Preparation of algal-oligosaccharide mixtures by bacterial agarases and their antioxidative properties

ABSTRACT:   Algal-oligosaccharide-lysates (AOL), derived from six agars and four algal polysaccharide extracts (APE), were treated with 100–500 activity units (AU) of MA103-agarases or MAEF108-agarases, and their antioxidative properties evaluated. Soluble total polyphenols (TP) were between 462.2 ± 1.6 gallic acid equivalents (GAE, µg/mL) and 70.6 ± 17.4 GAE. The DPPH radical scavenging capacity of all AOL went from 68.3 ± 0.7% to 0.5 ± 0.1%. The ferrous ion chelating capacity of all AOL went from 93.1 ± 0.2% to 21.7 ± 0.9%. Evaluation of the H₂O₂ scavenging capacity of all AOL was between 35.9 ± 5.4% and 0.1 ± 0.2%. The reducing power of all AOL went from 51.3 ± 2.6 to 3.2 ± 6.8 expressed as µg/mL ascorbic acid. In DPPH radical scavenging capacity, ferrous ion chelating capacity and reducing power etc., the AOL derived from the APE of Porphyra dentate (digested by 500 AU of MAEF108-agarases) were highest, in all test sets. However, the AOL derived from the APE of Monostroma nitidum (digested by 500 AU of MAEF108-agarases) had the highest H₂O₂ scavenging capacity in all test sets. The order of antioxidative activity performance of all AOL treated in this experiment, by these four antioxidative methods, is as follows: ferrous ion chelating capacity > DPPH radical scavenging capacity > H₂O₂ scavenging capacity > reducing power; this may be related to their polyphenols, small molecular weight polysaccharides or simple sugar constituents. In this study, it is demonstrated that various agarases derived from algal oligosaccharide mixtures possess good potential for use as a health food, due to their antioxidative capacity.     

两面针繁殖技术研究

[目的]探索两面针种子繁殖与扦插繁殖技术。[方法]以来自桂林和钦州两面针种子为试材,不处理为CK,研究了试剂处理、砂纸处理、润砂催芽对种子发芽的影响。同时也研究了不同水平的激素种类、激素浓度、处理时间、留叶程度、扦插基质对两地的插穗扦插质量的影响。[结果]不同产地的种子大小和千粒重具有显著差异,钦州的种子好于桂林的;两面针种子具有后熟特性,润砂处理的种子发芽最好,最高发芽率达到80%,冬季扦插效果比夏季好,基质与插穗留叶数量对扦插生根成活影响较大。激素种类对生根条数、生根总长的影响最大。[结论]温度和种子大小影响种子的发芽质量,适宜的留叶数量,扦插基质和枝条有利于扦插繁殖。     

Algal-oligosaccharide-lysates prepared by two bacterial agarases stepwise hydrolyzed and their anti-oxidative properties

Twenty algal-oligosaccharide-lysates (AOLs), derived from six agars and four algal polysaccharide extracts (APEs), were treated first with agarases with 250 or 500 agarase activity units (AU), which were produced from the agar-liquefying bacterial strain Pseudomonas vesicularis MA103, named MA103-agarases. The AOLs were then treated with agarases (250 or 500 AU) derived from the agar-softening bacterial strain Aeromonas salmonicida MAEF 108, named MAEF 108-agarases. Anti-oxidative properties of the AOLs were evaluated by five in vitro methods. The AOL obtained from the APE of Porphyra dentate, digested by 250 AU of MA 103-agarases, and by 250 AU of MAEF 108-agarases, designated as A₂₅₀-Por, showed better results than the 19 other AOLs. This result is in accordance with the level of soluble total polyphenols (STP) of A₂₅₀-Por, which was also higher than the remainder of the AOLs tested. The AOL derived from the APE of P. dentate, digested by 500 AU of MA103-agarases and then 500 AU of MAEF108-agarases, and designated as B₅₀₀-Por, displayed the second highest data in four potential evaluation methods, except in H₂O₂ scavenging capacity. In this study, certain agars or APEs digested by specific agarases can present an increasing antioxidative capacity. These agars include Bitek agar, Agar powder, Bacteriological, Agar Bacteriological, and Guanghui agar, plus APEs of Gracilaria sp. and Monostroma nitidum decomposed stepwise by two agarases. The fraction of polyphenols (<1 kDa) that were derived from A₂₅₀-Por showed anti-oxidative activities on α, α-diphenyl-β-picrylhydrazyl (DPPH) assay and reducing power determination, while the remaining four agar-lytic fractions obtained from A₂₅₀-Por did not exhibit anti-oxidative activity. This phenomenon may suggest that anti-oxidative properties of AOLs originate in polyphenols. Algal-oligosaccharide-lysates may have potential use as a health food.     

