Supercritical Fluid Extraction of Fucoxanthin from the Diatom Phaeodactylum tricornutum and Biogas Production through Anaerobic Digestion

Phaeodactylum tricornutum is the marine diatom best known for high-value compounds that are useful in aquaculture and food area. In this study, fucoxanthin was first extracted from the diatom using supercritical fluid extraction (SFE) and then using the extracted diatom-like substrate to produce bioenergy through anaerobic digestion (AD) processes. Factors such as temperature (30 °C and 50 °C), pressure (20, 30, and 40 MPa), and ethanol (co-solvent concentration from 10% to 50% v/v) were optimized for improving the yield, purity, and recovery of fucoxanthin extracted using SFE. The highest yield (24.41% w/w) was obtained at 30 MPa, 30 °C, and 30% ethanol but the highest fucoxanthin purity and recovery (85.03mg/g extract and 66.60% w/w, respectively) were obtained at 30 MPa, 30 °C, and 40%ethanol. Furthermore, ethanol as a factor had the most significant effect on the overall process of SFE. Subsequently, P.tricornutum biomass and SFE-extracted diatom were used as substrates for biogas production through AD. The effect of fucoxanthin was studied on the yield of AD, which resulted in 77.15 ± 3.85 LSTP CH₄/kg volatile solids (VS) and 56.66 ± 1.90 LSTP CH₄/kg VS for the whole diatom and the extracted P.tricornutum, respectively. Therefore, P.tricornutuman can be considered a potential source of fucoxanthin and methane and both productions will contribute to the sustainability of the algae-biorefinery processes.     

Pretreatment Techniques and Green Extraction Technologies for Agar from Gracilaria lemaneiformis

Optimizing the alkali treatment process alone without tracking the changes of algae and agar quality with each pretreatment process will not achieve the optimal agar yield and final quality. In this study, we monitored the changes of the morphology and weight of algae with each treatment process, and comprehensively analyzed the effects of each pretreatment process on the quality of agar by combining the changes of the physicochemical properties of agar. In conventional alkali-extraction technology, alkali treatment (7%, w/v) alone significantly reduced the weight of algae (52%), but hindered the dissolution of algae, resulting in a lower yield (4%). Acidification could solve the problem of algal hardening after alkali treatment to improve the yield (12%). In enzymatic extraction technology, agar with high purity cannot be obtained by enzyme treatment alone, but low gel strength (405 g/cm²) and high sulfate content (3.4%) can be obtained by subsequent acidification and bleaching. In enzyme-assisted extraction technology, enzyme damage to the surface fiber of algae promoted the penetration of low-concentration alkali (3%, w/v), which ensured a high desulfurization efficiency and a low gel degradation rate, thus improving yield (24.7%) and gel strength (706 g/cm²), which has the potential to replace the traditional alkali-extraction technology.     

Extraction of Fucoxanthin from Raw Macroalgae excluding Drying and Cell Wall Disruption by Liquefied Dimethyl Ether

Macroalgae are one of potential sources for carotenoids, such as fucoxanthin, which are consumed by humans and animals. This carotenoid has been applied in both the pharmaceutical and food industries. In this study, extraction of fucoxanthin from wet brown seaweed Undaria pinnatifida (water content was 93.2%) was carried out with a simple method using liquefied dimethyl ether (DME) as an extractant in semi-continuous flow-type system. The extraction temperature and absolute pressure were 25 °C and 0.59 MPa, respectively. The liquefied DME was passed through the extractor that filled by U. pinnatifida at different time intervals. The time of experiment was only 43 min. The amount of fucoxanthin could approach to 390 μg/g dry of wet U. pinnatifida when the amount of DME used was 286 g. Compared with ethanol Soxhlet and supercritical CO₂ extraction, which includes drying and cell disruption, the result was quite high. Thus, DME extraction process appears to be a good method for fucoxanthin recovery from U. pinnatifida with improved yields.     

Selection of Culture Conditions and Cell Morphology for Biocompatible Extraction of β-Carotene from Dunaliella salina

Biocompatible extraction emerges recently as a means to reduce costs of biotechnology processing of microalgae. In this frame, this study aimed at determining how specific culture conditions and the associated cell morphology impact the biocompatibility and the extraction yield of β-carotene from the green microalga Dunaliella salina using n-decane. The results highlight the relationship between the cell disruption yield and cell volume, the circularity and the relative abundance of naturally permeabilized cells. The disruption rate increased with both the cell volume and circularity. This was particularly obvious for volume and circularity exceeding 1500 µm³ and 0.7, respectively. The extraction of β-carotene was the most biocompatible with small (600 µm³) and circular cells (0.7) stressed in photobioreactor (30% of carotenoids recovery with 15% cell disruption). The naturally permeabilized cells were disrupted first; the remaining cells seems to follow a gradual permeabilization process: reversibility (up to 20 s) then irreversibility and cell disruption. This opens new carotenoid production schemes based on growing robust β-carotene enriched cells to ensure biocompatible extraction.     

