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.     

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.     

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。     

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.     

A new environmentally friendly method for astaxanthin extraction from <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

Few environmentally friendly solvents are available to extract food-grade astaxanthin. In this paper, some environmentally friendly solvents, such as lactic acid, ethyl lactate, and ethanol, were employed in cell disruption and astaxanthin extraction from Xanthophyllomyces dendrorhous. The extraction procedure was optimized, validated and compared with other conventional extraction techniques. This method gave the best result due to the highest extraction efficiency within short extraction time. The optimum extraction conditions were as follows: the yeast cell wall was disrupted by lactic acid at 65° C for 1 h and then extracted with ethyl lactate:ethanol (1:1, v/v) for 0.5 h. It was proved that the extraction efficiency was enhanced by the addition of the natural antioxidant α-tocopherol. This new method showed low chemical toxicity and gave high extraction efficiency, which had good prospects for mass production at the industrial scale.     

Pulsed electric fields- and ultrasound-assisted green extraction of valuable compounds from Origanum vulgare L. and Thymus serpyllum L.

This work compared the effects of pulsed electric fields (PEF) and ultrasound (US) technologies on the extent of cell disintegration of two Mediterranean herb tissues (Origanum vulgare L., Thymus serpyllum L.), as well as on the extractability of phenolic compounds during the subsequent hydroalcoholic extraction (0%–50% ethanol in water, v/v) for up to 4 h. The rate of phenolic compounds extraction decreased with time and was predicted rather satisfactorily (R² = 0.898–0.989) by the Peleg’s model. The application of either PEF or US treatment prior to solid–liquid extraction (SLE) has the potential to reduce duration and concentration of ethanol to achieve the same recovery yield of phenolic compounds. Under optimised PEF (3 kV cm⁻¹, 10 kJ kg⁻¹) and US (400 W, 20 min) treatment conditions, the extracts obtained from either PEF or US pretreated herb samples showed higher total phenolic yield (36% on average) and antioxidant activity (FRAP) (36% on average) as compared to the control extraction, especially when 25% ethanol was used as a solvent. GC/MS analyses revealed no evidence of degradation of individual phenolics due to either PEF or US application.     

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.     

Non-conventional high-pressure extraction process: A comparative study for astaxanthin recovery from Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is one of the most attractive natural sources of astaxanthin. The yeast produces the compound intracellularly, therefore, it is necessary to evaluate cell rupture methods using clean and efficient technologies for its extraction and it subsequent use in industries. At present work, two non-conventional high-pressure extraction methods were evaluated for astaxanthin recovery: Supercritical fluid extraction and microfluidisation. Results: Effect of pressure (15, 30, and 45 Megapascals; MPa), Temperature (313 and 343 °K) and usage of co-solvent were studied in supercritical extraction process, meanwhile the effect of microfluidisation process (five stages and 160 MPa) for cell rupture and its combination with conventional technics (lithic enzymes, glass beads and ultrasonication) for the recovery of astaxanthin were evaluated. Supercritical fluid extraction presented a higher astaxanthin recovery, 54% yield extraction, at 32.5 MPa and 313 °K, and using ethanol as co-solvent, compared to a 31% yield extraction by mean microfluidisation process at 160 MPa combined with 15 min of sonication (amplitude of 80%).     

The effect of Pulsed Electric Field as a pre-treatment step in Ultrasound Assisted Extraction of phenolic compounds from fresh rosemary and thyme by-products

Emerging extraction techniques, including pulsed electric field (PEF) and ultrasound (US), are attracting considerable interest in the recovery of bioactives. Though, limited work has focused on PEF application as pre-treatment for US assisted extraction to enhance the release of phenolics from herbs. Hence, the present study investigated the use of an optimized PEF pre-treatment to enhance the recovery of phenolics from fresh rosemary and thyme by-products in a subsequent US assisted extraction step. Total phenolic content (TPC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP) were assessed as an index of extraction efficacy. Qualitative and quantitative analyses were performed through liquid chromatography-mass spectrometry analyses to evaluate the influence of the methods on individual phenolic compounds and the formation of potential derivatives. The results indicated that in a number of cases PEF pre-treatment enhanced (p < 0.05) the recovery of phenolic compounds and antioxidant capacity compared to US individually.     

Effect of drying,storage temperature and air exposure on astaxanthin stability from Haematococcus pluvialis

Astaxanthin is a powerful antioxidant with various health benefits such as prevention of age-related macular degeneration and improvement of the immune system, liver and heart function. To improve the post-harvesting stability of astaxanthin used in food, feed and nutraceutical industries, the biomass of the high astaxanthin producing alga Haematococcus pluvialis was dried by spray- or freeze-drying and under vacuum or air at − 20 °C to 37 °C for 20 weeks. Freeze-drying led to 41% higher astaxanthin recovery compared to commonly-used spray-drying. Low storage temperature (− 20 °C, 4 °C) and vacuum-packing also showed higher astaxanthin stability with as little as 12.3 ± 3.1% degradation during 20 weeks of storage. Cost-benefit analysis showed that freeze-drying followed by vacuum-packed storage at − 20 °C can generate AUD$600 higher profit compared to spray-drying from 100 kg H. pluvialis powder. Therefore, freeze-drying can be suggested as a mild and more profitable method for ensuring longer shelf life of astaxanthin from H. pluvialis.     