北极礁膜室内人工育苗的研究

于2004年对大连地区北极礁膜M onostroma arcticum的生物学进行了初步研究,并在室内进行了合子采苗、孢子体培养和孢子采苗及幼苗培养等人工育苗试验。结果表明:水温为3℃时,北极礁膜的叶片长度达到16 cm;水温为5℃时,配子放散量达到2.4×108个/g;孢子体在水温低于19℃时生长较快,水温超过22℃时生长几乎停止。孢子体在度过夏季高温期后,当水温降至20℃以下时,开始形成孢子囊;当水温降至15℃时,孢子放散量达到6.1×105个/cm2;孢子附着到苗绳上后经过50 d的室内培养,培育出长度为1 cm的北极礁膜幼苗。     

盐度和营养盐对礁膜配子体发育的影响

礁膜(Monostroma nitidum)是我国重要的经济海藻之一,隶属于绿藻门,绿藻纲,石莼目,礁膜科,礁膜属。礁膜广泛分布于我国的东南沿海,生长在内湾静水处的岩石上或具有泥砂的石块上。日常所见的礁膜叶状体是配子体,为膜状,呈绿色或黄绿色,体软并具光泽。礁膜是绿藻中食用价值最高的一种,体软味美,我国南北沿海居民有食用。日本人也喜食“紫菜酱”(原料以礁膜为主,掺入少量紫菜,加入调味料,高压烹煮而成)。此外,礁膜还具有一定的药用价值,如清热化痰,利水解毒以及降低胆     

广西两面针种群分布特征

通过对广西两面针种群资源的实地调查,阐明了两面针在广西的地理分布、生态型分布及种群生境特点;并对两面针有效成分含量与土壤中各营养成分含量的相关性进行分析,结果表明:广西地区两面针有效成分含量仅与土壤有效磷含量成正相关,而与土壤pH值、有机质含量等都呈负相关。     

三种礁膜属藻类的分子遗传多样性和亲缘关系的RAPD分析

用RAPD技术对礁膜、袋礁膜和宽礁膜进行了DNA多样性分析,这3种礁膜的基因组DNA分子量均约23Kb。在使用的88个随机引物中,有43个引物在所有样品中都能重复性较好地扩增出条带清晰的多态性片段,多态引物比例为48.86%,袋礁膜、宽礁膜和礁膜多态位点比例分别为55.93%、65.28%和53.14%。礁膜与宽礁膜、宽礁膜与袋礁膜以及礁膜与袋礁膜种间遗传距离分别为0.4118,0.4148,0.4375。通过对遗传距离分析,结果表明采自3个不同海区的样本为同一礁膜属的3个种,与传统分类鉴定结果相吻合。UPGMA和NJ法构建的种间分子系统树一致,结果是宽礁膜和礁膜之间的亲缘关系最近,宽礁膜与袋礁膜之间的亲缘关系较远,礁膜与袋礁膜之间的亲缘关系最远,这与宽礁膜与礁膜共有形态特征较多,袋礁膜与宽礁膜、礁膜共有形态特征少相验证。     

两面针苗木分级标准的研究

运用随机抽样、主成分分析和逐步聚类分析等方法,对两面针当年生苗和1年生苗的分级标准进行调查研究。结果表明,地径和苗高对两面针苗木质量有很好的表达,可以作为苗木分级的质量指标;按照3个等级,提出了两面针当年生苗和1年生苗相应的分级标准;经实测该分级标准与苗木实际情况相符,可为生产提供参考。     

An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae

Preparation of protoplasts and their subsequent applications for both basic and applied research of marine macroalgae remains largely under developed due to lack of development of reliable methods with consistent yields of viable protoplasts. An improved enzyme preparation with a single commercial enzyme, e.g. 2% Cellulase Onozuka R-10 in 1% NaCl solution, was developed to produce protoplasts rapidly from different green algal genera of Ulva, Enteromorpha and Monostroma. The simple dissolution of enzyme powder in 1% NaCl resulted in exclusion of 2% Macerozyme R-10 from the mixture consisting of 2% Cellulase Onozuka R-10 with 3% NaCl earlier reported as superior for the same algae. Optimal conditions for the isolation of maximum yields of viable protoplasts were found to be with 2% Cellulase Onozuka R-10 incubated at 20 °C for 2 h in 1% NaCl solution with 0.8 M mannitol adjusted to pH 6.0. The protoplast yield with optimized enzyme mixture was as high as 102.8 × 10⁶ cells g^− 1 f. wt for M. oxyspermum while it was in the range of 74.4–88.6 × 10⁶ cells g^− 1 f. wt thallus for seven species of Ulva, and 82.5–95.4 × 10⁶ cells g^− 1 f. wt for three species of Enteromorpha. The regeneration rate of protoplasts isolated using this method ranged from 89 to 92% with normal morphogenesis. The seeding of nylon threads with isolated protoplasts of M. oxyspermum was successful and after 3–4 weeks the entire frame with nylon threads became thick green in color with tiny germlings in laboratory culture. Thus, the method described in the present study allow for rapid mass production of viable protoplasts that could be potentially used as a source for seed material for mariculture and for other applied phycological research.     

两面针与混伪品及近缘种DNA条形码鉴定研究

[目的]探讨DNA条形码技术对两面针真伪鉴定的可行性,为两面针种质资源的鉴定提供参考.[方法]对两面针及其常见混伪品和同属近缘种（10种31份样品）进行DNA提取、PCR扩增和双向测序,所得序列经CodonCodeAligner软件处理和人工校对,采用TaxonGap法比较rbcL、trnH-psbA和ITS2等3条序列的物种鉴定力大小;选择适用条形码在不同物种样品范围内进行分析,检查种内种间距离变化情况.[结果]ITS2和tmH-psbA两条序列对10个试验物种的鉴定效率均为100.0％,rbcL在实验物种范围内不存在最小种间遗传距离;ITS2序列在样品鉴定范围由12个物种33个数据扩大为33个物种132个数据时,鉴定成功率从100.0％下降至84.8％,种内变异大于种间变异的比例由71.4％上升至90.1％.[结论]ITS2和trn H-psbA序列可作为两面针真伪鉴定的标准条形码,实际应用中应注意确定物种鉴定样品数及种内充分取样.