Curcumin delivery nanoparticles based on Maillard reaction of Haematococcus pluvialis protein/galactose for alleviating acute alcoholic liver damage

The aim of this study is to investigate the feasibility of Maillard reaction products of Haematococcus pluvialis protein and galactose(HPP-GAL)for improving the bioactivities of curcumin(CUR)for alleviating alcoholic liver damage.CUR was embedded into HPP-GAL nanoparticles by the self-assembly of hydrogen bonding and hydrophobic interaction with the particle size around 200 nm.HPP-GAL enhanced the encapsulation efficiency and loading amount of CUR with the value of(89.21±0.33)%and(0.500±0.004)%,respectively.The stabilities of CUR under strong acid,salt ion stability and ultraviolet irradiation conditions were improved by the encapsulation.HPP-GAL-CUR nanoparticles exhibited excellent concentration-dependent in vitro antioxidant activities including DPPH and ABTS scavenging rates,and better protective effect on CUR against gastric acid environment as well as longer release of CUR in simulated intestinal fluid.In addition,the HPPGAL-CUR delivery system possessed liver targeting property due to the existence of GAL,which could effectively alleviate the alcohol-induced liver damage and the inflammation indexes by inhibiting the oxidative stress.Therefore,HPP-GAL-CUR nanoparticles might be a potential candidate system for the prevention of alcoholic liver damage in the future.

Metabolomics of astaxanthin hyperaccumulation in Haematococcus pluvialis under high light stress

Variation in metabolite profiles of Haematococcus pluvialis(a type of unicellular green algal)under light stress is a key issue of study at the present.To investigate the effect of light intensity on accumulation of astaxanthin in H.pluvialis,a 26-day batch culture experiment of H.pluvialis under the light intensity levels at 73,127,182,236,and 291μmol/(m^(2)·s)was conducted.Therefore,the optimal light intensity and the corresponding metabolic pathways of accumulation in H.pluvialis were determined.Results show that 236μmol/(m^(2)·s)was the optimum light intensity to induce astaxanthin accumulation,at which a maximum content of 9.01 mg/L was achieved on Day 24.A total of 132 metabolites were identified and quantified,of which 38 differential metabolites were highlighted and classified,including 3 fatty acids or intermediates,5 amino acids or derivatives,5 carbohydrates or intermediates,16nucleoside derivatives,and 9 other metabolites using LC-MS/MS technique.Subsequently,16 statistically significant differential metabolic pathways were enriched and annotated based on Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis between the control and the 236μmol/(m^(2)·s)treatment group(P<0.05).In addition,the bioprocesses included cellular basal metabolism and signaling systems,such as carbohydrate metabolism,amino acid metabolism,glycerol and derivatives metabolism,nucleotide and derivative metabolism,and inositol phosphate metabolism were activated and regulated under strong light stress conditions.Moreover,4 hub metabolites containing D-glucose-6-phosphate,L-tyrosine,glycerol-3-phosphate,and L-glutamine were identified,based on which the associated metabolic network was constructed.The study provided a metabolomic view of astaxanthin accumulation in H.pluvialis under strong light stress.

茴香醚对雨生红球藻(Haematococcus pluvialis LUGU)虾青素积累和脂肪酸合成的影响

雨生红球藻虾青素积累过程与脂肪酸合成密切相关。实验研究了添加不同质量浓度的茴香醚(butyl hydroxyanisole,BHA)对雨生红球藻Haematococcus pluvialis LUGU,生物量浓度、虾青素积累量以及脂肪酸合成的影响。结果显示,在茴香醚质量浓度为2 mg/L,虾青素积累量显著提高,最大含量可达31.56mg/g,比对照组(15.42 mg/g)提高到2.05倍,此时藻细胞内总脂肪酸的含量也达到最大值(12.27%),比同时刻对照组的含量(6.22%)提高到1.97倍,单不饱和脂肪酸成分相比于对照组也产生了较大变化。脂肪酸合成关键基因acp、kas表达量也分别上调16.77、27.53倍。研究表明,适当浓度的BHA不仅能够显著提高虾青素合成,而且影响了脂肪酸总量和组成。