Equivalent processing for pasteurization of a pineapple juice–coconut milk blend by selected nonthermal technologies

Identifying equivalent processing conditions is critical for the relevant comparison of food quality attributes. This study investigates equivalent processes for at least 5-log reduction of Escherichia coli and Listeria innocua in pineapple juice–coconut milk (PC) blends by high-pressure processing (HPP), pulsed electric fields (PEF), and ultrasound (US) either alone or combined with other preservation factors (pH, nisin, and/or heat). The two blends (pH 4 and 5) and coconut milk (pH 7) as a reference were subjected to HPP at 300–600 MPa, 20°C for 0.5–30 min; PEF at an electric field strength of 10–21 kV/cm, 40°C for 24 µs; and US at 120 µm amplitude, 25 or 45°C for 6 or 10 min. At least a 5-log reduction of E. coli was achieved at pH 4 by HPP at 400 MPa, 20°C for 1 min; PEF at 21 kV/cm, 235 Hz, 40°C for 24 µs; and US at 120 µm, 45°C for 6 min. As L. innocua showed greater resistance, a synergistic lethal effect was provided at pH 4 by HPP with 75 ppm nisin at 600 MPa, 20°C for 5 min; PEF with 50 ppm nisin at 18 kV/cm, 588 Hz, 40°C for 24 µs; and US at 45°C, 120 µm for 10 min. The total soluble solids (11.2–12.4°Bx), acidity (0.47%–0.51% citric acid), pH (3.91–4.16), and viscosity (3.55 × 10⁻³–4.0 × 10⁻³ Pa s) were not significantly affected under the identified equivalent conditions. HPP was superior to PEF and US, achieving higher ascorbic acid retention and lower color difference in PC blend compared to the untreated sample.     

Green ultra-high pressure extraction of bioactive compounds from Haematococcus pluvialis and Porphyridium cruentum microalgae

Microalgae are considered prolific sources of bioactive compounds that can be useful for nutraceuticals. In this study, the potential of ultra-high pressure extraction (UHPE) for the simultaneous cell disruption and extraction of bioactives from two microalgae species, Haematococcus pluvialis and Porphyridium cruentum, was evaluated. The variables studied to extract carotenoids for H. pluvialis were pressure (100–600 MPa) and number of cycles (1 and 3 cycles) whereas the variables studied to obtain bioactives such as B-phycoerythrin, carotenoids, and PUFAs for P. cruentum were pressure (100–600 MPa) and different extraction solvents (water, ethanol, ethyl acetate or ethanol/d-limonene), generally recognized as safe (GRAS). The UHPE results showed significant increase on the extraction of carotenoids (109.74–119.34 mg per g extract) from H. pluvialis using 1 cycle of 20 min regardless of the pressure used. For P. cruentum, an UHPE with water provided extracts enriched in B-phycoerythrin (up to 144.43 mg per g extract), while subsequent UHPE using ethanol, ethyl acetate or ethanol/d-limonene 1:1 (v/v) provided extracts enriched in carotenoids (up to 65.05 mg per g extract) and polyunsaturated fatty acids (mainly eicosapentanoic acid and linoleic acid). Therefore, UHPE proved to be a viable green alternative for the recovery of bioactives from microalgae biomass.Industrial relevance: Microalgae are promising sources of bioactives such as B-phycoerythrin, carotenoids and polyunsaturated fatty acids. The potential of ultra-high-pressure extraction (UHPE) has been demonstrated as a fast and viable eco-friendly alternative using GRAS solvents (water, ethanol, ethyl acetate or ethanol/d-limonene) for the simultaneous cell disruption and extraction of these bioactives from Haematococcus pluvialis and Porphyridium cruentum. The bioactives obtained with one step or two step-UHPE process can be used in an array of food, cosmetic and pharmaceutical applications.     

Impact of pulsed electric field pretreatment on yield and quality of lipid extracted from cephalothorax of Pacific white shrimp (Litopenaeus vannamei) by ultrasound-assisted process

Pacific white shrimp cephalothorax was subjected to pulsed electric field (PEF) pretreatment at different electric field strengths (4, 8, 12 and 16 kV cm⁻¹) and pulse numbers (120, 160, 200 and 240) to enhance the extraction yield of lipids. PEF-treated samples were subsequently subjected to lipid extraction using ultrasound-assisted extraction (UAE) process at ultrasound amplitude of 80% for 25 min in continuous mode. PEF-pretreated samples subjected to UAE rendered the highest lipid yield (30.34 g 100 g⁻¹ solids). PEF pretreatment resulted in reduced lipid oxidation as evidenced by the decreases in peroxide value (PV) and thiobarbituric acid reactive substances (TBARS). Lipids from PEF-pretreated samples extracted using UAE had higher content of PUFAs and carotenoids, including astaxanthin, astaxanthin monoester, astaxanthin diester, canthaxanthin and β-carotene. Overall, PEF pretreatment helped in enhancing the extraction yield of lipids and carotenoids from shrimp cephalothorax and reduce lipid oxidation to some extent.     