Role of exogenous phytohormones on growth and plumbagin accumulation in Plumbago indica hairy roots and conservation of elite root clones via synthetic seeds

The present study describes the role of different exogenous hormones on morphology and plumbagin production in Plumbago indica hairy roots. It was also aimed to conserve elite root clones via synthetic seed technology. Insertion of rolB gene in transformed roots was confirmed by polymerase chain reaction followed by southern blot analysis. Hairy roots were treated with single or in combination of different phytohormones viz. IAA, IBA, 2, 4-D, NAA, BAP, GA₃ and ABA. Cultures incubated with GA₃ (0.5 mg l⁻¹) yielded highest root growth due to formation of profuse lateral branching while NAA (0.5 mg l⁻¹) treatment caused highest plumbagin accumulation. Cultures incubated with 2, 4-D exhibited highest inhibitory effect in terms of both root growth and plumbagin production. All phytohormones were found to be effective at lower concentration. In combinatorial study, GA₃ + NAA (0.5 mg l⁻¹, each) was found optimum for root biomass and plumbagin production at earlier stage of culture. Different combinations of auxins and BAP induced different morphologies ranging from reduction of lateral branching to rapid disorganization of root matrix. The combinations of ABA and selected auxins were not found promising at any of selected concentration. Based on the effect of exogenous hormones on hairy root culture, elite root clones were selected and encapsulated with sodium alginate matrix. Uniform shaped alginate coated synthetic seeds were conserved up to 6 months exhibited high regeneration potential without disturbing plumbagin content.     

Microwave-Assisted Extraction of Phlorotannins from Fucus vesiculosus

Microwave-assisted extraction (MAE) was carried out to maximize the extraction of phlorotannins from Fucus vesiculosus using a hydroethanolic mixture as a solvent, as an alternative to the conventional method with a hydroacetonic mixture. Optimal MAE conditions were set as ethanol concentration of 57% (v/v), temperature of 75 °C, and time of 5 min, which allowed a similar recovery of phlorotannins from the macroalgae compared to the conventional extraction. While the phlorotannins richness of the conventional extract was slightly superior to that of MAE (11.1 ± 1.3 vs. 9.8 ± 1.8 mg PGE/g DW_extract), both extracts presented identical phlorotannins constituents, which included, among others, tetrafucol, pentafucol, hexafucol, and heptafucol structures. In addition, MAE showed a moderate capacity to scavenge ABTS^•+ (IC₅₀ of 96.0 ± 3.4 µg/mL) and to inhibit the activity of xanthine oxidase (IC₅₀ of 23.1 ± 3.4 µg/mL) and a superior ability to control the activity of the key metabolic enzyme α-glucosidase compared to the pharmaceutical drug acarbose.     

Pigment Composition of Nine Brown Algae from the Iberian Northwestern Coastline: Influence of the Extraction Solvent

Brown algae are ubiquitously distributed in the NW coastline of the Iberian Peninsula, where they stand as an underexploited resource. In this study, five solvents were applied to the extraction of pigments from nine brown algae, followed by their determination and quantification by HPLC-DAD. A total of 13 compounds were detected: Six were identified as chlorophylls, six were classified as xanthophylls, and one compound was reported as a carotene. Fucoxanthin was reported in all extracts, which is the most prominent pigment of these algae. Among them, L. saccharina and U. pinnatifida present the highest concentration of fucoxanthin (4.5–4.7 mg∙g⁻¹ dry weight). Ethanol and acetone were revealed as the most efficient solvents for the extraction of pigments, showing a maximal value of 11.9 mg of total pigments per gram of dry alga obtained from the ethanolic extracts of H. elongata, followed by the acetonic extracts of L. ochroleuca. Indeed, ethanol was also revealed as the most efficient solvent according to its high extraction yield along all species evaluated. Our results supply insights into the pigment composition of brown algae, opening new perspectives on their commercial exploitation by food, pharmaceutical, and cosmeceutical industries.     

