褪黑素对雨生红球藻虾青素和油脂合成的影响

以雨生红球藻(Haematococcus pluvialis) LUGU为对象,研究了高光照联合缺氮条件下,外源添加褪黑素(melatonin,MLT)对H. pluvialis生物量、虾青素积累量、油脂含量以及与虾青素、油脂合成相关酶基因表达量的影响。结果显示:缺氮胁迫、联合高光诱导条件下,外源添加10μmol/L MLT,虾青素积累量显著提高,最大虾青素积累量可达32. 37 mg/g,是对照组的2. 25倍;藻细胞内油脂的含量达到42. 84%,为对照组的1. 21倍。虾青素合成关键酶基因bkt及油脂合成关键酶基因fad的表达量均出现不同程度的上调。此外,研究了外源MLT对细胞内活性氧(ROS)水平的影响以及相关抗氧化酶的活性变化规律。ROS水平受到抑制,虾青素合成相关的抗氧化酶SOD、CAT和POD活性增强。研究表明,在高光照联合缺氮胁迫条件下,外源添加MLT有利于雨生红球藻积累虾青素,为强化虾青素的生物合成提供了理论依据。     

褪黑素对雨生红球藻虾青素积累和脂肪酸合成的影响

以雨生红球藻H.pluvialis LUGU为对象,研究了不同浓度的褪黑素(MLT)对藻细胞生长、虾青素积累、脂肪酸及脂肪酸合成相关酶活性的影响。结果显示:10μmol/L MLT诱导条件下培养13 d藻细胞中虾青素含量可达26.62 mg/g,是对照组(12.3 mg/g)的2.16倍;培养5 d脂肪酸合成量(15.32%)最大,是对照组的1.24倍,同时提高了虾青素酯含量;MLT诱导条件下,脂肪酸合成关键酶乙酰辅酶A羧化酶(ACCase)活性升高,而磷酸烯醇式丙酮酸羧化酶(PEPCase)活性降低。研究表明,外源添加适当浓度的MLT不仅可以促进雨生红球藻中虾青素的积累,而且提高了脂肪酸的合成量,改变了脂肪酸组成。     

多重因素对雨生红球藻虾青素积累的影响

虾青素独特的分子结构使其成为自然界最强的抗氧化剂,工业上成功利用雨生红球藻生产天然虾青素的生产模式均采取先藻细胞增殖、再虾青素积累的两步法,近年来用于积累虾青素的胁迫诱导方法仍然集中在高光照强度、高温、缺氮等传统典型胁迫条件,但开始倾向于这些方法的复合作用,同时也进行了异养培养、添加外源试剂、补充其他营养元素等多种尝试。     

雨生红球藻培养及虾青素累积条件探讨

探讨了雨生红球藻(Haematoccuspluvialis)712株的适宜培养条件及藻体诱导累积虾青素的培养基条件。重点研究了温度、pH和光照条件对雨生红球藻营养生长的影响,以及NaNO3、Fe2+盐和乙酸钠浓度对雨生红球藻诱导累积虾青素含量的影响。结果表明:24℃、1000～1500lx连续光照、pH8.0左右的生长条件适合雨生红球藻游动细胞增殖,使平均生长速率达到0.252/d。通过正交试验表明:缺氮培养基对于雨生红球藻细胞累积虾青素最为有利,虾青素含量达到6.72μg/mL,而FeSO4和乙酸钠浓度对虾青素的累积无显著性影响。     

外源乙烯利对雨生红球藻中虾青素积累的影响

本实验初步研究了一定浓度范围的乙烯利对雨生红球藻积累虾青素的影响。在对数生长期的藻液中分别加入一系列不同浓度的乙烯利溶液,然后转入胁迫培养(25℃,24h5000lux连续光照+营养盐饥饿),诱导细胞内虾青素的合成积累。在诱导过程中显微观察不同浓度乙烯利处理后细胞形态和色素积累的变化,并定期取样进行虾青素含量的测定。结果表明,添加0.05ml/L的乙烯利可以促进雨生红球藻积累虾青素,处理后的藻细胞比对照组的藻细胞提前17d完全变红,虾青素产量比对照提高了92.1%,达18.6mg/L藻液。另外还发现0.1ml/L的乙烯利对雨生红球藻积累虾青素有明显的抑制作用。高浓度的乙烯利(≥0.15ml/L)处理对雨生红球藻细胞存在致死作用。     

