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.

Three 5’-flanking Regions of crtO Encoding β-carotene Oxygenase in Haematococcus pluvialis

Three separate 5＇-flanking regions （1.1 kb, 1.9 kb and 2.2 kb） of crtO were cloned through walking upstream. Results of sequence analysis show that three 5＇-flanking regions of crtO might have some similar putative cis-acting elements such as ABA （abscisic acid）-responsive element （ABRE）, C-repeat/dehydration responsive element （C-repeat/DRE）, light-responsive element （G-box, GAG-motif, I-box and ATC-motif）, wound-responsive element （WUN-motif）, auxin-responsive element （TGA-element）, MeJA-responsive element （TGACG-element） and MYB binding site （MBS）, except for typical TATA box or CCAAT box. These findings might mean diversiform regulatory patterns of crtO being in astaxanthin biosynthesis of Haematococcus pluvialis.

Regulatory Analysis of IPP Isomerase Gene in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvia/is uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of astaxanthin in the cytosol. The enzyme, isopentenyl pyrophosphate （IPP） isomerase, plays a key role in astaxanthin biosynthesis of H. pluvialis. In this paper, two separate 5＇-flanking regions （1.8 kb and 2.5 kb） of IPP isomerase gene was cloned through walking upstream firstly. Results of sequence analysis =showed that two separate 5＇-flanking regions of IPP isomerase gene might have similar putative cis-acting elements such as ABA （abscisic acid）-responsive element （ABRE）, drought-responsive element （DRE/C-repeat）, light-responsive element （G-box, GAG-motif, I-box and ATC-motif）, heat-shock element （HSE）, wound-responsive element （WUN-motif）, SA （salicylic acid）-responsive element （TCA-element）, auxin-responsive element （TGA-element）, MeJA （methyl jasmonate）-responsive element （TGACG-element）, enhancer-like element involved in anoxic specific inducibility （GC-motif） and MYB binding sites （MBS and MRE）, except for typical TATA box or CCAAT box, which exhibit diversiform transcriptional patterns of IPP isomerase gene in astaxanthin biosynthesis of Haematococcus pluvialis.

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.

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.

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.

Cell cycles and proliferation patterns in Haematococcus pluvialis

Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation;far less attention has been paid to cell cycles and proliferation patterns.The purpose of this study was to clarify cell cycles and proliferation patterns in H.pluvialis microscopically using a camera and video recorder system.The complicated life history of H.pluvial i s can be divided into two stages:the motile stage and the non-motile stage.All the cells can be classifi ed into forms as follows:motile cell,nonmotile cell,zoospore and aplanospore.The main cell proliferation,both in the motile phase and non-motile phase in H.pluvialis,is by asexual reproduction.Under normal growth conditions,a motile cell usually produces two,sometimes four,and exceptionally eight zoospores.Under unfavorable conditions,the motile cell loses its fl agella and transforms into a non-motile cell,and the non-motile cell usually produces 2,4 or 8 aplanospores,and occasionally 20–32 aplanospores,which further develop into non-motile cells.Under suitable conditions,the non-motile cell is also able to release zoospores.The larger non-motile cells produce more than 16 zoospores,and the smaller ones produce 4 or 8 zoospores.Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase.There is,as yet,no convincing direct evidence for sexual reproduction.

Identification and Functional Characterization of a NovelΔ12 Fatty Acid Desaturase Gene from Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis accumulates large amounts of fatty acids in response to adverse conditions.However,the key fatty acid desaturase genes in H.pluvialis remain unknown.In this study,we cloned and functionally characterized aΔ12 fatty acid desaturase gene,and designated it as HpFAD2.The open reading frame of HpFAD2 consisted of 1137 base pairs and encoded 378 amino acids.The deduced polypeptide showed 70%identity to other endoplasmic reticulumΔ12 fatty acid desaturases,whereas it had only 44%identity to plastidΔ12 fatty acid desaturases.The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulumΔ12 fatty acid desaturases.Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2.Two additional fatty acids(C16:2 and C18:2)were detected in the yeast transformants.The results indicatedΔ12 desaturation activity and substrate preference for C18:1 over C16:1.The transcriptional levels of H.pluvialis HpFAD2 at different growth stages were measured by quantitative polymerase chain reaction(PCR),indicating that the HpFAD2 transcriptional levels were significantly higher in red cells than those in green cells.Our study brings more insight into the fatty acid biosynthetic pathway of H.pluvialis.

