Structure elucidation and in vitro rat intestinal fermentation properties of a novel sulfated glucogalactan from Porphyra haitanensis

This study was to investigate the structure and rat fecal microbial fermentation properties of a polysaccharide fraction(PHP2)isolated from the red marine alga Porphyra haitanensis.PHP2 was characterized as a sulfated glucogalactan,with a hypothetical backbone structure of→4)Gα(1→6)G4 Sβ(1→4)Glc(1→and a side chain of Man(1→6)Glc.PHP2 had an irregular spherical chain conformation.The 16 S r RNA sequence analysis revealed that PHP2 modulated the rat fecal micro-flora composition,with a similar effect to inulin,changing the dominant genus(Lactobacillus and Escherichia-Shigella)and promoting the growth of organisms that degrade sulfur-containing polysaccharides,such as Desulfovibrio,Ruminococcaceae_UCG-005,and Ruminococcus_2.PHP2 can promote production of acetic,propionic and butyric acid by rat fecal micro-flora.Prediction of metabolic function suggested that PHP2 could modulate cholesterol metabolism.The sulfated glucogalactan fermentation behavior may be associated with its monosaccharide composition,chain branching and chain conformation.PHP2 appeared to have considerable potential as functional food,and was associated with sulfur-containing polysaccharides in general.

Porphyra haitanensis polysaccharide(PH)attenuates cell hyperplasia via remodeling the cross-talk between Hippo/YAP and mTOR pathways

Porphyra polysaccharide is a special kind of nutrient showing multiple physiological functions including regulating cell proliferation,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to purify and characterize the Porphyra haitanensis polysaccharide(PH),investigate its physiological function,and demonstrate the underlying mechanisms.The purified PH was first characterized by Fourier-transform infrared spectroscopy.Then an intestinal epithelial cell model was established,in which PH significantly suppressed cell hyperplasia.Specifically,PH activated the Hippo/YAP pathway,which subsequently activated m TOR pathway,however m TOR activated Hippo in the absence of PH.Moreover,both the inhibition of Hippo by YAP1 knock-down and the suppression of m TOR by rapamycin impaired PH function.These results indicated that PH attenuated hyperplasia activity by remodeling the cross-talk between Hippo/YAP and m TOR pathways,which revealed potential targets and approaches for treating hyperplasia-related diseases and provided novel ways to utilize P.haitanensis as well as other related functional foods.

坛紫菜(Porphyra haitanensis)丝状孢子体EST的获取及其生物信息学分析

采用改进的一步法提取坛紫菜丝状孢子体的总RNA,构建了坛紫菜丝状孢子体的cDNA文库,对测序得到的EST进行分析并与条斑紫菜的EST相比较。结果表明,cDNA文库共包含2.5×105个克隆,测序得到490个EST,其中275个与已知功能基因同源,占56.1%,260个EST与已知的条斑紫菜EST同源,占53.0%。使用GO(Gene Ontology)分类方法对275个EST进行分类,其中226个可归属于细胞组分(Cellular component)、分子功能(Molecular function)和生物学过程(Biological process)三个大类,而其余49个则为未知功能的EST。挑选出与条斑紫菜同源性高的28个EST,这些EST所包括的密码子数为3221个,发现密码子第三位的平均GC含量远高于第一位的平均GC含量和第二位的平均GC含量。统计坛紫菜丝状孢子体3221个密码子的使用频率,发现同义密码子第三位碱基偏好使用G或C。

坛紫菜(Porphyrahaitanensis)自由丝状体的胶囊化冰冻保存

在适宜孢子囊枝发育的条件下培养坛紫菜自由丝状体 ,用 3 %褐藻酸钙包埋 (胶囊化 )制成胶球 ,经脱水后直接投入液氮中冰冻保存 .结果表明 ,胶囊化的坛紫菜丝状体在 10 h的脱水过程中可保持约 90 %的存活率 .当胶球含水量下降至 42 .9%～ 16.6%时 ,冰冻保存存活率可达 65 %左右 .与常规的两步法冰冻保存相比 ,胶囊化方法可大大简化操作程序 ,是保存坛紫菜自由丝状体的一种较为理想的方法 ,在藻类种质保存中有广阔的应用前景 .

