显示,抗牙鲆血清Ig单克隆抗体可与黏液Ig重链72 ku条带发生发应。     

花鲈免疫球蛋白的分离纯化及部分特性分析

分离纯化了花鲈(Lateolabrax japonicus)血清免疫球蛋白(Ig),并对其特性进行初步研究与分析.采用山羊-IgG(Goat-IgG)免疫花鲈并制备血清,分别采用硫酸铵分级沉淀法、A蛋白亲和层析法及Goat-IgG偶联亲和层析法提取花鲈Ig,对提取组份进行了蛋白浓度测定、间接ELISA效价测定、SDS-PAGE和Western-blotting分析.结果表明,采用硫酸铵分级沉淀法提取的花鲈Ig主要集中在硫酸铵饱和度为30%～50%的区间,其中45%饱和度硫酸铵富集的的抗体比活最高,ELISA效价为2×104/mg蛋白,比血清ELISA效价提高2.2倍;A蛋白柱亲和层析可得单一蛋白峰,洗脱峰集中在洗脱体积0.8～1.6 mL,处,占洗脱蛋白总量的76.2%,峰值的ELISA效价为1.37×105/mg蛋白,抗体得率为2.57%;Goat-IgG偶联Sephrose 4B亲和柱层析也可得单一蛋白峰,洗脱峰集中在洗脱体积1.4～2.8 mL处,占洗脱蛋白总量的72.4%,峰值的ELISA效价为1.39×105/mg蛋白,抗体得率为2.63%.以上3种提取方法均可得到79 kD的重链和29 kD轻链,表明可得到纯化的免疫球蛋白;采用鼠抗花鲈Ig血清和单抗AA5经Western-blotting证实提取成份为花鲈Ig.本研究表明,A蛋白柱和Goat-IgG偶联柱亲和层析均可用于花鲈Ig的提取,其中A蛋白柱法获得的洗脱峰值更为集中,抗体效价高,且提取过程不需要Goat-IgG免疫鱼体,是一种更为有效的免疫球蛋白提取方法.     

条斑紫菜蛋白酶解多肽的抑菌活性

以条斑紫菜为原料,分别提取水溶、酸溶、碱溶、盐溶性蛋白质,并用6种蛋白酶酶解,将所得24种酶解物采用琼脂孔穴扩散法对金黄色葡萄球菌(Staphylococcus aureus)、四联微球菌(Micrococcus tetragenus)、枯草杆菌(Bacillus subtilis)以及大肠杆菌(Escherichia coli)4种菌进行抑菌活性测定。结果显示,水溶性胃蛋白酶酶解物的抑菌活性最优。将水溶性蛋白质经硫酸铵沉淀以及超滤分级,比较不同饱和度硫酸铵沉淀所得蛋白质酶解物以及不同分子量多肽的抑菌效果。结果显示,水溶性蛋白在硫酸铵饱和度为40%、50%时沉淀所得蛋白的胃蛋白酶酶解物中,相对分子质量小于5 k Da的级分抑菌效果最为明显。此外,对该级分进行了抑菌活性影响因素的初步研究,结果表明该级分热稳定性以及酸碱稳定性均较好,有望开发为天然的食品防腐剂。     

远洋金枪鱼钓船的超低温制冷系统

1　超低温的必要性金枪鱼是一种高蛋白、低脂肪的大型鱼类 ,大部分出口至日本作生鱼片或罐头。加工生鱼片要求鱼肉非常新鲜 ,其程度主要表现在色泽上。新鲜的金枪鱼肉为鲜红色 ,如果保存不当就会发生褐变。褐变的主要原因是鱼肉中肌红蛋白呈鲜红色 ,发生氧化成为氧化肌红蛋白时呈褐色 ,变色程度与氧化肌红蛋白生成率相关。例如当氧化肌红蛋白占全部肌红蛋白的 2 0 %以下时 ,则鱼肉呈现鲜红色 ,当氧化肌红蛋白占 30 %时呈暗红色 ,占5 0 %时呈褐红色 ,占 70 %以上时呈褐色 ,其口味和商品价值大幅度下降。在一般的冻结冷藏温度下 (如 - 2 3℃ ,…     

