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。     

菲律宾蛤仔酶解产物的抑菌活性

以菲律宾蛤仔为原料,比较其胃蛋白酶、酸性蛋白酶、菠萝蛋白酶、胰蛋白酶和木瓜蛋白酶酶解产物的抑菌活性;利用正交实验优化抑菌活性最强的酶解产物的制备工艺,并测定最优酶解条件下酶解产物的相对分子质量分布。结果表明,菲律宾蛤仔胃蛋白酶酶解产物的抑菌活性最强,对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酿酒酵母和副溶血弧菌均有一定的抑菌活性,其中对金黄色葡萄球菌、枯草芽孢杆菌的抑菌活性较高,对四联微球菌则未见明显的抑菌活性。胃蛋白酶的最优酶解工艺条件为：酶解时间2h,温度35℃,pH 2.0,加酶量3 000U/g,料水比1∶4;该条件下酶解产物的相对分子质量主要分布在5000以下。     

魁蚶蛋白胰蛋白酶酶解产物的抑菌活性

采用胰蛋白酶对魁蚶蛋白进行不同时间的酶解,比较酶解产物的抑菌活性。选取抑菌活性较好的胰蛋白酶酶解产物进行细菌呼吸抑制研究。通过RP-HPLC对该酶解产物进行分离纯化,采用质谱分析其氨基酸序列。结果表明,胰蛋白酶酶解0.5h所得到的酶解产物对金黄色葡萄球菌及希瓦氏菌具有较好的抑菌活性。其抑制金黄色葡萄球菌和希瓦氏菌的HMP途径。RP-HPLC分离纯化得到了4个抑菌活性较好的组分。对其中第2组分进行质谱分析得到3个目标多肽,其m/z分别为679、792及905,将这3个目标多肽导入De novo Explorer(TM)Software(AB SCIEX;Version 4.1)进行分析,获得了候选抑菌肽氨基酸序列。     

鼠尾藻多酚分级组分的抑菌活性研究

采用平板生长抑制法对鼠尾藻Sargassum thunbergiikuntze多酚化合物各分级组分进行抑菌活性研究。结果表明,在一定浓度范围内各分级组分对副溶血弧菌、哈维氏弧菌、沙蚕弧菌、金黄色葡萄球菌、大肠杆菌、鳗弧菌、溶藻弧菌和四联微球菌等受试菌株均有抑菌活性。其中,分级组分I(Mr5.0×103)的抑菌效果优于其他组分,对副溶血弧菌、哈维氏弧菌和沙蚕弧菌的最低抑菌浓度(MIC)值为900μg/ml,对金黄色葡萄球菌、大肠杆菌、鳗弧菌和溶藻弧菌的MIC值为1 800μg/ml,对四联微球菌的MIC值为3 600μg/ml。另外,组分I(Mr5.0×103)显示出较好的热稳定性,在pH 3~4时对受试菌的抑菌活性最强。     

南极磷虾酶解产物中一种含氟多肽的研究

通过酶解法从南极磷虾肌肉酶解产物中分离、纯化含氟多肽,并采用LC-MS/MS和氨基酸序列分析对其结构进行研究。结果表明,采用胰蛋白酶酶解所得南极磷虾多肽粗提物的得率约为20%,其总氟含量为(731.73±1.01)μg·g^-1。粗提物精制后,制备得到纯度较高的6个多肽(分别命名为F1、F2、F3、F4、F5、F6),采用离子色谱法筛选出含氟量最高的多肽F6,测定其结合肽氟含量为(266.11±0.40)μg·g^-1。将F6的氨基酸序列片段通过NCBI-Nucleotide-Euphausiacea蛋白质数据库检索,按匹配值得分高于100分可筛选鉴定得到4种蛋白质[Clock,Tropomyosin,ATP synthase subunit 6(mitochondrion),Arginine kinase],通过对4种蛋白的分析发现,南极磷虾蛋白结合氟通过与Clock蛋白磷酸化结合的可能性最大。研究结果对今后探究南极磷虾中含氟蛋白的安全评价提供了技术支撑,对南极磷虾应用价值的进一步开发具有重要意义。     

