运用雨生红球藻生产虾青素的研究进展

正雨生红球藻属于水生单细胞藻类物质,按照虾青素含量、形态以及颜色等,可将其分为后壁孢子、孢子、游动细胞与不动细胞四种。为提高雨生红球藻虾青素提取量,以雨生红球藻生产虾青素相关专利申请概况介绍为例,对运用雨生红球藻进行虾青素生产的研究进展展开解读,期望能够为雨生红球藻应用提供一些理论方面支持。     

雨生红球藻中虾青素的研究进展

虾青素是一种具有众多生理功能的类胡萝卜素,经济价值极高,但是在自然界中含量较少,其最佳生物来源-雨生红球藻中含量也不超过5%,且该藻的细胞壁较厚,虾青素提取不充分,造成部分虾青素浪费。因此,如何充分提取雨生红球藻中的虾青素已成为研究热点。就虾青素的生理功能、提取方法、雨生红球藻的破壁方法展开论述。     

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。     

雨生红球藻中虾青素提取方法的比较研究

比较了微波法、低温研磨萃取法和碱提法提取雨生红球藻中虾青素的效果,又进一步比较了这3种方法提取的虾青素在总抗氧化能力(T-AOC)和清除羟自由基(·OH)能力等方面的差别.为从雨生红球藻中提取虾青素的商业化生产提供了一些参考和指导.     

雨生红球藻虾青素的制备及生物活性研究进展

虾青素是类胡萝卜素的含氧衍生物,人工养殖的雨生红球藻是天然虾青素的最好来源。虾青素具有多种生物活性,在疾病的预防和治疗中起重要作用。文章介绍了虾青素的提取与稳定性以及在人体中的吸收与代谢,主要对雨生红球藻虾青素的抗氧化活性以及在炎症、糖尿病、心血管疾病、免疫调节以及抗癌等方面的研究进展和应用现状进行了综述。     

雨生红球藻中虾青素含量快速测定方法

雨生红球藻中虾青素以虾青素单酯、二酯以及少量游离虾青素的形式存在,为了准确测定虾青素含量,通常需要将提取的虾青素酯水解转化为游离虾青素,再利用HPLC进行定量,操作耗时,不利于生产过程的快速监测。基于系统研究分光光度法直接测定细胞提取物中的混合虾青素含量和提取-酶解-HPLC法测定的关系,发现分光光度法估算的虾青素含量与HPLC法测定的准确含量之间具有良好的线性关系(r2=0. 997)。基于此建立了雨生红球藻虾青素快速测定方法,并对提取条件进行了优化。雨生红球藻粉(约5 mg)利用1 m L二甲基亚砜和6 m L丙酮进行1次提取,准确定容后,测定474 nm处的吸光度,根据吸光度与HPLC法虾青素含量间线性关系计算雨生红球藻中虾青素的含量。该方法操作简单,仅需10～20 min,测定准确,适于生产和流通环节的所需要的快速测定领域。     

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。     

微生物法生产虾青素的研究进展

虾青素是一种萜烯类不饱和化合物,具有很强的抗氧化活性,同时还具有抗癌、增强免疫、护眼、保护心脑血管等其他诸多生理功能。天然虾青素主要来源于藻类、酵母以及海洋生物中。目前应用于市场的虾青素主要包括雨生红球藻虾青素、酵母虾青素以及化学合成虾青素。雨生红球藻的培养时间长,生长条件要求苛刻,使得虾青素价格昂贵;化学合成虾青素为混合构型虾青素,抗氧化能力低于天然虾青素,安全性较差。该文介绍了虾青素的结构性质、虾青素来源和生产方法,着重讲述红法夫酵母（Phaffia rhodozyma）生产虾青素的生物合成途径、发酵培养条件以及虾青素的破壁提取和纯化方法,为虾青素工业化生产提供一定理论基础。     

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。     

响应面法优化雨生红球藻中虾青素的提取条件

以雨生红球藻粉为原料,选取乙酸乙酯:乙醇(1:1,v/v)为提取溶剂,以虾青素提取率为评价指标,在单因素实验的基础上,利用响应面法对雨生红球藻中虾青素的提取条件进行了优化。结果表明,影响虾青素提取条件的强弱分别为温度>液固比>时间,通过计算回归方程优化得到的最佳提取条件为:温度50℃,时间100 min,液固比1.5:1。通过与荆州公司提供的提取方法和碱提法对比,应用此方法虾青素提取率分别提高了44.31%和86.80%。     

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.     

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.     

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.     

胁迫条件下褪黑素诱导雨生红球藻虾青素合成

雨生红球藻(Haematococcus pluvialis)在胁迫条件下可大量积累虾青素,已成为天然虾青素的主要来源。通过解析外源褪黑素(Melatonin,MLT)调控雨生红球藻在缺氮联合高光照胁迫条件下的防御效应,以期建立虾青素高效合成的技术体系。结果表明,胁迫条件下外源MLT的诱导显著促进了虾青素的积累,最高质量分数达到32.37 mg/g,较对照组增加了2.25倍。此外,外源MLT提高了胞内NO和丝裂原活化蛋白激酶(MAPK)的含量,同时上调了虾青素合成关键酶基因dxs和chy的表达水平。研究表明,外源MLT诱导高光缺氮胁迫下雨生红球藻中虾青素的高效合成可能与MLT调控藻细胞内的NO、MAPK含量和虾青素合成关键酶基因dxs和chy的表达水平相关。     

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.     

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.     

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.     

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.     

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.