坛紫菜(Porphyra haitanensis)叶绿体DNA的快速提取

以阴干的坛紫菜叶状体为材料,通过酶解等方法获取完整的叶绿体,再裂解叶绿体并酚仿抽提获得叶绿体DNA.限制性内切酶HindⅢ和EcoRⅠ双酶切及RAPD检测结果显示:叶绿体DNA与基因组DNA的酶切图谱及RAPD图谱均有比较明显的差异.多次重复实验证明,按此法提取的叶绿体DNA,其得率稳定,并可用作PCR扩增的模板及酶切图谱的构建等.该方法操作简便,过程快捷,可作为大型海藻叶绿体DNA提取的参考方法.

坛紫菜(Porphyra haitanensis)丝状藻体生长增殖的优化调控培养条件研究

丝状藻体是紫菜生活史中的重要阶段,其生长增殖与环境条件密切相关.本研究考察了与坛紫菜丝状藻体生长增殖相关的多个影响因子,探讨了可促进坛紫菜丝状藻体生长、达到生长速率快的优化调控培养条件.具体操作实施过程为:初始丝状藻体来自成熟紫菜叶状体的果孢子释放,基于8因子3水平的正交实验设计方案[采用正交表L27(313)],确定了丝状藻体生长对多个环境因子的响应以及优化的调控培养条件.结果表明,除开pH、复合维生素浓度和NaHCO3浓度外,其他因子对丝状藻体生长均有显著的影响(p≤0.043 8).获得的优化调控培养条件为:温度18℃、光强27μmol/m2·s-1、盐度25、pH值8.5、复合维生素浓度15μg/dm3、NaHCO3浓度2 mg/dm3、每天光照时数18 h、营养液浓度为f培养介质.优化调控培养条件下的丝状藻体生长速率高达12.07%,平均值±标准偏差SD为(10.70±0.88)%/d.

坛紫菜(Porphyra haitanensis)化学成分的研究

利用硅胶色谱、Sephadex LH-20凝胶色谱、HPLC等技术,从坛紫菜(Porphyra haitanensis)乙醇提取物首次分离纯化得到了7个化合物,通过质谱(MS)及核磁共振波谱(1H NMR、13C NMR)等现代波谱技术鉴定为:1,8-二羟基-9,10-蒽醌(1)、邻苯二甲酸二正丁酯(2)、邻苯二甲酸二异丁酯(3)、β-谷甾醇(4)、鲨肝醇(5)、(E)-N-2(1,3-二羟基-4-十八烯基)-十六酰胺(6)、胆甾醇(7)。其中,化合物1对金黄色葡萄球菌(Staphyloccocus aureus)的最低抑菌浓度(MIC)为80μg/mL。

大气CO_2浓度升高缓解高浓度NH_4^+对坛紫菜(Porphyra haitanensis)生理特性的胁迫作用

研究不同CO2浓度与NH4+浓度交互作用对坛紫菜(Porphyra haitanensis)生长与光合作用的影响。经过7 d培养,结果表明120 μmol·L-1 NH4+浓度最有利于坛紫菜的生长,此时坛紫菜的光合色素含量、最大光化学量子产量(Fv/Fm),最大相对电子传递速率(r ETRm)也最高;同时,升高的CO2浓度促进了坛紫菜的生长与光合作用。NH4+浓度高于120μmol·L-1,坛紫菜的生长随NH4+浓度增高而降低;坛紫菜的光合活性也受到抑制。但是,在高CO2浓度培养条件下,坛紫菜生长与光合活性随NH4+浓度增高而降低的速率变慢。本研究表明,大气CO2浓度升高能缓解高浓度NH4+对坛紫菜所造成的生理胁迫作用。

坛紫菜(Porphyra haitanensis)叶状体细胞对4种有机溶剂的敏感性比较

分析了坛紫菜叶状体细胞对甲醇、乙醇、异丙醇、1, 3 丙二醇等4种有机溶剂的敏感性.结果表明:体细胞对1, 3 丙二醇敏感,在实验最低剂量(0. 2μL/mL)下对紫菜体细胞就表现出较强的致死作用;对甲醇有较强的耐受性,在最高的1. 0μL/mL处理浓度组,生长发育状况与对照组无明显差别;乙醇在浓度高于0. 6μL/mL时强烈抑制体细胞的分裂;异丙醇随着浓度的升高表现出使体细胞向细胞团方向分化,而叶状体的形成则受到抑制.本实验结果表明,在用外源化合物处理紫菜叶状体细胞时,甲醇在1. 0μL/mL浓度范围内可作为合适的有机溶剂.

