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.     

Pressurized liquids as an alternative process to antioxidant carotenoids' extraction from Haematococcus pluvialis microalgae

In this work, extraction of antioxidant carotenoids from Haematococcus pluvialis microalga, has been studied combining pressurized liquid extraction (PLE), using hexane and ethanol as extracting solvents, and analytical techniques such as thin layer chromatography (TLC) and HPLC with DAD. The effect of the extraction temperature (50, 100, 150 and 200 °C) and the polarity of the solvent have been studied in terms of in vitro antioxidant activity and chemical composition considering two different morphological cells (green vegetative cells and red cysts). Results demonstrate that the extraction temperature had a positive influence in the extraction yield while its effect in the antioxidant activity was negative, lowering the activity of the extracts with an increase of the extraction temperature. The best yields were obtained with ethanol at the higher extraction temperature while the best antioxidant activity was also achieved using ethanol but at lower temperatures. Chemical composition was determined by TLC and HPLC with DAD. Several compounds were identified in the samples and concentration of astaxanthin was obtained. Results pointed out that the extracts contained different carotenoids in both, the green and the red phase, and that depending on its contribution a stronger antioxidant activity would be expected.     

Green processes based on the extraction with pressurized fluids to obtain potent antimicrobials from Haematococcus pluvialis microalgae

In the present work, the antimicrobial activity of different pressurized liquid extracts obtained from Haematococcus pluvialis microalga was tested against several microorganisms of importance for the food industry (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger). Extractions were performed with hexane and ethanol at four different temperatures (50, 100, 150 and 200 °C) for 20 min. The results showed that extracts obtained with both solvents (hexane and ethanol) from the green motile cells of the microalgae (green phase) presented a low antimicrobial activity against all the microorganisms tested. However, the antimicrobial activity of the extracts obtained from the red hematocysts without flagella (red phase) was totally different depending on the solvent used for the extraction. Hexane extracts showed an antimicrobial activity quite similar to that obtained with the green microalgae, while the antimicrobial activity of ethanol extracts was much higher. This fact seems to indicate that compounds related to antimicrobial activity of this microalga are found in higher quantities in the red phase of the microalgae and could be relatively polar compounds. Moreover, ethanol extracts from the red phase obtained at 100 °C presented the highest antimicrobial activity. In order to identify the compounds responsible for the antimicrobial activity, a GC-MS characterization of the extracts obtained with both hexane and ethanol at 100 °C, for Haematococcus pluvialis in the green and red phases was also performed. Therefore, the highest antimicrobial activity of the ethanol extract corresponding to red Haematococcus can be associated with the presence in this extract of short-chain fatty acids, which have been previously described to possess antimicrobial activity.     

Effect of high hydrostatic pressure on the structure of the soluble protein fraction in Porphyridium cruentum extracts

High hydrostatic pressure (HHP) treatments are trending as “green” stabilization and extraction process. The extraction of B-phycoerythrin from microalgae is getting more and more interest due to its numerous potentialities in foods, cosmetics and medicine. Thus, the effects of high pressure on the structural characteristics of B-phycoerythrin extracted from Porphyridium cruentum are explored in this paper.Spectrophotometric methods allowed to measure B-phycoerythrin content (UV–visible) and gave an indication on the protein structure (fluorescence). Micro-DSC analysis and electrophoresis complemented this structural investigation for all the protein fractions of P. cruentum extracts.Applying high hydrostatic pressure treatments up to 300 MPa during 5 min had no significant effect on B-phycoerythrin content and structure in P. cruentum extracts. Nevertheless, conformational changes of the protein are suggested by fluorescence yield decrease at 400 MPa, and protein aggregation of B-phycoerythrin, observed by Micro-DSC and electrophoresis, occurred at 500 MPa.Industrial relevanceThe HHP process is an emerging technology for the microbiological stability of various food matrices, including the proteins of microalgae as natural colorant. The target pressure to stabilize is around 400 MPa. High hydrostatic pressure can be used on P. cruentum extracts up to 300 MPa without any change in protein structure, as the threshold of protein aggregation is observed at 400 MPa. The observed changes of the proteins structure after applying HHP above 400 MPa can have a strong impact at macroscopic scale on the food matrices: increase of turbidity, change of texture, stability of emulsion.     