胺鲜酯提高雨生红球藻(Haematococcus pluvialis LUGU)虾青素含量的作用

探究添加不同浓度的胺鲜酯(diethyl aminoethyl hexanoate,DA-6)在雨生红球藻Haematococcus pluvialis LUGU培养过程中对生物量、虾青素含量、活性氧(ROS)水平以及叶绿素浓度的作用。结果表明,0.1 mmol/L DA-6可以略微增加生物量(0.52 g/L),但相对于同一天对照组,可以显著(p<0.05)提高虾青素含量,最大含量可达24.94 mg/g,是对照组(16.43 mg/g)的1.52倍,此时藻细胞内ROS水平以及叶绿素浓度相比于对照组均出现了降低。研究表明,DA-6诱导可以促进虾青素含量的提高,这为虾青素产业化生产提供了一种有效的策略。

黄腐酸对Haematococcus pluvialis LUGU虾青素积累和lcy基因表达量的影响

雨生红球藻是虾青素的主要来源,为探明黄腐酸(FA)对雨生红球藻Haematococcus pluvialis LUGU的影响,将不同质量浓度的FA添加至对数生长期的藻液中置于胁迫条件下培养,并分别对细胞生物量浓度、虾青素积累质量浓度以及番茄红素β-环化酶(lcy)基因表达量进行了测定。结果表明,黄腐酸质量浓度为10 mg/L,藻细胞的生物量产率达到最大80.28 mg/(L·d),比对照组提高5.44%;而虾青素最大质量浓度为20.82 mg/L,是在FA质量浓度为5 mg/L时测得,比对照组提高了86.89%。RT-PCR分析显示,虾青素合成的关键基因lcy的表达受FA的影响,当添加5 mg/L和10 mg/L的黄腐酸时lcy基因最大的表达量分别为对照的1.2倍和0.7倍。实验证明FA诱导下的雨生红球藻虾青素的积累含量和lcy基因表达量呈正相关。

Strain H_2-419-4 of Haematococcus pluvialis induced by ethyl methanesulphonate and ultraviolet radiation

Two strains H2-410 and H2-419 were obtained from the chemically mutated survivors of wild Haematococcus pluvialis 2 by using ethyl methanesulphonate (EMS). Strains H2-410 and H2-419 showed a fast cell growth with 13% and 20% increase in biomass compared to wild type, respectively. Then H2-419-4, a fast cell growth and high astaxanthin accumulation strain, was obtained by exposing the strain H2-419 to ultraviolet radiation (UV) further. The total biomass, the astaxanthin content per cell, astaxanthin production of H2-419-4 showed 68%, 28%, and 120% increase compared to wild H. pluvialis 2, respectively. HPLC (High Performance Liquid Chromatography) data showed also an obvious proportional variation of different carotenoid compositions in the extracts of H2-419-4 and the wild type, although no peak of carotenoids appeared or disappeared. Therefore, the main compositions in strain H2-419-4, like its wild one, were free of astaxanthin, monoester, and diester of astaxanthin. The asexual reproduction in survivors after exposed to UV was not synchronous, and different from the normal synchronous asexual reproduction as the mother cells were motile instead of non-motile. Interestingly, some survivors from UV irradiation produced many mini-spores (or gamete?), the spores moved away from the mother cell gradually 4 or 5 days later. This is quite similar to sexual reproduction described by Elliot in 1934. However, whether this was sexual reproduction remains questionable, as no mating process has been observed.

Stability and changes in astaxanthin ester composition from Haematococcus pluvialis during storage

In this paper,we investigated the effects of temperature,oxygen,antioxidants,and corn germ oil on the stability of astaxanthin from Haematococcus pluvialis under different storage conditions,and changes in the composition of astaxanthin esters during storage using high performance liquid chromatography and spectrophotometry.Oxygen and high temperatures(22–25°C) significantly reduced the stability of astaxanthin esters.Corn germ oil and antioxidants(ascorbic acid and vitamin E)failed to protect astaxanthin from oxidation,and actually significantly increased the instability of astaxanthin.A change in the relative composition of astaxanthin esters was observed after 96 weeks of long-term storage.During storage,the relative amounts of free astaxanthin and astaxanthin monoesters declined,while the relative amount of astaxanthin diesters increased.Thus,the ratio of astaxanthin diester to monoester increased,and this ratio could be used to indicate if astaxanthin esters have been properly preserved.If the ratio is greater than 0.2,it suggests that the decrease in astaxanthin content could be higher than 20%.Our results show that storing algal powder from H.pluvialis or other natural astaxanthin products under vacuum and in the dark below 4°C is the most economical and applicable storage method for the large-scale production of astaxanthin from H.pluvialis.This storage method can produce an astaxanthin preservation rate of at least 80%after 96 weeks of storage.