Sequence analysis of the ITS region and 5.8S rDNA of Porphyra haitanensis

The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8S rDNA were 158 bp and the sequences of ITS2 ranged from 673 bp to 681 bp.The sequences of the ITS had a high level of homology(up to 99.5%) with that of P.haitanensis(DQ662228) retrieved from GenBank,but were only approximately 50% homologous with those of other species of Porphyra.The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis.These results suggest that the three cultivated strains of P.haitanensis evolved conservatively and that the ITS showed evolutionary consistency.However,the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations.Therefore,the relationship of Porphyra interspecies phyletic evolution could be judged,which provides the proof for Porphyra identification study.However,proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

In vitro Antioxidant Effects of Porphyra haitanensis Peptides on H_2O_2-Induced Damage in HepG2 Cells

In this study,protein from Porphyra haitanensis was used as raw material to prepare an antioxidant peptide,and its antioxidant activity was evaluated in vitro.A model of H_2O_2-induced oxidative damage in Hep G2 cells was established,and the effects of Porphyra haitanensis hydrolysates (PHHs) on superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected.Finally,the structure of PHHs was identified by ESI-MS/MS.The results showed that the 1,1-diphenyl-2-pyridylhydrazine(DPPH)-free radical-scavenging ability of PHHs was the strongest (59.28%at 1.0 mg m L~(-1)) when hydrolyzed with an acidic protease for 4 h.PHHs with different concentrations had protective effects on H_2O_2-induced damage to Hep G2 cells,and the protective effect was enhanced with increasing concentrations.When the level was 400μg m L~(-1),the cell survival rate was as high as 88.62%.Moreover,PHHs can significantly reduce oxidative damage to Hep G2 cells by H_2O_2,improve SOD activity,and reduce MDA content.The tetrapeptide Asp-Lys-Ser-Thr,with a molecular weight of 448 Da,was identified as an important fraction of PHHs by high-resolution mass spectrometry.

SUBUNIT COMPOSITION AND CHROMOPHORE CONTENT OF R-PHYCOERYTHRIN FROM PORPHYRA HAITANENSIS

R-phycoerythrin from Porphyra haitanensis exists in two aggregaion states with different molecularweights. A more highly aggregated form, RPE I,Was chromatographed on Bio-Rex 70 column with ureasolution （pH 3.0） as eluent, and the molecular weights of the 3 subunits （α,β,γ） obtained were determinedon SDS-PAGE at 18000, 19200 and 30000, respatively. α subunit carried two phycoerythrobilin （PEB）,β subunit, three PEB and one phycourobilin （PUB）,γ subunit, one PEB and three PUB chromophores.The molar ratio of α,β, and γ subunits of RPE I wn 6:6:1, and their subunit composition was con-fired to be （αβ）6γon account of the molecular weight of RPE I, 232000.A lower aggregated form, RPE Ⅱ, contained α and β subunits similar to those of RPE I, but itssubunit composition was the （αβ） monomer of RPE.

COMPARATIVE STUDIES ON STRUCTURAL FEATURE OF AGAR POLYSACCHARIDES FROM PORPHYRA HAITANENSIS GROWN IN SOUTH AND NORTH CHINA

The structural feature of agar polysaccharides from Porphyra haitanensis grown in south China and transplanted to the north was investigated by fractioriation on DEAE - Sephadex A 50, chemical analysis, and infrared and 13C - NMR spectroscopy.The agars composed mainly of Charged molecules were eluted from DEAE - Sephadex A 50 with 1.0 mol/L- NaCl solution from the southern P. haitanensis and with 0.5 mol/L NaCl from the northern one. The 13C-NMR spectra showed that agarobiose [(1 -3)- B D - galactopyranosyl - (1 -4)-3,6-anhydro-a-L-galactopyranose] and the biological precursor of agarobiose [(1-3} B-D-galactopyra nosyl- (1-4)- 6- sulfate-a- L-galactopyranose ] were the major disaccharide repeating units in the charged fractions. The content of the biological precur sor in the agar polysaccharides from southern P. haitanensis was higher than that in the northern one, the content of the biological precursor extracted from cold water was higher than that from hot water, and the content of 6-