光裸星虫对盐度和温度的耐受性研究

试验结果表明,(1)盐度从20突变为5时,其成活率为60%;盐度由20突变至50时,成活率为0.求得光裸星虫96 h半致死盐度上限为41.7.(2)盐度由25每隔12 h上升5至盐度50时,其成活率由100%降至60%,盐度逐渐由25降至5,光裸星虫成活率由100%降至85%.(3)当水温由25 ℃急骤降至20、15、10、5 ℃时,光裸星虫的成活率依次为100%、100%、100%、70%;当水温由25 ℃急骤升至30 ℃时,光裸星虫的成活率仅为10%;当突变温度急骤升至35 ℃时,其成活率为0.求得其96 h急性半致死温度上限为27.5 ℃.(4)水温由22 ℃每隔12 h逐渐升至25、30、35、40 ℃时,光裸星虫成活率下降幅度不大,分别为100%、100%、85%、85%.水温由22 ℃每隔12 h逐渐降至20、15、10、5 ℃时,其成活率均为100%.根据试验结果可知,光裸星虫属广温、广盐种.     

鳙鱼肉中土腥昧物质的测定方法

本研究通过微波蒸馏提取、固相微萃取(SPME)富集和气相色谱-质谱联用(GC/MS)分析的方法,实现了对鳙(Aristichthys nobilis)鱼肉中土腥味物质的定量测定.实验对微波蒸馏提取、SPME法富集鱼肉中土腥味物质的条件进行了优化,确定最佳条件为微波功率350W,10g样品蒸馏时间为8min,氮气流速60mL/min;萃取时间30min,萃取温度60℃,NaCl加入量4:1(V:W),搅拌速度1 500 r/min.结果表明,使用SPME富集土味素(Geosmin)时,回收率为95.4%.检测限为1.0 ng/L,线性范围为5～100 ng/L;采用优化后的微波蒸馏-SPME-GC/MS方法测定鳙鱼肉中的土味素时,回收率为57%;经该法测定10月份青岛市场市售鳙鱼肉中土味素平均含量为5.4μg/L.     

用鼠红细胞膜纯化中华绒螯蟹血清凝集素的方法

本研究建立鼠红细胞膜吸附法纯化中华绒螯蟹(Eriocheir sinensis)血清凝集素的方法,并与硫酸铵盐析、凝胶过滤层析和离子交换层析等其他提取方法进行比较。实验结果表明,中华绒螯蟹血清经鼠红细胞膜吸附后,再由EDTA把结合的凝集素从膜上解离,可以获得纯化的高活性凝集素。该纯化物在PAGE中出现2个清晰的区带,一个是大分子的b带,另一个则是由3种大小相近的小分子组成的c区带;在SDS-PAGE中,则显示出3个分子量接近、约为100 000的条带。经50％饱和硫酸铵盐析或用Sephadex G200凝胶过滤层析法可浓缩但不能纯化中华绒螯蟹血清凝集素。经DEAE-Sephadex A50离子交换层析法可获得2个蛋白峰,其中只有1个峰有凝集活性,在PAGE中表现为a蛋白带。凝集活性较高、分子量较大的蛋白带。上述实验结果表明,应用鼠红细胞膜法可纯化中华绒螯蟹血清凝集素。     

pH调节法分离凡纳滨对虾壳中β虾青蛋白的研究

采用pH调节法对凡纳滨对虾（Litopenaeus vannamei）虾壳中影响虾壳红变的β虾青蛋白进行分离回收,以蛋白质回收率、纯度和二级结构含量为分离特性参数,以1.0为pH变化梯度,研究pH调节法从虾壳中提取β虾青蛋白的规律。结果显示,pH调节法的回收率为47.5%,所得蛋白质纯度为78.23%、分子量为45 000 Da。该蛋白质在pH3.0和11.0时有最大溶解度,分别为60.5%和55.7%;在pH5.0时溶解度最低为15.4%;等电点为5.6。通过圆二色光谱图分析得知β虾青蛋白是一种以α螺旋为主要二级结构存在的物质,pH调节法所得α螺旋含量为70.1%。     

泥鳅体内凡隆气单胞菌拮抗细菌CMA 1的分离筛选及其抑菌活性

本研究从泥鳅养殖水体、底泥、健康泥鳅和患病泥鳅肠道等环境中采集样品,从中分离纯化出对凡隆气单胞菌具有拮抗作用的细菌57株.在TSB平板上采用牛津杯法进行体外拮抗实验,筛选出一株对凡隆气单胞菌具有明显拮抗作用的菌株CMA-1,经形态观察、生理生化鉴定和16SrDNA序列系统发育分析,将该菌鉴定并命名为Bacillus methylotrophicus CMA-1.菌株CMA-1生长24 h后进入稳定期,36 h时发酵上清液抑菌活性达到最大.对菌株CMA-1的发酵上清液采用硫酸铵分级沉淀,测得75％硫酸铵饱和度下的沉淀粗提物抑菌活性最高.     