黄腐酸对雨生红球藻(Haematococcus pluvialis LUGU)虾青素积累的影响

通过形态学以及18S r RNA的分析,筛选鉴定出了一株新的雨生红球藻Haematococcus pluvialis LUGU,并以其作为研究对象,研究了不同质量浓度的黄腐酸(FA)对胁迫培养条件下细胞生长和虾青素产量的影响。结果表明,5 mg/L和10 mg/L的FA不仅促进了藻细胞的生长(生物量浓度分别比对照组增加了4.26%和10.58%),而且能够显著提高虾青素的含量(最大含量分别达到20.82和12.23 mg/L)。其中,5 mg/L的FA诱导效果最为明显,虾青素产量(37.46 mg/g)比对照组(20.90 mg/g)提高79.23%。实验表明,黄腐酸在适当浓度下能够显著促进藻细胞内虾青素的积累,可作为一种有效的诱导子应用于虾青素的生产。

Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD),peroxidase (POD),catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O2ˉ).The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H.pluvialis during exposure to reactive oxygen species (ROS) such as Oˉ2.Astaxanthin reacted with ROS much faster than did the protective enzymes,and had the strongest antioxidative capacity to protect against lipid peroxidation.The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells.Astaxanthin-enriched red cells had the strongest antioxidative capacity,followed by brown cells,and astaxanthin-deficient green cells.Although there was no significant increase in expression of protective enzymes,the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin,which quenched Oˉ2 before the protective enzymes could act.In green cells,astaxanthin is very low or absent;therefore,scavenging of ROS is inevitably reliant on antioxidative enzymes.Accordingly,in green cells,these enzymes play the leading role in scavenging ROS,and the expression of these enzymes is rapidly increased to reduce excessive ROS.However,because ROS were constantly increased in this study,the enhance enzyme activity in the green cells was not able to repair the ROS damage,leading to elevated MDA content.Of the four defensive enzymes measured in astaxanthin-deficient green cells,SOD eliminates Oˉ2,POD eliminates H2O2,which is a by-product of SOD activity,and APX and CAT are then initiated to scavenge excessive ROS.

Concomitant NH_4^+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.

Optimization of the Extraction Process of Astaxanthin from Haematococcus pluvialis with Oil Dissolution Method

[ Objective ] This study aimed to optimize the extraction process of astaxanthin from Haematococcus pluvialis with oil dissolution method. [ Method ] Small amounts of acetone or ethanol were separately added into soybean oil for astaxanthin extraction. The extraction efficiency of astaxanthin from H. pluvialis with different methods was compared. [ Result] The extraction efficiency of astaxanthin from H. pluvialis with acetone, acetone ＋ soybean oil, ethanol ＋ soybean oil, soybean oil was 20.46, 21.65, 20.85 mg/g and 13.05 mg/g, respectively. According to the results, acetone ＋ soybean oil led to the highest extraction rate, which was approximately twice that of soybean oil and higher than that of acetone. [ Conclusion ] This study laid the foundation for large-scale production of astaxanthin.

Sodium acetate can promote the growth and astaxanthin accumulation in the unicellular green alga Haematococcus pluvialis as revealed by a proteomics approach

Haematococcus pluvialis is an ideal natural source of strong antioxidant astaxanthin.Sodium acetate(NaAc)was proven an effective organic carbon source for improving algal growth and astaxanthin production;however,the underlying mechanism remains obscure.To reveal the mechanism of NaAc at the green vegetative stage of H.pluvialis,the physiochemical characteristics and the global protein expression profiles obtained using a tandem mass tag labeling approach were compared between the control(CK)and two NaAc-addition groups.Results show that after NaAc addition,the biomass,nitrate consumption rate,and activities of three carbohydrate metabolism enzymes of H.pluvialis were significantly increased,and the net photosynthetic rate and chlorophyll content decreased.In addition,astaxanthin,total carbohydrates,and total lipids were accumulated,and some red cells appeared in the NaAc5 group.Moreover,317 differentially expressed proteins(DEPs)with the most altered expression patterns were screened out in the CK vs.NaAc5 comparison in our proteomics study.All the DEPs involved in carbohydrate metabolism and lipid metabolism were significantly increased,while most of the photosynthesis-related proteins were depressed in the two NaAc-treated groups.The proteomics results were verified and supported by parallel reaction monitoring approach and physiochemical data.Our findings demonstrate that NaAc promoted the tricarboxylic acid cycle,glyoxylate cycle,and amino acid and lipid synthesis,and inhibited the photo synthe sis-related activities,which consequently speeded up the growth and astaxanthin accumulation in this alga.