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.     

Effect of pulsed electric fields and high pressure on improved recovery of high-added-value compounds from olive pomace

Olive pomace is considered a solid by-product and a rich source of valuable compounds such as polyphenols, flavonoids with antioxidant properties, and proteins. Nonthermal technologies, which cause alterations to cell permeability, are being explored to assist conventional recovery techniques. The aim of this study was to assess the effect of pulsed electric fields (PEF) and high pressure (HP) on improved recovery yield of the high-added-value compounds or to shorten the extraction time of these compounds. Olive pomace (Tsounati cv) was pretreated with PEF (1.0 to 6.5 kV/cm, 0.9 to 51.1 kJ/kg, and 15 µs pulse width) or HP (200 to 600 MPa and 0 to 40 min). Evaluation of the intracellular compounds extracted via solid–liquid extraction (50% ethanol–water solution) was performed. More intense PEF and HP conditions resulted in a significant increase of the phenolic concentration up to 91.6% and 71.8%, respectively. The increased antioxidant capacity of each extract was correlated to phenolic compound concentration. The protein concentration that was achieved with PEF pretreatment was doubled; however, HP-pretreated extracts reached 88.1% higher yield than untreated for pressures up to 200 MPa. HP and PEF pretreatment decreased extraction completion time t₉₈ (needed time to recover the equal amount of phenolics and proteins of untreated after 60 min of conventional extraction) to 12 min and lower than 1 min, respectively. To conclude, both pretreatments are effective in improving the conventional extraction process for increased yield recovery of high-added-value compounds from olive pomace.     

Enzymatic cell wall degradation combined with pulsed electric fields increases yields of water-soluble-protein extraction from the green marine macroalga Ulva sp.

Marine macroalgae are an attractive source of alternative protein. However, protein extraction from macroalgae is challenging. In this work, we investigated a combination of enzymatic cell wall degradation and high voltage Pulsed Electric Fields (PEF), to enhance yields of water-soluble-protein extraction from the green marine macroalga Ulva sp. The combined process showed a considerably higher protein extraction yield (19.6 ± 0.33%) compared to that of PEF alone (10.8 ± 0.37%) and enzyme pretreatment alone (9.7 ± 0.42%). Moreover, the water-soluble-protein extract obtained by PEF, which followed enzymatic cell wall degradation, had significant antioxidant activity. These results indicate that PEF combined with enzymatic pretreatment could contribute to protein extraction yields from Ulva sp., as a part of sustainable seaweed biorefinery.Industrial relevanceAlthough several previous works reported on methods for protein extraction from seaweeds for food application, the commercialization of the seaweed proteins is challenging due to multiple challenges in the extraction process development. In this work we show that a combination of enzymes for cell wall degradation with high voltage pulsed electric fields for membrane permeabilization lead to higher yields of water-soluble proteins. Both enzyme treatment and PEF are scalable processes, which do not modify proteins chemically, potentially leading to higher quality of the extract in comparison to standard alkaline extraction with a need to treat chemical waste.     

Production and stability of water‐dispersible astaxanthin oleoresin from Phaffia rhodozyma

The process of extracting the astaxanthin oleoresin from pretreated Phaffia rhodozyma cells was optimised using a Box‐Behnken response surface design. Microwaving the cells at 105 W for 1 min followed by ethyl acetate extraction was the best pretreatment, and the optimal extraction conditions were 65 °C for 24 min using a solvent–solid ratio of 19:1. The order of the ability to disperse the astaxanthin oleoresin was propylene glycol> Tween 80 > Tween 20 > α‐cyclodextrin, β‐cyclodextrin. It was determined that the degradation of the colour of the water‐dispersible oleoresin followed a first‐order kinetics model. The greatest stability was observed at pH 4 and at the lowest temperature evaluated (40 °C). The thermal degradation of the pigment occurs in two steps, the first one from 0 to 1.5 h, with an E_aI = 10.31 kJ mol^?1, and the second one from 1.5 to 5 h, with an E_aII = 30.06 kJ mol^?1     

Pulsed electric energy and ultrasonication assisted green solvent extraction of bio-molecules from different microalgal species

The effects of physical treatments (pulsed electrical fields (PEF), high voltage electrical discharges (HVED) and ultrasonication (US)) on aqueous extraction of carbohydrates and proteins, and ethanolic extraction of chlorophyll a from three microalgal species (Nannochloropsis sp., P. tricornutum and P. kessleri) have been studied. The total energy consumption of 530 kJ/kg suspension was applied for each treatment. For studied species, HVED was the most effective for extraction of carbohydrates, while US was the most effective for extraction of proteins and chlorophyll a. The observed differences for studied species can reflect the more fragile cell wall structure for P. tricornutum as compared with Nannochloropsis sp. or P. kessleri. The applied PEF, HVED and US treatments along with combinations of aqueous extraction of carbohydrates and proteins, and ethanolic extraction of pigments can be used in future implementations of selective extraction of valuable bio-molecules from microalgae.