茴香醚对雨生红球藻(Haematococcus pluvialis LUGU)虾青素积累的影响

利用不同诱导条件促进雨生红球藻Haematococcus pluvialis积累虾青素已成为虾青素生产和研究的热点。实验研究了不同质量浓度的茴香醚(BHA)对胁迫培养条件下雨生红球藻Haematococcus pluvialis LUGU生长和虾青素积累的影响。结果表明,2 mg/L的BHA能够显著提高虾青素的含量,最大含量可达29.03 mg/g,比对照组(14.30 mg/g)提高到2.03倍。而在相同的茴香醚诱导条件下,初始藻细胞的不同生长状态对细胞的生长及虾青素的合成也有很大的影响:处于对数生长期后期(14 d)的细胞更有利于虾青素的积累,其最大虾青素含量达到29.3 mg/g,分别比前期、中期和稳定期提高到2.9、1.01和1.72倍。实验表明茴香醚是虾青素诱导的有效诱导子,本研究也可为两阶段法诱导生产虾青素提供依据。     

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。     

3-羟基丁酸对雨生红球藻PSⅡ光化学活性与色素合成的影响

利用试验生态学方法,研究3-羟基丁酸对雨生红球藻光系统Ⅱ（photosystem Ⅱ,PSⅡ）光化学活性与色素合成的影响。结果发现,3-羟基丁酸对雨生红球藻PSⅡ光反应中心造成胁迫损伤,细胞PSⅡ光化学最大光能转化效率（Fv/Fm）、有效光能转化效率（Yield）和表观电子传递速率（electron transfer rate,ETR）的参数均出现不同程度降低,其中0.02、0.10 μg/L 处理组的作用最明显;3-羟基丁酸造成雨生红球藻叶绿素a 含量波动变化并最终下降至较低水平;3-羟基丁酸能够促进雨生红球藻合成并积累虾青素,其中0.01 μg/L 浓度的促进作用最强,虾青素最高产量为7.57 mg/L;3-羟基丁酸影响了雨生红球藻自身资源在光合作用与抗逆合成之间的权衡分配。研究结果为使用3-羟基丁酸作为外源诱导物促进雨生红球藻生产虾青素提供了依据。     

人工调控雨生红球藻积累虾青素的研究进展

红球藻是富含营养价值和药用价值的藻类食品。雨生红球藻是自然界中生产天然虾青素的理想来源,虾青素作为一种高效的纯天然抗氧化剂,在清除自由基、抗衰老、抗肿瘤和免疫调节等方面显示出良好的生物活性。利用雨生红球藻生产虾青素具有广阔的发展前景,已成为近年来国际上研究的热点。此外,天然虾青素产量有限,大部分虾青素都是人工调控生产的。因此,本文综述了国内外人工调控对雨生红球藻积累虾青素的方法,对我国提高雨生红球藻生产虾青素具有重要意义。     

雨生红球藻虾青素积累条件优化研究

虾青素具有很强的抗氧化能力,雨生红球藻在目前已知生物中虾青素含量最高。该文采用响应面法研究温度、pH值、NaCl浓度、钨酸钠浓度对雨生红球藻虾青素积累的影响。结果表明,雨生红球藻虾青素积累的最佳工艺条件为温度27℃、pH 9.6、NaCl浓度1.7 g/L、钨酸钠浓度5.2 mmol/L。各因素对雨生红球藻虾青素积累影响的顺序为pH值＞温度＞钨酸钠浓度＞NaCl浓度。     

雨生红球藻天然虾青素提取研究进展

文章介绍了天然虾青素的生物来源、功能及应用研究的新进展,对比分析了雨生红球藻的不同破壁方法和虾青素的提取方法,重点介绍了近期国内外对雨生红球藻虾青素高效分离提取的新方法,并对其未来发展方向进行了展望。     

Enlarged and astaxanthin-accumulating cyst cells of the green alga Haematococcus pluvialis

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2′,7′-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.     

