盐胁迫诱导雨生红球藻合成虾青素的机理

为研究盐胁迫诱导雨生红球藻合成虾青素的机理,分析了在添加氯化钠(HS)和未添加氯化钠(CK)的培养液中,细胞内氮和碳代谢的变化。结果表明:HS中硝酸还原酶(NR)和1,5-二磷酸核酮糖羧化酶(Rub isco)活性迅速下降。虾青素在第4天开始合成时,二者分别降至初始值或最高值的46.5%和25.7%。相比之下,在对照(CK)中,NR和Rub isco活性仍然很高,仅下降了26.1%和25.6%,细胞内没有虾青素积累。上述数据表明盐胁迫条件下NR活性被抑制,细胞内氮源供应不足(氮饥饿)并进一步抑制了Rub isco的合成,导致CO2固定量减少(碳饥饿)。为了生存,藻细胞开始合成虾青素。     

强光照对雨生红球藻合成虾青素的影响

在不同的光照强度下研究了雨生红球藻细胞内虾青素的合成与初级代谢的关系.在强光(HL)和中等强度(ML)的光照条件下,雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶 (Rubisco)和硝酸还原酶(NR)的活性第1天大幅度提高,2天后又迅速下降.与此同时,硝酸盐浓度也快速降低.当虾青素在第4天(HL)和第6天(ML)开始合成时, HL中Rubisco和NR活性以及NO3-浓度分别下降了75.5%,71.5% 和96.2%,而ML中则下降了76.5%,74.7% 和94.3%.相比之下,在低光照(LL)条件下,实验结束时三个指标仅下降了25.9%,29.8% 和56.8%,细胞中没有虾青素积累.结果表明强光提高了Rubisco 和 NR活性,导致硝酸盐浓度迅速降低而最终又抑制了这两种酶的活性,造成雨生红球藻光合作用效率下降即"碳饥饿".在此状态下,为了生存,细胞内合成虾青素的相关基因被激活,藻细胞开始合成并积累虾青素.     

雨生红球藻合成虾青素过程中分泌铵离子的研究

雨生红球藻在在缺氮(NF)的条件下,随着虾青素的合成,肽链内切酶(EP)的活性上升而蛋白质含量下降,天冬酰氨酸含量在前4天增加然后又减少,铵离子浓度则持续上升。相比之下,在氮充足(NR)的培养液中,藻细胞不合成虾青素,蛋白质和天冬酰氨酸的含量以及EP的活性基本稳定,培养液中也没有检测到铵离子。上述结果表明:①降解的蛋白质为虾青素的合成提供了碳源,②EP参与了蛋白质的降解反应,③为避免铵离子的毒害作用,蛋白质降解所产生的部分氨临时贮存在天冬酰氨酸中,而其余的则分泌到细胞外。     

雨生红球藻的新繁殖方式及其藻蓝蛋白的合成

雨生红球藻(Haematococcus pluvialis)是一种能够合成强抗氧化剂-虾青素的单细胞绿藻,然而由于其生长速率缓慢,制约了虾青素产量的提高。在对雨生红球藻细胞周期的研究中,首次发现了雨生红球藻在高温和低光照条件下的一种特殊繁殖方式,该繁殖方式比已知的营养繁殖和无性繁殖速度快。繁殖过程中厚壁孢子萌发产生大量的小细胞,并且这些小细胞中含有以前在绿藻中从未发现的捕光色素蛋白—藻蓝蛋白(最大吸收波长λmax=621 nm,最大荧光发射波长λmFax=643 nm)。这一发现对于提高天然虾青素的产量和藻蓝蛋白的生产以及藻类的进化有着重要的意义。     

利用雨生红球藻生产虾青素的小试研究

以BG11为基本培养基,小试研究了环境因子的调控对雨生红球藻(Haematoccus pluvialis,FACHB-712)生长和虾青素积累的影响。通过单因子实验改变营养方式、pH值、温度、光照时间和光照强度,测定藻液光密度和虾青素含量等指标。结果表明:添加1g/L葡萄糖作为碳源可明显提高藻种生物量;pH=8.0左右,22～27℃,高光强(200μmol·m-2·s-1)连续光照(24h)的生长条件最适合雨生红球藻游动细胞的增殖,可以作为大规模培养雨生红球藻生产虾青素的实验依据。     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

转光膜在雨生红球藻养殖上的应用

通过对雨生红球藻在不同光质条件下生长的比较,确定了红色光有利于藻生长,进而用2.5 L气升式光照反应器在转光膜及普通PE膜下培养藻进行对比,结果显示雨生红球藻生物量、色素、光合活性等几项生物指标在转光膜条件下明显高于普通PE膜. 在气升式反应器内培养的藻细胞,接种9 d,虾青素含量可达3.57 mg/L,叶绿素浓度达到12.42 mg/L,干重提高8.8%以上.     