Extraction and analysis of antioxidant components from Origanum dictamnus

Three alternative extraction procedures were carried out in order to separate the antioxidant components and isolate an efficient extract from Origanum dictamnus. Procedure A included sequential extractions with petroleum ether (PE), diethyl ether (DE) and ethanol; procedure B sequential extractions with PE and ethyl acetate (EAc); procedure C a single step extraction with ethanol. The most efficient radical scavengers, according to the DPPH method, were isolated in ethanol extract of procedure A (mainly rosmarinic acid), followed by ethanol extract of procedure C. However, both ethanol extracts had low solubility in oil and could not protect it. EAc and DE extracts, containing mainly apigenin and epirosmanol ethyl ether, presented lower radical scavenging activity but were very effective against autoxidation of cottonseed oil (a concentration of 200 ppm was adequate to stabilize it). PE extract protected the oil effectively at concentration of 350 ppm, while being the least active against DPPH.Industrial relevanceThis study examines Origanum dictamnus as a potential antioxidant additive for edible oils and fats. Three different extraction procedures of the raw material with non-toxic organic solvents were used to recover the sum of the extractable compounds and specific fractions respectively. The extraction procedures, accompanied with analytical techniques, led to the characterization of the antioxidant properties and the composition of the plant as well as the isolation of very efficient oil-soluble antioxidant fractions.     

Novel foods with microalgal ingredients – Effect of gel setting conditions on the linear viscoelasticity of Spirulina and Haematococcus gels

Microalgae represent an alternative and innovative source of natural ingredients that can be used in the development of novel food products. Biologically active compounds (e.g. carotenoids) are naturally encapsulated within microalgal cells, being able to resist harsh technological conditions involved in food technology processes. The aim of this work was to study the effect of adding Haematococcus pluvialis and Spirulina maxima microalgal biomass on the linear viscoelastic behaviour of vegetarian food gels prepared from pea protein, κ-carrageenan and starch. The gelation process was monitored in situ through dynamic oscillatory measurements, under different thermal profile conditions. Increasing temperature (70–90 °C, 5 min) resulted in more structured gels, while the effect of time (5–30 min, at 90 °C) was less pronounced. The effect of heating and cooling rates on gel setting was also studied. Haematococcus gels were highly structured and less dependent on gel setting conditions. Spirulina gels presented lower values of viscoelastic functions than the control (gel matrix without microalgae), but this was overcome when using lower heating/cooling rates.     

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.     

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.     

Effect of different solvents on volatile and non-volatile constituents of red bell pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and their in vitro antioxidant activity

The fresh fruit of Capsicum annuum L. (red bell pepper) was successively extracted using n-hexane, chloroform, ethyl acetate, ethanol and their percentage yield was calculated. The effectiveness of each extract on chemical composition and antioxidant activity was studied. The qualitative phytochemical evaluation of each extract of bell pepper was done by HPTLC and Gas chromatography–mass spectroscopy (GC-MS) analysis. The total content of phenols, flavonoids and carotenoids were estimated by standard chemical methods. Further, the antioxidant potential of each extract was measured via DPPH and reducing power assays. Gas chromatography–mass spectroscopy analysis showed that the majority of compounds were related to phenols and flavonoids. Further analysis of the extract by HPTLC verified the presence of different types of phenolic compounds in addition to flavonoids and carotenoids. Among the different solvent extracts analyzed, total phenolic content was higher in ethanol extract (7.136?±?0.03%, w/w) whereas ethyl acetate extract showed the presence of higher flavonoid content (4.0521?±?0.03%, w/w). The ethanol and ethyl acetate extracts of the fruit of C. annuum exhibited the highest radical scavenging activity with inhibition percentage of 53.66 and 49.55% at a concentration of 254 µg/ml. Based on the biochemical analysis and phytochemical screening, we conclude that C. annuum possess potent antioxidant potential and this ability of the extract is attributed to the presence of rich polyphenolic compounds.     