Molecular characterization of southern bluefin tuna myoglobin (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

The primary structure of southern bluefin tuna Thunnus maccoyii Mb has been elucidated by molecular cloning techniques. The cDNA of this tuna encoding Mb contained 776 nucleotides, with an open reading frame of 444 nucleotides encoding 147 amino acids. The nucleotide sequence of the coding region was identical to those of other bluefin tunas (T. thynnus and T. orientalis), thus giving the same amino acid sequences. Based on the deduced amino acid sequence, bioinformatic analysis was performed including phylogenic tree, hydropathy plot and homology modeling. In order to investigate the autoxidation profiles, the isolation of Mb was performed from the dark muscle. The water soluble fraction was subjected to ammonium sulfate fractionation (60–90 % saturation) followed by preparative gel electrophoresis. Autoxidation profiles of Mb were delineated at pH 5.6, 6.5 and 7.4 at temperature 37 °C. The autoxidation rate of tuna Mb was slightly higher than that of horse Mb at all pH examined. These results revealed that tuna myoglobin was unstable than that of horse Mb mainly at acidic pH.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.     

Effects of acid and alkaline pretreatment on the discoloration rates of dark muscle and myoglobin extract of skinned tilapia fillet during iced storage

Discoloration profiles of dark muscle of skinned tilapia fillets were examined during iced storage after pretreatment with lactic acid (LA) or sodium carbonate (SC). During the subsequent storage, the a* values decreased gradually, and changed more rapidly when the pH was lower than 6.3. The fillet pretreated with 10% (v/v) LA exhibited the highest metmyoglobin formation ratio (MetMb%), followed by the fillet pretreated with 5% (v/v) LA, the control fillet, and the fillet pretreated with 10% (w/v) SC. The sample pretreated with 10% LA showed a marked decrease in the a* value. Discoloration of the control was not observed until the ninth day of iced storage, and no discoloration was observed up to the 11th day for the fillet pretreated with 10% SC. These fillet discoloration profiles were subsequently verified using the myoglobin (Mb) fraction prepared from the dark muscle. MetMb% of the Mb fraction gradually increased during storage, and this increase accelerated at pH values of <6.3. Discoloration of the Mb fraction also showed a similar tendency, and no significant discoloration was observed at pH values of >6.5. These results suggest that pH greatly affects the discoloration rate of the dark muscle of skinned fillet, and the critical pH for the accelerated autooxidation of tilapia Mb is in the range 6.3–6.5.     

Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish

ABSTRACT:   In the present study, the cDNA encoding myoglobin (Mb) of bigeye tuna Thunnus obesus was cloned and its amino acid sequence deduced in order to investigate the relationship between the primary structure and thermostability of scombridae fish Mb. An open reading frame of bigeye tuna Mb cDNA contained 444 nucleotides encoding 147 amino acids. The primary structure of bigeye tuna Mb was highly conserved when compared with those of bluefin tuna and yellowfin tuna Mb, the sequence identity being 95.2–100.0%. It also showed relatively high identity (82.3–89.1%) with the counterparts of scombridae fish. Myoglobin was then isolated from the dark muscle of four scombridae fish including bigeye tuna. Differential scanning calorimetry and circular dichroism measurements on these Mb revealed that the thermostability of bigeye tuna Mb was lowest and that of skipjack Katsuwonus pelamis Mb highest among the scombridae fish Mb examined. The α-helical contents of scombridae fish Mb at 10°C were in the range of 39.8–44.8%, clearly lower than that of horse Mb (55.3%), suggesting instability of fish Mb. The melting temperatures of these Mb fell in the range of 75.7–79.9°C, lower than that of horse Mb (84.2°C). These results strongly suggest the instability of fish Mb.     