DYNAMIC CHANGES OF INORGANIC NITROGEN AND ASTAXANTHIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS

This study on dynamic changes of culture color, astaxanthin and chlorophylls, inorganic N including N NO - 3, N NO - 2 and N NH + 4 in batch culture of Haematococcus pluvialis exposed to different additive nitrate concentration showed (1) ast/chl ratio was over 0.8 for brown and red algae, but was usually less than 0.5 for green and yellow algae; (2) N NO - 3, in general, was unstable and decreased, except for a small unexpected increase in nitrate enriched treatment groups; (3) measurable amounts of N NO - 2 and N NH + 4 were observed respectively with three change modes although no external nitrite and ammonia were added into the culture; (4) a non linear correlation between ast/chl ratio (or color) changes and the levels of N NO - 3 , N NO - 2 , N NH + 4 in H. pluvialis culture; (5) up and down variation of the ast/chl ratio occurred simultaneously with a perceptible color change from yellow to brown (or red) when N NO - 3, N NO - 2 and N NH + 4 fluctuated around 30, 5, 5 μmol/L respectively; (6) existence of three dynamic modes of N NO - 3, N NO - 2 and N NH + 4 changes, obviously associated with initial external nitrate; (7) the key level of total inorganic N concentration regulating the above physiological changes during indoor cultivation was about 50 μmol/L; and (8) 0.5-10 mmol/L of nitrate was theoretically conducive to cell growth in batch culture.

Effects of iron electrovalence and species on growth and astaxanthin production of Haematococcus pluvialis

To increase the cell concentration and the accumulation of astaxanthin in the cultivation of Haematococcus pluvialis, effects of different iron electrovalencies （Fe^2＋-EDTA and Fe^3＋-EDTA） and species （Fe-EDTA, Fe（OH）x^32x and FeC6H5O7） addition on cell growth and accumulation of astaxanthin were studied. Results show that different iron electrovalencies have various effects on cell growth and astaxanthin accumulation of H. pluvialis. Compared with Fe^3＋-EDTA, Fe^2＋-EDTA stimulate more effectively the formation of astaxanthin. The maximum astaxanthin content （30.70 mg/g biomass cell） was obtained under conditions of 18 μmol/L Fe^2＋-EDTA, despite the lower cell density （2.3×10^5 cell/ml） in such condition. Fe^3＋-EDTA is more effective than Fe^2＋-EDTA in improving the cell growth. Especially, the maximal steady-state cell density, 2.9×10^5 cell/ml was obtained at 18 μmol/L Fe^3＋-EDTA addition. On the other hand, all the various species of iron （EDTA-Fe, Fe（OH）x^32x, FeC6H5O7） are capable to improve the growth of the algae and astaxanthin production. Among the three iron species, FeC6H5O7 performed the best. Under the condition of a higher concentration （36 μmol/L） of FeC6H5O7, the cell density and astaxanthin production is 2 and 7 times higher than those of iron-limited group, respectively. The present study demonstrates that the effects of the stimulation with different iron species increased in the order of FeC6H5O7, Fe（OH）x^32x and EDTA-Fe.

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.

Variation in Rubisco and other photosynthetic parameters in the life cycle of Haematococcus pluvialis

Cells of Haematococcus pluvialis Flot. et Will were collected in four different growth phases. We quantified the initial and total enzyme activity of ribulose-1,5-bisphosphate carboxylase （Rubiseo） in crude extracts, and the relative expression of large-subunit ribulose-1,5-bisphosphate caboxylase / oxygenase （rbcL） mRNA. We measured the ratio of photosynthetic rate to respiration rate （P/R）, maximal effective quantum yield of photosystem II （Fv/Fm）, electron transport rate （ETR）, actual photochemical efficiency of PSI1 in the light （ФPSII）, and non-photochemical quenching （NPQ）. Green vegetative cells were found to be in the most active state, with a relatively higher P/R ratio. These cells also displayed the lowest NPQ and the highest Fv/Fm, ETR, and ФPSII, indicating the most effective PSII. However, both Rubisco activity and rbcL mRNA expression were the lowest measured. In orange resting cysts with relatively lower P/R and NPQ, Rubisco activity and rbcL expression reached a peak, while Fv/Fm, ETR, and ФPSII were the lowest measured. Taking into account the methods of astaxanthin induction used in industry, we suggest that Rubisco may participate in astaxanthin accumulation in H. pluvialis. A continuous and sufficient supply of a carbon source such as CO2 may therefore aid the large scale production of astaxanthin.