Rosmarinic Acid from Eelgrass Shows Nematicidal and Antibacterial Activities against Pine Wood Nematode and Its Carrying Bacteria

Pine wilt disease (PWD), a destructive disease for pine trees, is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus and additional bacteria. In this study, extracts of Zostera marina showed a high nematicidal activity against PWN and some of the bacteria that it carries. Light yellow crystals were obtained from extracts of Z. marina through solvent extraction, followed by chromatography on AB-8 resin and crystallization. The NMR and HPLC analysis showed that the isolated compound was rosmarinic acid (RosA). RosA showed effective nematicidal activity, of which the LC₅₀ (50% lethal concentration) to PWN at 24 h, 48 h and 72 h was 1.18 mg/g, 1.05 mg/g and 0.95 mg/g, respectively. To get a high yield rate of RosA from Z. marina, single factor experiments and an L₉ (3⁴) orthogonal experiment were performed. This extraction process involved 70% ethanol for 3 h at 40 °C. The extraction dosage was 1:50 (w/v). The highest yield of RosA from Zostera was 3.13 mg/g DW (dried weight). The crude extracts of Zostera marina (10 mg/mL) and RosA (1 mg/mL) also showed inhibitory effects to some bacterial strains carried by PWN: Klebsiella sp., Stenotrophomonas maltophilia, Streptomyces sp. and Pantoea agglomerans. The results of these studies provide clues for preparing pesticide to control PWD from Z. marina.     

Biological Properties of Fucoxanthin in Oil Recovered from Two Brown Seaweeds Using Supercritical CO2 Extraction

The bioactive materials in brown seaweeds hold great interest for developing new drugs and healthy foods. The oil content in brown seaweeds (Saccharina japonica and Sargassum horneri) was extracted by using environmentally friendly supercritical CO₂ (SC-CO₂) with ethanol as a co-solvent in a semi-batch flow extraction process and compared the results with a conventional extraction process using hexane, ethanol, and acetone mixed with methanol (1:1, v/v). The SC-CO₂ method was used at a temperature of 45 °C and pressure of 250 bar. The flow rate of CO₂ (27 g/min) was constant for the entire extraction period of 2 h. The obtained oil from the brown seaweeds was analyzed to determine their valuable compounds such as fatty acids, phenolic compounds, fucoxanthin and biological properties including antioxidant, antimicrobial, and antihypertension effects. The amounts of fucoxanthin extracted from the SC-CO₂ oils of S. japonica and S. horneri were 0.41 ± 0.05 and 0.77 ± 0.07 mg/g, respectively. High antihypertensive activity was detected when using mixed acetone and methanol, whereas the phenolic content and antioxidant property were higher in the oil extracted by SC-CO₂. The acetone–methanol mix extracts exhibited better antimicrobial activities than those obtained by other means. Thus, the SC-CO₂ extraction process appears to be a good method for obtaining valuable compounds from both brown seaweeds, and showed stronger biological activity than that obtained by the conventional extraction process.     

Preparative Separation of Sulfur-Containing Diketopiperazines from Marine Fungus Cladosporium sp. Using High-Speed Counter-Current Chromatography in Stepwise Elution Mode

High-speed counter-current chromatography (HSCCC) was successively applied to the separation of three sulfur-containing diketopiperazines (DKPs) (including two new compounds cladosporin A (1) and cladosporin B (3), and a known compound haematocin (2)) from a marine fungus Cladosporium sp. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at (1:1:1:1, v/v) and (2:1:2:1, v/v), in stepwise elution mode, was used for HSCCC. The preparative HSCCC separation was performed on 300 mg of crude sample yielding 26.7 mg of compound 3 at a purity of over 95%, 53.6 mg of a mixture of compounds 1 and 2, which was further separated by preparative-HPLC yielding 14.3 mg of compound 1 and 25.4 mg of compound 2 each at a purity of over 95%. Their structures were established by spectroscopic methods. The sulfur-containing DKPs suppressed the proliferation of hepatocellular carcinoma cell line HepG2. The present work represents the first application of HSCCC in the efficient preparation of marine fungal natural products.     

A Novel Lipid Extraction Method from Wet Microalga Picochlorum sp. at Room Temperature

A novel method using ethanol was proposed for extracting lipids from wet microalga Picochlorum sp. at room temperature and pressure. In this study, Central Composite design (CCD) was applied to investigate the optimum conditions of lipid extraction. The results revealed that the solvent to biomass ratio had the largest effect on lipid extraction efficiency, followed by extraction time and temperature. A high lipid extraction yield (33.04% of the dry weight) was obtained under the following extraction conditions: 5 mL solvents per gram of wet biomass for 37 min with gentle stirring at room temperature. The extraction yield was comparable to that obtained by the widely used Bligh-Dyer method. Furthermore, no significant differences in the distribution of lipid classes and fatty acid composition were observed according to different extraction methods. In conclusion, these results indicated that the proposed procedure using ethanol could extract lipids from wet biomass efficiently and had giant potential for lipid extraction at large scale.     