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.     

Cyphomandra betacea Sendt. phenolics protect LDL from oxidation and PC12 cells from oxidative stress

Cyphomandra betacea Sendt. (tamarillo) is a subtropical fruit containing rich contents of anthocyanins and carotenoids. The antioxidant activity was investigated using a crude ethanol extract of C. betacea fruit and its partitioned fractions, i.e. the ethyl acetate, butanol and water fractions. The ethyl acetate fraction exhibited the highest DPPH scavenging activity, Trolox equivalent antioxidant capacity (TEAC) and total phenol content. C. betacea phenolics in ethyl acetate fraction inhibited copper-induced LDL oxidation equally to or more effectively than dl-α-tocopherol, as measured by decreased formation of thiobarbituric acid-reactive substances (TBARS), conjugated diene and relative electrophoretic mobility (REM). Furthermore, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction assay showed that C. betacea phenolics in ethyl acetate fraction prevent oxidative stress-induced cell death in neuronal PC12 cells in a dose-dependent manner via attenuation of ROS production. In conclusion, C. betacea phenolics are potent antioxidants which can inhibit LDL oxidation in vitro and ROS production in PC12 cells.     

Comparison of different methods for extracting polyphenols from Ipomoea batatas leaves,and identification of antioxidant constituents by HPLC-QTOF-MS2

This study aimed to compare the extraction efficiency of dynamic high pressure microfluidization-assisted extraction (DHPMAE) and other commonly used extraction methods on the antioxidant activities (AAs) of sweet potato leaves (Ipomoea batatas L., SPL), and to identify the antioxidant compounds by HPLC-QTOF-MS². Two highly consumed commercial varieties, orange-fleshed and cream-fleshed sweet potato leaves (OFL and CFL), were taken as materials. OFL was evidenced to be a better antioxidants resource than CFL due to higher polyphenols and AAs. DHPMAE had the best extraction efficiency on total phenolics and total flavonoids, with the values in OFL extract at 16.35 mg GAE/g DM and 16.51 mg RE/g DM, respectively. DHPMAE extract also exhibited the strongest DPPH· and ABTS·⁺ scavenging ability, chelating ability and reducing power. Multiple regression analysis revealed that polyphenols played an important contribution to the AAs of SPL extracts. After successive fractionation of DHPMAE extract by chloroform, ethyl acetate, and n-butanol, the ethyl acetate fraction showed the highest total phenolic (569.38 mg GAE/g Extract) and total flavonoid (43.15 mg RE/g Extract) content, as well as the highest AAs. HPLC-QTOF-MS² analysis of the ethyl acetate fraction resulted in identifying 37 compounds including 20 phenolic acids, 12 flavonoids, 3 organic acids, 1 nucleoside and 1 ester, and 20 of them were firstly detected in SPL. Caffeoylquinic acid derivatives and flavonoids were the most abundant antioxidant compounds. The presented results suggested that DHPM might be a promising assisted technology in extracting polyphenols from plants, and SPL could be utilized for the development of functional food.     

Assessment of Carotenoids Recovery through Cell Rupture of <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> CBS 2636 Using Compressed Fluids

The increasing demand for carotenoids by industries has drawn attention to their bio-production. Since pigments are intracellular, extraction steps are then needed after cell cultivation. In this work, different strategies for extraction of carotenoid pigments from Sporidiobolus salmonicolor (CBS 2636) were investigated. The cell rupture was carried out using dimethyl sulfoxide (DMSO), two pure compressed fluids, supercritical carbon dioxide and propane, and also a combination of pressurized fluid treatment followed by liquid DMSO. Dichloromethane, ethanol, ethyl acetate, and acetone were tested for the carotenoids extraction. Results obtained show that when multiple solvents were used a synergetic effect on the extent of carotenoids recovery was verified. Maximum concentration of total carotenoids (2,875 μg/L) was obtained in the treatment using supercritical CO₂ (300 bar/120 min) followed by dimethyl sulfoxide to disrupt the cell, and then the extraction with a solution of acetone/methanol (7:3, v/v).     