Changes of pigments and lipids composition in Haematococcus pluvialis vegetative cell as affected by monochromatic red light compared with white light

Much attention has been paid on studies of astaxanthin accumulation process in H aematococcus pluvialis industry. However, growth of H. pluvialis in motile vegetative stage is still the most important and problematic part in the whole cultivation process, such as low growth rate and cell yields. Motile vegetative cells are extremely sensitive to various stresses which make it difficult to maintain the cells of this state to grow. Previous reports showed that motile vegetative cells may have higher biomass yields ifapplied monochromatic red light. However, metabolic responses of these cells are not completely understood, which constraints application of this illumination protocol in industry. The aim of this study was to examine how critical biochemical changes of H. pluvialis motile vegetative cells were af fected by red light when compared with white light. Variation of photosynthetic pigments composition and lipids were mainly studied. By comparing growth process of cultures in red light and white light, prominent variation of pigments composition and lipids changes were observed. The results showed that, even though cell proliferation was the same during exponential growth phase, variation of photosynthetic pigment composition and lipids occurred. The final biomass of cell number was higher in red light group than in white light group. The variations were significant different. Increase or decrease of major photosynthetic membrane lipids to some extent did not influence photosynthesis of the vegetative cells during this phase. However, vegetative cells under polychromatic white light other than monochromatic red light need further metabolic process to adjust its pigment composition and lipids, possibly this is energetically and biochemically unfavorable for motile vegetative cells to growth under white light, a light condition normally not considered as a stress.

Transient Expression of VHB and GLUT1 in the Green Alga Haematococcus pluvialis Using Agrobacteriummediated Transformation

The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis using the vectors hosting the genes coding for VHB （ Vitreoscilla hemoglobin） and GLUT1 （glucose transporter 1 ） is reported here. The greenish yellow fluorescence of EGFP was observed when the zeocin-resistant cells were viewed with a fluorescent microscope. The functional expression of EGFP showed that the Agrobacterium-mediated transformation could efficiently transfer the exogenous gene into H. pluvialis. RT-PCR was used to successfully amplify the mRNA of VHB and GLUT1 genes from transformed cells, while Southern blots indicated the integration of VHB and GLUT1 genes into the genome of H. pluvialis. Transferring VHB and GLUT1 genes into this alga would pave the way for manip- ulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.

雨生红球藻(Haematococcus pluvialis)破壁方法对虾青素提取率的影响

采用匀浆法、冻融温差法、超声波法、直接研磨法和低温研磨法等5种破壁方法,研究了雨生红球藻提取虾青素时破壁方法对虾青素提取率的影响。结果表明,对雨生红球藻进行破壁是必要的。其中匀浆法、冻融温差法、超声波法、直接研磨法和低温研磨法等的最佳破壁条件为:匀浆法破壁时间22min,水为介质;冻融温差法破壁温度为-70℃,时间为12h,冻融2次,水为介质;超声功率400W,每次超声时间5s,共超声25min;直接研磨法研磨时间1min;加液氮低温研磨法破壁2次,每次时间0·5min;虾青素的提取率依次为0·76%、0·93%、1·03%、1·51%和3·21%。匀浆法和超声波法破壁由于要使用溶剂或介质,对后续的虾青素提取和分离会有影响,而直接研磨法在破壁过程中产生高温,降低了虾青素的生理活性,所以它们都不是雨生红球藻破壁的最佳方法。加液氮低温研磨法在破壁过程中不添加化学试剂,不产生污染,能最大限度地保留虾青素的生理活性,是所选方法中最好的一种。

无机碳与雨生红球藻(Haematococcus pluvialis)细胞调节物质

以单细胞雨生红球藻为材料 ,采用酸碱滴定和CO2 加富通气培养微藻的方法 ,对旧液中HCO-3 和CO2 -3 浓度变化以及对红球藻细胞生长的影响进行了研究。结果表明 ,旧液具有限制红球藻细胞生长和诱导细胞转化的作用。同时 ,旧液中无机碳离子浓度明显高于新液。培养液中富含CO2 -3 时 ,各细胞数量与CO2 -3 浓度呈正相关 ,相关系数为 0 .88。溶液中仅有HCO-3 时 ,各细胞数与HCO-3 浓度也呈正相关性。因此 ,排除了CO2 -3 和HCO-3 作为旧液中的调节物质 ,限制红球藻细胞生长和诱导细胞转化的可能性。旧液乙酸乙酯提取物生物检测实验表明 ,在粗提取物中有降低细胞增长和诱导细胞转化的活性 ,表明调节物质能溶于有机相 ,也反过来证实无机碳离子不是旧液中的调节物质。DNA含量和倍性分析结果表明 ,红球藻游动细胞DNA复制可以加倍后不经过原生质分裂就可以再次进行 ,因此推测旧液中的调节物质对原生质分裂过程产生抑制作用 ,而不对DNA复制过程产生抑制作用。