Subunit composition and chromophore content of R－phycocyanin and allophycocyanin from Porphyra haitanensis

R-phycocyanin (RPC) of Porphyra haitanensis (T. J. Chang et B. F. Zheng )' was chromatographed on Bio-Rex 70 column with urea solution (pH 3. 0) as an eluent, and a and β two subunits were isolated.Their molecular weights were determined on SDS-PAGE at 18 400 and 20 500, respetively,while those of a and β subunits of allophycocyanin (APC) at 18 800 and 19 700, respectively,and those of RPC and APC were at 117 000 and 122 000,respectively.Both the molar ratio of a and β subests of RPC and APC were 1:1, and the subunit composition was confirmed to be (αβ )3.It was ascertained that in RPC αsubunit contains one chromophore phycocyanobilin (PCB) and β subunit has one chromophore PCB and one phycoerythrobilin (PEB), while in APC both α and βsubunits contain one PCB.

The UV-absorbing compounds in gametophytic blades of Porphyra haitanensis and the effect on formation of sporangial branchlets of conchocelis

Endogenous levels of UVAC in different parts of matured gametophytic blades of Porphyra haitanensis and the effect of exogenous UVAC on sporangial branchlet formation are studied. The UVAC value in vegetative (36. 78 mg/g) and reproductive (29.25 mg/g) parts of female thallus is higher than their counterparts in male thallus (34.68 mg/g and 20.46 mg/g). The UVAC value of reproductive parts is higher than vegetative parts of same sexual thallus. The optimal UVAC concentration for sporangial branchlet formation is 0.1 mg/g.

DEVELOPMENT OF SUSPENDED CONCHOCELIS OF PORPHYRA HAITANENSIS (BANGIALES, RHODOPHYTA)

Ths Mar. 1993 to Aug. 1994 study on suspended conchocelis of Porphyra haitanensisshowed tha there were three pattems for developmnt of vegetative filaments: filaments to filaments by"budding"; filaments to sporangial branchlets by "budding", or cell swelling. There were also three pat-terns for sporangial branchlet development: vegetatively propagting, changing into conbhospores, or dy-ing. Each developmntal stage had one or mere different developmntal directions between vegetative fila-ments and Sporangial branchlets. Developmnts from conchosporangial branchlets to conchospores were se-quential and irreversible. Although sporangial brachlets formed at 29℃ could give rise to filaments, theycould not propagae as healthily under the same condihons as those formed at 25℃ did. Probaby the cru-cial peried of plant cell differenhation is in the late stage of spompal branchlets. In line with the devel-opmental directions of different stages, the authors reghated the developmnt of conchocelis to get idealdifferent developmental smps materials to obtain very develoPmentally homogeneous stages, including fil-aments and sporanglal branchiets.

坛紫菜多糖对微冻南美白对虾仁肌原纤维蛋白氧化和结构特性的影响

该研究通过探讨坛紫菜多糖浸泡的南美白对虾仁在微冻过程中肌原纤维蛋白氧化特性和结构特性的变化规律,从蛋白质角度揭示坛紫菜多糖抗冻保鲜的潜在机制。将虾仁分别用5 mg/mL坛紫菜多糖,蒸馏水(空白对照)和5 mg/mL焦磷酸钠(阳性对照)浸泡处理后于-3℃微冻贮藏,每5 d测定指标。结果表明,与对照组相比,坛紫菜多糖浸泡虾仁可显著延缓总巯基含量下降,防止羰基含量和表面疏水性上升,阻碍平均粒径增加和Zeta电位绝对值的下降(P<0.05),抑制肌原纤维蛋白的氧化和聚集。圆二色光谱分析表明,坛紫菜多糖显著抑制肌原纤维蛋白α-螺旋相对含量的降低(P<0.05)。内源荧光光谱分析表明,坛紫菜多糖能缓解肌原纤维蛋白中发色基团暴露,维持蛋白空间结构稳定性。扫描电镜结果显示,坛紫菜多糖能保持虾肌肉组织微观结构的完整性。该研究为坛紫菜多糖作为新型绿色无磷抗冻保鲜剂添加到水产品冷冻过程中保护蛋白质特性提供理论依据。

