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。     

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.     

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.     

富含虾青素的红球藻粉对大鼠的生理影响

目的观察红球藻粉对大鼠生理的影响。方法连续30 d灌胃给予Wistar大鼠0.4 g/kg,0.8 g/kg和1.6g/kg剂量红球藻破壁藻粉,观察其行为和生理变化。结果动物外观和行为无异常反应,没有发生死亡和明显毒性反应,体重迅速增加,雌雄个体间出现明显分化,雄性增重速度快、摄食量大。单因子统计显示红球藻粉对大鼠体重无明显影响(P>0.0 5),但配对统计表明红球藻粉对大鼠体重有不同程度作用。该作用与摄食量无关,而与剂量和大鼠性别有一定关系,低剂量使雌雄大鼠增重;中剂量使雄性减重而对雌性无影响;高剂量使雌性增重而对雄性无影响。红球藻粉对大鼠血液学及血清各项生化指标均无明显影响(P>0.05)。结论国产红球藻粉无明显的毒性反应,食用安全。     

Astaxanthin preparation by lipase-catalyzed hydrolysis of its esters from Haematococcus pluvialis algal extracts

Five of 8 fungal lipases screened were found to effectively hydrolyze astaxanthin esters from Haematococcus pluvialis algal cell extracts. Among these, an alkaline lipase from Penicillium cyclopium, expressed in Pichia pastoris, had the highest enzymolysis efficiency. Tween80 was shown to be an effective emulsifier in this lipase hydrolysis system for the 1st time. A series of experiments were performed to find optimal conditions for hydrolysis (pH, temperature, reaction time, lipase dosage). In the optimal reaction system, Tween80 and H. pluvialis extracts (mass ratio 1:1) were emulsified and added to the above lipase at a dosage of 4.6 U/μg (relative to total carotenoids), in phosphate buffer (0.1 M, pH 7.0), and incubated at 28 °C for 7 h, with agitation at 180 rpm. The free astaxanthin recovery ratio under these conditions was 63.2%.     

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.     

Enzymatic enrichment of astaxanthin from <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cell extracts

An industrially available preparation of astaxanthin (Ax) from Haematococcus pluvialis contained 41.6 wt% acylglycerols and 24.9 wt% FFA in addition to 14.6 wt% Ax, which was a mixture of free and FA ester forms (free Ax/Ax monoesters/Ax diesters=4.9∶80.3∶14.8, by mol). Enrichment of Ax by a two-step process was attempted. The first step was hydrolysis of acylglycerols with Candida rugosa lipase: A mixture of 1.0 kg H. pluvialis cell extracts, 1.0 L water, and 50 U/g-reaction mixture of the lipase was agitated at 30°C for 42 h. The degree of hydrolysis of acylglycerols reached 94.4%, but Ax esters were not hydrolyzed. Removal of FFA from the resulting oil layer by molecular distillation enriched the content of Ax esters to 40.8 wt5 (named Ax40). The second step was enzymatic conversion of Ax esters to free Ax, which successfully proceeded in the presence of ethanol (EtOH). When a mixture of 50.0 g Ax40, 8.2 g EtOH (5 molar equiv. against FA), 58.2 mL water, and 1500 U/g-mixture of Pseudomonas aeruginosa lipase was stirred at 30°C for 68 h, the free Ax content increased to 89.3 mol%. Free Ax was efficiently recovered by precipitation with n-hexane. The purity of Ax was thereby raised to 70.2 wt% with a 63.9% overall recovery of the initial content in the cell extracts.     

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.