Ionic liquid as an effective solvent for cell wall deconstructing through astaxanthin extraction from Haematococcus pluvialis

Haematococcus pluvialis is a proficient source of natural antioxidant astaxanthin. However, the efficient extraction of astaxanthin from this microalga remains a great challenge due to the presence of the tough and non-hydrolysable cell walls. In this study, ionic liquid (IL) pretreatment was used for deconstruction of cell wall method. Imidazolium-based ILs exhibited higher cell disruption capability than pyridinium-based and ammonium-based ILs. After the ILs determination, 1-butyl-3-methylimidazolium chloride ([Bmim] Cl) was the most efficient method for cell wall deconstruction that leads to the highest astaxanthin extractability. More than 80% astaxanthin was extracted from H. pluvialis under mild conditions (pretreatment with 40% IL aqueous solution at 35 °C, followed by methanolic extraction at 50 °C). In addition, [Bmim] Cl showed the excellent recyclability, and negligible loss of astaxanthin during IL pretreatment was observed. The present work demonstrates that the combination of IL pretreatment and organic solvent extraction was an energy efficient and eco-friendly process for the astaxanthin recovery from H. pluvialis.     

Highly efficient preparation of free all-trans-astaxanthin from Haematococcus pluvialis extract by a rapid biocatalytic method based on crude extracellular enzyme extract

A highly efficient, rapid, green and safe procedure for the preparation of free all-trans-astaxanthin from Haematococcus pluvialis algal extract, by a crude extracellular enzyme extract, was reported. The free all-trans-astaxanthin obtained by the biocatalysed method had fewer side products compared to the saponification procedure. Through single-factor experiments and a Box–Behnken design, it was possible to find the optimal biocatalytic conditions for the hydrolysis of 2 mg of H. pluvialis oil with 14.7 mg (protein content) of lyophilised crude extracellular enzyme extract obtained from Pseudomonas aeruginosa. The reaction was carried out in 30 min at pH 9.16 and 36 °C, in 5.5 mL total reaction volume, under nitrogen atmosphere and dark conditions. The hydrolysis ratio of the astaxanthin esters was 98.72%, and the production of free all-trans-astaxanthin was 82.83 μg per mg of H. pluvialis oil. The method herein reported was simpler than other enzymatic methods previously described and allowed saving of time and costs.     

Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis

Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from Haematococcus pluvialis was carried out, for several experimental conditions, using a semi-continuous apparatus. The microalga was previously freeze-dried and ground with a ball mill. The effects of pressure (200 and 300 bar), temperature (40 and 60 °C), degree of crushing, as well as the use of ethanol as a co-solvent (10%) on the extraction efficiency were assessed. Organic solvent extractions, using acetone, were also carried out in a vortex, on ground cells mixed with very small glass beads. Supercritical extraction from the completely crushed alga was compared with acetone and the highest recovery of carotenoids (92%) was obtained at the pressure of 300 bar and the temperature of 60 °C, using ethanol as a co-solvent.The extraction recovery increased with the pressure at 60 °C. On the other hand, the increase in temperature, at 300 bar, led to a slight improvement. The main carotenoid of Haematococcus pluvialis is the esterified astaxanthin (about 75%). Other carotenoids present are lutein, astaxanthin (free), β-carotene and canthaxanthin. All of them were recovered through supercritical fluid extraction with values higher than 90%, with the exception of canthaxanthin (about 85%), at a pressure of 300 bar and a temperature of 60 °C.     