不同采收阶段的坛紫菜营养成分分析及预测模型

为获取不同采收阶段坛紫菜的区分方法,以4个采收阶段坛紫菜为原料,测定其色泽、营养物质、氨基酸等营养成分指标。依据坛紫菜采收阶段与营养成分指标的相关性建立模型,预测坛紫菜适宜的采收阶段。结果表明,随着坛紫菜采收阶段的增加,L*和b*值显著上升(P<0.05),而a*值呈下降趋势;蛋白质、必需氨基酸、非必需氨基酸和总氨基酸含量先减小后增加,脂肪和粗纤维分别呈现下降和上升趋势,灰分含量先上升后下降。此外,三水坛紫菜中鲜味氨基酸的TAV值最高,四水坛紫菜中甜味氨基酸的TAV值最高,只在四水坛紫菜中检测到C16∶1和C18∶3,采收后期坛紫菜的矿物质元素含量高于采收前期。根据除蛋氨酸外的必需氨基酸含量拟合的回归曲线,可准确鉴定不同海域的头水坛紫菜。本研究建立了坛紫菜采收阶段与相关品质指标的关联模型,为不同采收阶段坛紫菜的鉴定筛选及高值利用提供了技术参考。

紫菜(Porphyra)遗传差异的ISSR分析

采用ISSR标记技术对来自不同产地的6个坛紫菜(Porphyra haitanensis)无性系和两个条斑紫菜(P.yezoensis)无性系进行了遗传差异的分析。结果表明,7条ISSR引物在8个紫菜系中共扩增出59条片段,全部表现出多态性。根据Nei等的相似性系数得出8个紫菜系间的遗传距离在0.274—0.746之间。用UPGMA方法作出的系统树中各紫菜基本上是按照产地进行聚类,从而推测紫菜的遗传差异可能与地域分布相关。本文结果也证明了ISSR与RAPD、AFLP等标记技术一样适用于紫菜的遗传多样性分析。

低分子量坛紫菜多糖纳米硒的制备、表征及其体外抗氧化活性

目的:制备低分子量坛紫菜多糖纳米硒(SPS-SeNPs),检测其形态特征及抗氧化活性。方法:以多糖浓度、反应时间、反应温度作为实验因素,采用双波长比色法测定SPS-SeNPs中纳米硒(SeNPs)的粒径变化,以SPS-SeNPs溶液的吸光度比值(A410/490)为评价指标,采用单因素及正交试验考察SPS-SeNPs的制备工艺。使用扫描电子显微镜、透射电子显微镜、X射线衍射和X射线光电子能谱分析对SPS-SeNPs进行结构表征。研究其总抗氧化活性、还原能力、DPPH自由基清除率。结果:制备SPS-SeNPs最佳条件为:多糖浓度2 mg/mL,反应温度为40℃,反应时间为0.5 h,该条件下制备出的SPS-SeNPs溶液吸光度比值为2.0487±0.0082。SPS-SeNPs中的SeNPs呈现出具有良好分散性的球形形态。确定了SPS-SeNPs的元素组成百分比为:C元素(42.00%)、O元素(41.35%)、Se元素(16.65%)。证实了SPS-SeNPs中的Se为0价态的无定形硒。通过稳定性考察发现SPS-SeNPs能在4℃避光保存30 d仍保持稳定。与亚硒酸钠(Na2SeO3)和低分子量坛紫菜多糖(SPS)相比,SPS-SeNPs表现出良好的总抗氧化能力、还原能力和DPPH自由基清除能力(IC50=31.45±1.16μg/mL)。结论:SPS-SeNPs结合了多糖和纳米硒的优点,既具有多糖的抗氧化性,又具有纳米硒的高生物活性。这为进一步开发和利用坛紫菜粗多糖提供了重要的理论依据。