南极大磷虾共附生细菌多样性及其防御功能研究#br#

为研究南极大磷虾(Euphausia superba)中共附生微生物的功能性,并探讨南极大磷虾与其相关微生物的相互作用关系,采用宏基因组测序技术分析了南极大磷虾中共附生微生物的物种多样性,同时采用细菌纯培养技术获得可培养菌株,对菌株进行了抑菌活性、耐氟化钠特性分析。宏基因组测序结果表明,在南极大磷虾眼柄、尾部和肠道中,变形菌门(Proteobacteria)的群落相对丰度最高,分别占各组织细菌总量的41.4%、35.8%、59.5%;在南极大磷虾胃部,厚壁菌门(Firmicutes)的相对丰度最高,占细菌总量的32.2%。微生物多样性分析表明,南极大磷虾眼柄和尾部的微生物多样性和丰度高于胃部和肠道组织,肠道和胃部的微生物组成差异较大。采用细菌纯培养技术共获得60株细菌,其中有一株细菌对革兰氏阳性菌[单增李斯特菌LFM2813(Listeria monocytogenes LFM2813)、金黄色葡萄球菌LFM3263(Staphylococcus aureus LFM3263)、蜡样芽孢杆菌LFM2805(Bacillus cereus LFM2805)]有抑菌活性,并鉴定为马胃葡萄球菌(Staphylococcus equorum NJ2),该细菌的抑菌产物具有耐高温和蛋白酶处理后失活的细菌素基本特征;同时,可培养细菌的最大氟化钠耐受浓度为1%,且耐受菌株占比为10%。研究初步揭示了南极大磷虾不同组织的微生物多样性和相对丰度,并探讨了微生物对宿主的防御功能,可为进一步开发利用南极大磷虾资源提供参考。     

酶解工艺优化对南极磷虾蛋白功能特性的影响

南极磷虾是一种资源量巨大,营养价值高的极地海洋生物,其蛋白质含量高,氨基酸组成符合联合国粮农组织(FAO)和世界卫生组织(WHO)规定的优质蛋白标准,是一种具有良好开发利用潜力的优质蛋白。以南极磷虾蛋白为原料,开展酶解对南极磷虾蛋白功能特性(吸水性、吸油性、乳化性和起泡性)的影响研究。南极磷虾蛋白功能特性的评价方法采用食品蛋白功能特性评价国际通用方法。酶解能够改善南极磷虾蛋白的吸油性、乳化性、起泡性及泡沫稳定性,能够靶向性地改善南极磷虾蛋白的功能特性,提高南极磷虾蛋白及其衍生品现实应用的可行性。研究表明,酶解加工能够改善南极磷虾蛋白的功能特性,拓展南极磷虾蛋白的应用领域和范围。     

南极磷虾粉作为鲽形目鱼饲料蛋白源的营养价值评价

分析商品南极磷虾粉、自制酶解磷虾粉的主要营养成分(氨基酸和脂肪酸)组成,同时采用生化方法比较了5种鲽形目鱼肉蛋白的氨基酸组成。以5种鲽形目鱼肉蛋白为参比蛋白,利用必需氨基酸指数、氨基酸比值系数分和关联度分析法评价南极磷虾粉作为鲽形目鱼饲料蛋白源的营养价值,同时对2种磷虾粉的氟含量进行了分析。结果显示,鲽形目鱼肉(干样)中含有16种常见氨基酸,其中,7种为人体必需氨基酸,4种为呈味氨基酸(总量分别为28.52%–38.03%、25.26%–33.56%);5种鲽形目鱼氨基酸组成均符合 FAO/WHO 的理想模式。南极磷虾粉和酶解磷虾粉的粗蛋白分别为60.84%和68.60%,粗脂肪分别为12.08%和10.79%,达到了规定鱼粉的一级品甚至特级品的指标。酶解处理后蛋白含量显著升高(P<0.05),脂肪含量显著下降(P<0.05),灰分含量无差异(P>0.05)。从必需氨基酸指数(EAAI)来看,EAAI 均大于0.95,磷虾粉的必需氨基酸与鲽形目鱼的必需氨基酸拟合度较高。从氨基酸比值系数分(SRC)和关联度分析法来看,2种磷虾粉对于鲽形目鱼的必需氨基酸平衡性较好,相关系数与鱼粉相差不大。氟含量方面,酶解后磷虾粉的氟含量[(331.21±6.70) mg/kg]显著降低(P<0.05),低于欧盟标准(350 mg/kg)。综合来看,2种磷虾粉氨基酸平衡性较好、营养价值较高,是优质蛋白源。     