从雨声红球藻中提取虾青素的皂化工艺优化

目的:优选从雨声红球藻提取虾青素的皂化工艺。方法:以虾青素的提取得率为考察目标,通过单因素法、正交设计法优化从雨声红球藻提取虾青素的皂化工艺。结果:筛选最佳皂化工艺为是加入10倍雨生红球藻量的4%KOH无水乙醇溶液,在10℃下皂化10min。结论:筛选出来的皂化工艺稳定可行、操作简单。     

虾青素的生产方法

近年来,虾青素作为高级营养保健品和药品的潜力倍受关注,其产品定位主要在强化免疫系统功能、抗癌、抗炎、保护视网膜免受紫外线辐射和光氧化损伤等方面。 虾青素的生产方法一般有如下几种：从水产品加工废弃物中提取虾青素、利用藻类如雨生红球藻等生产虾青素、利用红法夫酵母发酵生产虾青素和化学合成法等等。     

红发夫酵母细胞内蛋白质、脂肪酸与虾青素合成关系的研究

为研究红发夫酵母细胞内虾青素合成与蛋白质和脂肪酸代谢之间的关系,实验测定了发酵过程中蛋白质、脂肪酸和虾青素含量的变化。实验结果表明:红发夫酵母细胞内蛋白质的含量在整个发酵周期内变化非常显著,在0～16h(延滞期)和58～76h(指数生长后期)上升,而在16～58h(指数期)和76～124h(稳定期后期)下降。脂肪酸的含量变化趋势与蛋白质的基本相似,但是在稳定期表现出先快速上升然后迅速下降的变化特征。相比之下,虾青素含量的变化规律与蛋白质和脂肪酸相反。上述变化规律表明红发夫酵母细胞内虾青素的合成与蛋白质和脂肪酸的代谢密切相关。在延滞期和稳定前期蛋白质和脂肪酸的快速合成会减少流向虾青素合成方向的碳通量,使虾青素含量减少;而在稳定期后期底物耗尽出现碳饥饿时,蛋白质和脂肪酸的降解又为虾青素的进一步合成提供了碳骨架,提高了虾青素的含量。     

雨生红球藻合成虾青素过程中分泌铵离子

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.     

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 syn-thesis 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) aspar-agine 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 (O2 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.     

Stability,structure, and antioxidant activity of astaxanthin crystal from Haematococcus pluvialis

Crystallized anthaxanthin was prepared through the cleaning crystallization method and the stabilities during thermal, lighting, and pH treatment, and antioxidant activities in vitro were evaluated. The structural characterizations of astaxanthin crystal were verified by ¹H, ¹³C NMR, and XRD analysis. The stability data showed that the astaxanthin crystal was more sensitive to heat, light, and pH compared to oleoresin. The sucrose had no outstanding influence on the astaxanthin crystal, while the stability of astaxanthin oleoresin slightly increased with the increase of sugar concentration. The XRD and nuclear magnetic resonance spectra elucidated that the astaxanthin crystal was all trans structure. The astaxanthin crystal showed the dominant 1,1′-diphenyl-2-picrylhydrazyl (IC₅₀ 18.65 μmol L⁻¹), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt˙⁺(21.1 μmol L⁻¹), •OH (IC₅₀ 49.46 μmol L⁻¹) and ferric reducing antioxidant power (IC₅₀ 81.60 μmol L⁻¹) activities. The investigated results would be helpful for improving the development of astaxanthin crystal in food products.     

混炼方式对螺旋纳米碳纤维增强天然橡胶复合材料力学性能的影响

采用干法和湿法两种混炼工艺制备了螺旋纳米碳纤维(HCNFs)/炭黑(CB)/天然橡胶(NR)复合材料,通过扫描电镜、拉伸试验机和应变扫描仪分别对所制备复合材料的界面形貌、力学性能和Payne效应进行了测试分析,考察了混炼方式对复合材料宏观力学性能及Payne效应的影响。结果表明,与纯CB填料相比,在干湿两种混炼方式下,添加适量的HCNFs(1～6份)能提高HCNFs/CB/NR复合材料的300%定伸应力、扯断伸长率、拉伸强度和硬度。与干法混炼相比,湿法混炼能明显增强HCNFs/CB/NR复合材料的Payne效应,并提升在HCNFs高添加量(2～6份)条件下的拉伸强度和扯断伸长率,这主要源于湿法混炼能够有效改善HCNFs在橡胶基质中的分散性。     

Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health.