Modeling of Biomass Production of Haematococcus pluvialis

Microalgae cultivation is justified by the production of high-value fine chemicals and biofuels, essential to reduce the emissions of gases that cause global warming. This paper presents a study of the growth of microalgae Haematococcus pluvialis considering light conditions from 2000 to 10,000 lux, temperature 22?C and pH in the 6.5-12.5 range. The experiments were performed in 4 liter flat plate photobioreactors using the Rudic culture medium. The biomass growth was measured by counting cells in a Neubauer chamber. Both the light intensity and the pH of the medium influenced the rate of growth of the microalgae. A model with exponential behavior was proposed to describe the production of microalgae biomass over time. A nonlinear autoregressive model based on an Artificial Neural Network was used to predict the dynamic behavior of the pH during the growth of the microalgae at different light intensities. Simulations were carried out to analyze the behavior of biomass production at other light intensities within the range considered.

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.

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis： Effects of Cultivation Parameters

The green alga, Haematococcus pluvlalis Flotow is used as a source of the ketocarotenoid astaxanthin for application in fish aquaculture, pharmaceutical and cosmetic industries. Ceils of the green alga were induced by the application of different light and starvation conditions to evaluate the effect in astaxanthin accumulate. The conditions used for the Induction were high light intensity （170 μmol·m^-2·s^-1）, iron starvation, sulfur starvation and phosphate starvation. The results show that stresses applied in culture, which interfere with cell division, trigger the accumulation of astaxanthin. Notably, sulfur starvation results in a massive accumulation of this commercially important carotenoid.

Accurate quantification of astaxanthin from Haematococcus crude extract spectrophotometrically

The influence of alkali on astaxanthin and the optimal working wave length for measurement of astaxanthin from Haematocoecus crude extract were investigated, and a spectrophotometric method for precise quantification of the astaxanthin based on the method of Boussiba et al. was established. According to Boussiba＇s method, alkali treatment destroys chlorophyll. However, we found that： 1） carotenoid content declined for about 25% in Haematococcus fresh cysts and up to 30% in dry powder of Haematococcus broken cysts after alkali treatment; and 2） dimethyl sulfoxide （DMSO）-extracted chlorophyll of green Haematococeus bares little absorption at 520-550 nm. Interestingly, a good linear relationship existed between absorbance at 530 nm and astaxanthin content, while an unknown interference at 540-550 nm was detected in our study. Therefore, with 530 nm as working wavelength, the alkali treatment to destroy chlorophyll was not necessary and the influence of chlorophyll, other carotenoids, and the unknown interference could be avoided. The astaxanthin contents of two samples were measured at 492 nm and 530 nm; the measured values at 530 nm were 2.617 g/100 g and 1.811 g/100 g. When compared with the measured values at 492 nm, the measured values at 530 nm decreased by 6.93% and 11.96%, respectively. The measured values at 530 nm are closer to the true astaxanthin contents in the samples. The data show that 530 nm is the most suitable wave length for spectrophotometric determination to the astaxanthin in Haematococcus crude extract.

乙醇-硫酸铵双水相体系萃取虾青素的研究

虾青素具有突出的生理功能和巨大的开发价值。本文研究了乙醇-硫酸铵双水相体系用于雨生红球藻(Haematococcus pluvialis)虾青素粉中虾青素制备的可行性。结果表明:雨生红球藻虾青素粉中含游离虾青素及杂色素。雨生红球藻虾青素粉乙醇提取液与20%或30%硫酸铵溶液形成的双水相可以使部分虾青素形成沉淀集中在上下相之间。当乙醇提取液∶20%硫酸铵(v/v)为1∶1.5时,虾青素沉淀率可达到70%以上,且沉淀中虾青素的纯度较高,提取液中的杂色素基本不沉淀而主要分布在上相。这说明乙醇-硫酸铵双水相体系可用于从雨生红球藻虾青素粉中制备虾青素。