南极磷虾的特性和利用

南极磷虾是地球上最大的蛋白质资源。由于特殊的生态环境,南极磷虾表现出独特的生物学特征:脂类营养成分含量丰富,富含不饱和脂肪酸、虾青素和磷脂;肌肉蛋白中氨基酸组成全面,配比合理;内源酶种类多、活性高。但南极磷虾肌肉中氟含量高,不能直接供人类食用,必须经过精深加工后才能成为有价值的食品。根据南极磷虾的生物学特征,结合多年的水产品精深加工经验,笔者提出了综合开发利用南极磷虾的思路。     

南极磷虾粉作为鲟鱼饲料蛋白源的营养和氟含量的分析

通过分析商品南极磷虾粉、自制冻干南极磷虾粉和南极磷虾肽粉的主要营养成分和氨基酸含量,以6种鲟鱼肌肉为参比蛋白,利用3种营养评价指标(必需氨基酸指数、氨基酸比值系数分和关联度分析法)评价了这3种磷虾粉作为鲟鱼蛋白质饲料的营养价值;同时对3种南极磷虾粉的氟含量进行了检测分析。结果显示,3种南极磷虾粉平均粗蛋白含量在61.73%～67.13%,粗脂肪含量在3.56%～4.56%,灰分含量在15.56%～17.15%,达到了一级品甚至是特级品的指标。总氨基酸含量达51.46%～58.75%,10种必需氨基酸含量为23.69%～28.51%,三者必需氨基酸总量与粗蛋白的百分比为45.09%～47.94%;呈味氨基酸总量为18.60%～21.19%,占总氨基酸的比例达到了36.07%～36.17%。从必需氨基酸指数方面看,3种南极磷虾粉都为优质的蛋白源。同时从氨基酸比值系数分和关联度分析法,3种南极磷虾粉对于鲟鱼的必需氨基酸平衡性较好,相关系数和鱼粉相差不大,且要高于豆粕。氟含量方面,自制的冷冻冻干南极磷虾粉氟含量最高(2 382.83±112.43 mg.kg-1),其次为商品南极磷虾粉(1 122.73±62.82 mg.kg-1),而经过酶解后南极磷虾肽粉的氟含量(65.19±5.08 mg.kg-1)则显著下降,低于欧盟标准(350 mg.kg-1)。综合来看,3种南极磷虾粉氨基酸平衡性较好,营养价值较高,氟含量可通过一定方法降低,南极磷虾是一种具有较大开发利用前景的优质资源。     

海参水煮液酶解多肽的抗氧化活性

以海参水煮液的冻干粉为原料,选取中性蛋白酶、胃蛋白酶和胰蛋白酶分别酶解制备多肽,测定其对·OH、O2-·和DPPH·自由基的清除能力,并与合成抗氧化剂TBHQ对比.结果表明,3种蛋白酶酶解制得的海参水煮液多肽对·OH、O2-·和DPPH·自由基的清除能力均随样品质量浓度的升高而增强;其中,中性蛋白酶酶解制得的海参水煮液多肽清除·OH、O2-·和DPPH·自由基的IC50值依次为8.565、6.658和2.015mg/ml,其对O2·的清除能力优于TBHQ.经液相色谱法测定,中性蛋白酶、胃蛋白酶和胰蛋白酶酶解制得的海参水煮液多肽分子量分别分布在＜2 500、＜3 500和＜1 000 D的小分子肽中.     