Suppressive effect of ATP on autoxidation of tuna oxymyoglobin to metmyoglobin

The discoloration of tuna meat proceeds during frozen storage at around ?20 °C. On the other hand, the discoloration of highly fresh tuna meat could effectively be suppressed even if stored at ?20 °C. However, the suppressive mechanism of the discoloration is not well understood. Here the effects of ATP on the autoxidation rate and molecular structure of tuna myoglobin are reported. The autoxidation rate of southern bluefin tuna Thunnus maccoyii myoglobin at 25 °C was suppressed in the presence of ATP especially in acidic pH range. Mixing ATP with myoglobin induced a spectral perturbation in the soret region of myoglobin. This spectral perturbation was observed as a function of the ATP concentration. Quenching of myoglobin fluorescence was also caused by ATP, saturating at around 0.5 mM ATP. According to dynamic light-scattering measurements, the molecular weights of tuna Mb changed from 15.5 to 11.3 kDa with ATP and zeta-potential measurements gave also a negative surface charge without ATP and a positive one with ATP, respectively. The above results indicate that ATP-induces changes in the conformational structure of myoglobin. The effects of ATP on myoglobin could thus provide a possible mechanism to regulate the autoxidation of myoglobin.     

pH- and temperature-dependent denaturation profiles of tuna myoglobin

Attempts have been made to elucidate the denaturation profiles of tuna myoglobin (Mb) in comparison with horse Mb. Intensive absorbance characterization was carried out for derivatives (deoxy, oxy, met forms) of Mb. The wavelength of the maximum absorbance of tuna Mb was shorter only by 1 nm for deoxy and metMb, while it was 4 nm shorter for the second peak of metMb. Percentage Mb denaturation (PMD) was measured under a combination of pH (5.6, 6.5, 7.4) and temperature (70, 75, 80 °C). Tuna Mb was almost completely denatured even during the initial incubation at 75 and 80 °C at all pHs examined. During the incubation at 70 °C, the PMD values for tuna Mb were 88.5, 52.1, and 67.7% at pH 5.6, 6.5, and 7.4, respectively. The denaturation of tuna Mb proceeded even at 55 °C, but denaturation rates were very slow at pH 6.5. On the other hand, horse Mb was found to be very stable at pH 6.5 and 7.4 at all temperatures examined, except at pH 6.5 and 80 °C. At pH 5.6, the PMD values of horse Mb gradually increased, especially at 75 and 80 °C. These different Mbs showed quite different denaturation profiles.     

Expression and characterization of rainbow trout Oncorhynchus mykiss recombinant myoglobin

Fish Physiology and Biochemistry - Recombinant expression system was established for rainbow trout myoglobin (Mb) considering its unique primary structure of having one unusual deletion and two...     

欧洲鳗血清免疫球蛋白纯化及部分特性分析

采用饱和硫酸铵分步盐析法结合柱层析提取，纯化欧洲鳗血清免疫球蛋白（Ｉg) ,实验证实欧洲鳗Ｉg主要分布在硫酸铵饱和度３０％－５０％的区间内，Ｓephacry1-S200进一步提纯的Ｉg主要存在于第一个蛋白峰，而Ｓepharose-4B柱层析提纯的Ｉg则存在于第二个蛋白峰，ＤＥＡE-52脑子交换柱层析可进一步纯化Ｓepharose-4B柱层析的产物，ＥＬＩＳＡ和Western-blot分别证实上述提取物具有抗体的免疫学活性，ＳＤＳ－ＰＡＥＧ分析纯化的Ｉg，显示了洲鳗Ｉg重链约为６８kD，轻链约为２６kD.     

Myosin and actin denaturation in frozen stored kuruma prawn Marsupenaeus japonicus myofibrils

Myosin and actin denaturation in kuruma prawn myofibrils stored frozen (0.1 M NaCl, pH 7.5) at ?20 °C was investigated. The inactivation profile of Ca²⁺-ATPase in the myofibrils was identical to that for myosin, indicating that myosin in myofibrils was not protected by actin. The presence of myosin detached from actin in the soluble fraction was proven by ammonium sulfate fractionation in the absence and presence of Mg-ATP. Actin denaturation in myofibrils was further confirmed by its increased susceptibility to chymotryptic degradation. In the frozen myofibrils, actin denatured more rapidly quicker than myosin: actin had completely denatured by storage day 1, followed by a gradual denaturation of myosin. Both myosin and actin in the frozen stored myofibrils retained their high salt-solubility, which decreased slowly during the frozen storage period. The presence of aggregated inactivated myosin in the salt-soluble fraction was proven by precipitation at 40 % saturation of ammonium sulfate in the presence of Mg-ATP, leaving active monomeric myosin in the soluble fraction. Almost no actin denaturation was observed with heated myofibrils.