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.     

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。     

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.     

Citharexylum solanaceum fruit extracts: Profiles of phenolic compounds and carotenoids and their relation with ROS and RNS scavenging capacities

Citharexylum solanaceum is a native fruit from Brazil, which both bioactive compounds and antioxidant potential were not yet investigated. Thus, the freeze-dried extracts of seed and pulp + skin of C. solanaceum fruits were obtained after solid-liquid extraction with ethanol and their bioactive compounds composition, namely phenolic compounds and carotenoids, were determined. The antioxidant capacity of both extracts against physiologically relevant reactive oxygen species (ROS) and reactive nitrogen species (RNS) was further investigated. Both C. solanaceum extracts showed high contents of phenolic compounds; however, pulp + skin extract presented 2.4-times more phenolic compounds (33.54 mg/g) than the seed extract (14.09 mg/g). Verbascoside (phenylpropanoid) was the major compound identified in both extracts (11–25 mg/g). Regarding the carotenoid composition, all-trans-lutein (14–42 μg/g) and all-trans-β-carotene (13–44 μg/g) were the major compounds in both extracts. The high content of phenolic compounds and carotenoids in pulp + skin extract might explain its higher scavenging capacity against all the ROS/RNS as compared to seed extract. In general, both extracts showed better scavenging capacity for the RNS than for the ROS. Our results indicate that C. solanaceum fruits can be explored as an important natural source of antioxidant compounds.     

Industrial potential of carotenoid pigments from microalgae: Current trends and future prospects

Microalgae are rich source of various bioactive molecules such as carotenoids, lipids, fatty acids, hydrocarbons, proteins, carbohydrates, amino acids, etc. and in recent Years carotenoids from algae gained commercial recognition in the global market for food and cosmeceutical applications. However, the production of carotenoids from algae is not yet fully cost effective to compete with synthetic ones. In this context the present review examines the technologies/methods in relation to mass production of algae, cell harvesting for extraction of carotenoids, optimizing extraction methods etc. Research studies from different microalgal species such as Spirulina platensis, Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., Botryococcus braunii and Diatoms in relation to carotenoid content, chemical structure, extraction and processing of carotenoids are discussed. Further these carotenoid pigments, are useful in various health applications and their use in food, feed, nutraceutical, pharmaceutical and cosmeceutical industries was briefly touched upon. The commercial value of algal carotenoids has also been discussed in this review. Possible recommendations for future research studies are proposed.     

Industrially scalable complex coacervation process to microencapsulate food ingredients

Microencapsulation by conventional complex coacervation, though highly effective and achievable at the bench-scale, is challenging to scale-up because of the complexity of the process. A novel, industrially-scalable microencapsulation process by in situ complex coacervation during spray drying (the ‘CoCo process’) is introduced, where the multiple steps are collapsed into one, to form dry complex coacervated (CoCo) microcapsules by spray drying. The CoCo process was used to encapsulate d-limonene in CoCo microcapsules using alginate and gelatin as wall materials. Insoluble CoCo particles were produced without chemical cross-linking, with extents of complex coacervation of 75 ± 6% and 64 ± 6% for CoCo particles with and without d-limonene, respectively. Up to 82.7% of d-limonene was retained during spray drying; moreover, the CoCo matrix exhibited excellent barrier properties, retaining up to 80.0% of total d-limonene over 72-day storage in sealed vials at room temperature.Industrial relevanceCommercialization of microencapsulation of bioactives by complex coacervation in agricultural and food applications is hindered by the high-cost and time-intensive multistep process consisting of emulsification, coacervation, shell hardening and drying. In this work, we overcome these limitations by developing an industrially scalable in situ complex coacervation process during spray drying (‘CoCo process’). One-step complex coacervation during spray-drying opens the door to cost-effective, high-throughput, high-volume production of bioactive-containing microcapsules. The protective matrix microcapsules formed by this novel process stabilize and protect the bioactive, while allowing controlled release of the cargo for various applications in the food and many other industries.