雨生红球藻(Haematococcus pluvialis)基因工程选择试剂的筛选

采用细胞计数法,研究了重要经济微藻——雨生红球藻对10种基因工程常用选择试剂的敏感性。结果表明,雨生红球藻对除草剂草丁膦和抗生素Zeocin比较敏感,50μg/ml的草丁膦和25μg/ml的Zeocin可明显地抑制雨生红球藻生长,尤其是50μg/ml的草丁膦能导致细胞两周内全部死亡;高浓度的卡那霉素和硫酸链霉素也可抑制雨生红球藻的生长;雨生红球藻对包括氯霉素和G418在内的其他6种试剂不敏感,即使处理浓度高达1000μg/ml。本文结果提示草丁膦可能是雨生红球藻基因工程适宜的选择试剂,其相应的抗性基因bar可能成为雨生红球藻遗传转化的选择标记基因。

温度对雨生红球藻(Haematococcus pluvialis)生物量和虾青素产量的影响

在用环形培养池模拟系统培养雨生红球藻的过程中,研究了温度对雨生红球藻生物量及虾青素产量的影响。结果表明,在15～25℃的范围内,不同温度下雨生红球藻生物量和虾青素含量及产量都经历了一个上升—最高—下降的过程。25℃与22℃时红球藻的虾青素产量、虾青素含量(干重)均显著高于其他温度的(P<0.01),但两者间差异不显著(P>0.05)。15℃时,红球藻生物量、虾青素含量和虾青素产量均最低,分别为1.4g、0.54%和2.49mg/L;25℃时,红球藻生物量和虾青素产量最高,分别为2.68g和13.53mg/L;22℃时,虾青素含量最高,为1.52%。

雨生红球藻(Haematococcus pluvialis-6B)培养条件研究

系统比较了９种培养基对雨生红球藻Ｈａｅｍａｔｏｃｏｃｃｕｓｐｌｕｖｉａｌｉｓ－６Ｂ生长的影响，从中选出较适合于其生长的培养基ＳＭ；并研究了Ｈ．ｐｌｕｖｉａｌｉｓ－６Ｂ的光温需求，得出Ｈ．ｐｌｕｖｉａｌｉｓ－６Ｂ生长的适宜光照强度（７０～９０μｍｏｌｑｕａｎｔａ·ｍ－２·ｓ－１）和适宜温度（１９～２２℃）；同时，还了解了通气对Ｈ．ｐｌｕｖｉａｌｉｓ－６Ｂ生长的影响，发现当通气量为１５０ｍＬ·ｍｉｎ－１空气时，Ｈ．

Fe^(3+)对雨生红球藻(Haematococcus pluvialis)生长及荧光特性的影响

为探求适宜雨生红球藻CG-06株生长的Fe3+浓度和Fe3+对藻细胞荧光特性的影响,以BBM为基础培养基,选用EDTA-FeNa(Ⅲ)为Fe3+源,设置0、1.79、8.95、17.9、35.8、71.6μmol.L-16种Fe3+浓度梯度,实验测定藻的生长并分析不同Fe3+对藻细胞叶绿素荧光和77 K低温荧光等的影响。结果表明:适合雨生红球藻CG-06株生长的Fe3+浓度为8.95μmol.L-1,Fe3+浓度较高时,雨生红球藻的生长受到抑制,Fe3+浓度低于1.79μmol.L-1将产生低铁限制。W alz-PAM测定数据显示,在铁不足或高铁抑制条件下,雨生红球藻光系统Ⅱ活性明显下降,开放态的反应中心数目减少,光合作用受到抑制。77 K低温荧光光谱显示,在铁不足或高铁抑制条件下,710 nm荧光峰降低,684 nm和694 nm荧光峰相对增强,说明能量在两个光系统分配上发生变化,能量更多的分配给光系统Ⅱ,限制了光系统Ⅰ的活性。在高铁抑制条件下,CP47蛋白荧光峰降低,CP43蛋白荧光峰增强,推测存在D1蛋白降解。