对虾组织膜蛋白制备方法及其多肽组成

选取凡纳缤对虾(Litopenaeus vannamei)的4种组织(鳃、血淋巴、肝胰腺、肌肉)，着重对膜蛋白的提取方法及其多肽组成进行分析。在对虾组织膜蛋白提取过程中，使用了5种蛋白酶抑制剂(苯甲基磺酰氟PMSF、胃酶抑制剂、亮抑酶肽、胰凝乳蛋白酶抑制剂、胰蛋白酶抑制剂)，以减少组织自身的酶将蛋白降解的可能。电镜观察，可见纯度较高的、细胞膜结构卷曲形成的小泡样、封闭的结构。使用Gel-Pro软件对提取产物的SDSPAGE图谱分析，确定了这几种组织的特征多肽分子量分别为75．0kD、70．5kD、26．7kD和71．2kD。     

Evaluation of the bioactivities of water-soluble extracts from twelve deep-sea jellyfish species

Many polypeptides isolated from shallow water cnidarian species have been utilized as valuable biochemical tools in both basic and applied biological sciences. Deepwater cnidarian species might be another potential resource for novel biochemical tools. However, because of limited access to cnidarian samples from deep-sea environments, bioactive polypeptides have never before been reported from this group. In this study, we collected twelve deep-sea jellyfish species (nine hydrozoans and three scyphozoans) using a plankton net that was specially designed for collecting deep-sea organisms, and prepared water-soluble extracts, presumably containing polypeptides, of these jellyfishes. The extracts were subjected to cytotoxicity, hemolytic activity, and crustacean lethal toxicity tests. In the cytotoxicity test, six out of the nine tested hydrozoan species showed activity. In the hemolytic activity test, only three hydrozoans showed activity and none of the scyphozoan jellyfishes showed activity. In the crustacean lethality test, two hydrozoan jellyfishes and all three of the tested scyphozoan jellyfishes showed lethal activity. These results revealed a high incidence of water-soluble bioactive substances occurring in these deep-sea jellyfishes. Furthermore, all the heat-treated and the methanol-treated crude jellyfish extracts lost their bioactivities. Thus, it is likely that the bioactive compounds in the water-soluble extracts were unstable polypeptides (proteins). This is the first published report on bioactivities in extracts from deep-sea jellyfishes.     

Protein content and freezing avoidance properties of the subdermal extracellular matrix and serum of the Antarctic snailfish,Paraliparis devriesi

The Antarctic snailfish, Paraliparis devriesi (Liparididae), occupies an epibenthic habitat at a depth of 500–650 m in the subzero waters of McMurdo Sound, Antarctica. This species has watery (97%) gelatinous subdermal extracellular matrix (SECM) comprising a mean of 33.8% of the body weight, the largest known proportion of any adult fish. The protein concentration of the SECM was found to be 6–7 mg ml^–1 (0.6–0.7% w/v). Separation of the polypeptides of the SECM by SDS-PAGE revealed 11 polypeptides ranging in relative molecular mass (M_r) from 67,000 to 13,000, with other unresolved polypeptides of less than 13,000. The isoelectric points of these proteins ranged from 4.85 to 8.05. Partial N-terminal amino acid sequence data were obtained for four of the major SECM polypeptides. The N-terminal amino acid sequences of three of these were not identical to or homologous with any other known sequences, whereas the N-terminal sequence of one polypeptide (M_r 51,000) was identical to partial sequence from the apolipoprotein A-I precursor of Atlantic salmon (Salmo salar). Although not isolated from either SECM or serum, melting point-freezing point behavior of body fluids suggest that Paraliparis possess modest amounts of a noncolligative antifreeze compound. Since relatively small amounts of antifreeze are present in the serum and even less in the SECM, freezing avoidance results from the combined effects of antifreeze and the elevated osmolality of body fluids. There are no special adaptations to prevent freezing in the superficially located high water content SECM.     

Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos

Our earlier studies indicated the high expression of a UV-damaged-DNA binding activity in zebrafish (Danio rerio) embryos at 12?h postfertilization (hpf). Two 30- to 35-kDa polypeptides homologous to the N-terminal lipovitellin 1 (Lv1) domain of the 150-kDa zebrafish vitellogenin 1 (zfVg1) were identified as the damage recognition factors in zebrafish extracts, and the metal-chelating agent 1,10-phenanthroline (OP) was found to inhibit the embryonic UV-damaged-DNA binding activity. This study further explored the DNA damage-sensing components in 12 hpf zebrafish extracts. UV-damaged-DNA binding proteins were enriched from zebrafish extracts by isoelectrofocusing. Both OP-sensitive and OP-stimulated, UV-damaged-DNA binding activities were detected in fractionated zebrafish extracts. Two-dimensional gel electrophoresis of proteins captured by an immobilized oligonucleotide carrying a UV-induced (6-4)photoproduct (6-4PP) revealed a 25-kDa polypeptide as the major 6-4PP-binding factor in an OP-stimulated fraction. Three 25-kDa factors that bound weakly to 6-4PPs were also isolated. The four polypeptides having pIs between 7.0 and 7.3 were unreactive to an anti-zfVg1 antibody targeting the Lv1 domain. Mass spectral analysis showed the appearance of amino acid sequences LPIIVTTYAK and IPEITMSK in all 25-kDa polypeptides and sequences exactly matching those contained in the four factors exist only in the C-terminal Lv2 domain of zfVg1, reflecting the origination of these factors from enzymatic cleavage of the Lv2 domain at slightly different positions. The OP-stimulated fraction produced a much stronger UV-dependent DNA incision activity in the presence than in the absence of OP, suggesting the association of these factors with DNA damage repair under metal-deficient conditions.     

海产品蛋白酶水解多肽研究进展

对海产品蛋白质进行脱脂、酶水解并进一步分离纯化,制备生物功能活性多肽。生产出的海产品水解多肽具有降血压、抗氧化、抗肿瘤、提高机体免疫力等多种生物活性。综述海洋鱼、贝类水解活性肽的制备流程以及生物活性功能。     

Antifreeze proteins in the urine of marine fish

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6–33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.     

Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) skin and gill

Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 μg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.     

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases XbaI, KpnI and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism.     

刺参肠、性腺酶解多肽体外抗氧化作用研究

刺参（Apostichopus japonicus）肠和性腺是刺参加工过程中的副产物,为丰富其高值化利用的基础理论,研究了刺参肠、性腺酶解过程中可溶性蛋白和氨基酸态氮质量浓度变化,分析了酶解多肽对11-二苯基苦基苯肼（DPPH·）、羟基自由基（·OH）和超氧阴离子自由基（O2^-·）的体外清除效果。结果显示,刺参肠和性腺经生物酶水解后,水解度分别为53.63%和63.40%,酶解液中可溶性蛋白质量浓度分别为4.62 mg·mL-1和5.01 mg·mL-1,氨基酸态氮质量浓度分别为0.43 mg·mL-1和0.56 mg·mL-1。刺参肠和刺参性腺酶解多肽清除DPPH·的半抑制质量浓度（IC50）分别为3.31 mg·mL-1和0.88 mg·mL-1,清除·OH的IC50分别为9.53 mg·mL-1和8.81 mg·mL-1,清除O-2·的IC50分别为6.42 mg·mL-1和3.22 mg·mL-1。刺参肠和刺参性腺酶解多肽具有一定的体外抗氧化效果,应用前景广阔。