The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis

The efficiency of various techniques pulsed electric field (PEF), ultrasound (US), high‐pressure microfluidisation (HPMF), hydrochloric acid (HCl) and ionic liquids (ILs) for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis was compared. The results indicated that ILs, HCl and HPMF treatment were shown the most efficient for cell disruption with more than 80% astaxanthin recovery. While the cell wall integrity of H. pluvialis cyst cell was less affected by US and PEF treatment. It was found that imidazolium‐based ILs showed the greater potential for cell disruption than pyridinium‐based and ammonium‐based ILs. Among all the ILs examined, 1‐butyl‐3‐methylimidazolium chloride ([Bmim] Cl) exhibited efficient cell disruption and capability of astaxanthin recovery at mild condition (pretreatment with 40% IL aqueous solution at 40 °C, followed by extraction with methanol at 50 °C) without extensive energy consumption and special facility requirement. In addition, recyclability of ILs was excellent.     

Potential health-promoting effects of astaxanthin: a high-value carotenoid mostly from microalgae

The ketocarotenoid astaxanthin can be found in the microalgae Haematococcus pluvialis, Chlorella zofingiensis, and Chlorococcum sp., and the red yeast Phaffia rhodozyma. The microalga H. pluvialis has the highest capacity to accumulate astaxanthin up to 4–5% of cell dry weight. Astaxanthin has been attributed with extraordinary potential for protecting the organism against a wide range of diseases, and has considerable potential and promising applications in human health. Numerous studies have shown that astaxanthin has potential health‐promoting effects in the prevention and treatment of various diseases, such as cancers, chronic inflammatory diseases, metabolic syndrome, diabetes, diabetic nephropathy, cardiovascular diseases, gastrointestinal diseases, liver diseases, neurodegenerative diseases, eye diseases, skin diseases, exercise‐induced fatigue, male infertility, and HgCl₂‐induced acute renal failure. In this article, the currently available scientific literature regarding the most significant activities of astaxanthin is reviewed.     

Preparation of stable microcapsules from disrupted cell of Haematococcus pluvialis by spray drying

Haematococcus pluvialis, including astaxanthin, disrupted by high‐pressure homogenisation was microencapsulated with Maillard reaction products as wall materials by spray drying. The microcapsules were characterised by scanning electron microscope, size analysis and also the storage stability. The optimised cell disruption process for H. pluvialis based on response surface optimisation was 70 MPa of pressure, 7.38% of H. pluvialis concentration and homogenisation once with a disruption rate of 98.96 ± 0.12%. The optimised spray drying process consisted of a wall‐to‐core material ratio of 2.4:1, inlet temperature of 180 °C and outlet temperature of 80 °C with a microencapsulation rate and microcapsule production rate of (92.1 ± 0.1)% and (97.7 ± 0.2)%, respectively. Characterisation and stability test showed that this microencapsulation process ensured the stability of astaxanthin.     

Mechanisms of breakdown of Haematococcus pluvialis cell wall by ionic liquids,hydrochloric acid and multi-enzyme treatment

The robust cell wall structure of Haematococcus pluvialis (H. pluvialis) consists of polysaccharides and tough non-hydrolysable sporopollenins, which makes it difficult to extract superpotent antioxidant, astaxanthin from these cells. Therefore, breakdown of cell wall is a key step in the overall process of astaxanthin recovery. In this study, the mechanism of three well-established chemical techniques for cell disruption of H. pluvialis cysts [ionic liquids (IL), hydrochloric acid (HCl) and multiple enzymes (multi-enzyme, ME)] on deconstruction of the cyst cell wall of H. pluvialis was explored and characterised by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS) analyses. The results demonstrated that the three cell wall breakdown techniques exhibited high extraction efficacies for the recovery of astaxanthin from H. pluvialis [IL (86.71 ± 2.06%), HCl (80.52 ± 2.28%) and ME (71.08 ± 2.49%)]. However, their performances on disrupting the trilayered cell walls of H. pluvialis were significantly different, which were confirmed by distinct morphologies of the treated cell walls visualised by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Meanwhile, the results of FTIR confirmed that, to some extent, cellulose, hemicellulose and lignin in the cell walls were hydrolysed by HCl, IL and ME treatments. However, ME exhibited a less hydrolytic effect on lignin than HCl and IL. Moreover, XRD and NMR analyses implied that the amorphous region of cell wall was susceptible to hydrolysis/breakdown by the three techniques.