Combination of high-speed countercurrent chromatography and reversed phase C18 chromatography for large-scale isolation of cyanidin-3-O-β-d-glucoside from black rice bran extract

Cyanidin-3-O-β-d-glucoside (C3G), the best known and most investigated anthocyanin, has many pharmacological properties. Black rice bran is an outstanding source for the separation of C3G due to its high C3G content and a simple anthocyanin composition. The present study described a large-scale isolation of C3G from the crude black rice bran extract by combining high-speed countercurrent chromatography (HSCCC) and reversed phase C₁₈ column chromatography. After HSCCC enrichment with the solvent system water-n-butanol-tert-butyl-methyl-ether (TBME)-acetonitrile-trifluoroacetic acid (TFA) (5:2:2:1:0.1%), the C3G content was concentrated about 16-fold and reached to 563.23 mg/g with a recovery 96.89%. Further purification was carried out using reversed phase C₁₈ column chromatography and 1.43 g of pure C3G (purity 94.38%) was obtained from 3.0 g of anthocyanin-enriched extract. The method is time-saving, low risk of sample denaturation, high sample recovery, and so is suitable for large-scale preparation of C3G for further studies of bioactivities.     

Differences in the functionality and characterization of kafirins extracted from decorticated sorghum flour or gluten meal treated with protease

Kafirins have been extracted from several types of sorghums due to their potential use for production of gluten-free products. Nevertheless, the extraction of these proteins from wet-milled sorghum gluten meal (SGM) has not yet been explored. In this study, we investigated the differences in composition, color, molecular structures, functionality and in vitro protein digestibility of kafirin extracts obtained from dry-milled flour or SGM obtained from decorticated white sorghum treated with and without endopeptidic protease. Kafirins were extracted using aqueous ethanol and metabisulfite. Kafirin extracts from SGM presented higher protein purity (95% vs 86%), lower fat content (0.7% vs 2.0%), in vitro protein digestibility (89% vs 85%), and better water holding (2.8 vs 1.9 g/g) and fat absorption capacities (2.4 vs 1.6 g/g) compared to extracts from ground decorticated sorghum. Color was not affected by treatments. SDS-PAGE showed differences in the low molecular weight patterns of kafirin extracts obtained from SGM whereas FTIR analyses showed reduction of α-helical and β-turn percentages and β-sheet increment after extraction. The proposed protease treatment increased free amino nitrogen and emulsifying index of kafirins, but did not affect other functional properties. Thus, SGM represents a potential new feedstock for the extraction of food-grade kafirins.     

Response Surface Methodology for Ultrasound-Assisted Extraction of Astaxanthin from Haematococcus pluvialis

Astaxanthin is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has exhibited various biological activities including prevention or amelioration of cardiovascular disease, gastric ulcer, hypertension, and diabetic nephropathy. In this study, ultrasound-assisted extraction was developed for the effective extraction of astaxanthin from Haematococcus pluvialis. Some parameters such as extraction solvent, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 48.0% ethanol in ethyl acetate, the liquid-to-solid ratio was 20:1 (mL/g), and extraction for 16.0 min at 41.1 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of astaxanthin was 27.58 ± 0.40 mg/g. The results obtained are beneficial for the full utilization of Haematococcus pluvialis, which also indicated that ultrasound-assisted extraction is a very useful method for extracting astaxanthin from marine life.     

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A₅₆₅/A₂₈₀) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K₂HPO₄ (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.     

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Characterization and In Vitro digestibility of rice protein prepared by enzyme-assisted microfluidization: Comparison to alkaline extraction

Microfluidization followed by density-based separation was employed to extract protein from broken rice by disrupting protein-starch agglomerates. Follow-up enzyme treatments (amylase and glucoamylase) were performed to further improve the purity of the protein-rich fraction. High protein recovery (81.87%) and purity (87.89%) were obtained. The protein composition, solubility, structural properties, and in vitro digestibility of rice proteins prepared by enzyme-assisted microfluidization (EM-RP) and alkaline extraction (AE-RP) were compared. EM-RP was mainly composed of glutelin, which had low solubility and native structure. By contrast, large quantities of prolamin and globulin appeared in the AE-RP except glutelin, leading to the richness of glutamic acid/glutamine, leucine, aromatic and charged amino acids in the AE-RP. Compared to AE-RP, EM-RP showed higher digestibility due to the richness of glutelin (an easy-to-digest protein), as evidenced by higher nitrogen release during pepsin-trypsin digestion. The presence of prolamin (an indigestible protein) in AE-RP decreased protein digestibility although alkaline extraction improved its hydrolysis. These results suggest that enzyme-assisted microfluidization could be an effective technique to non-destructively and selectively extract rice glutelin.     

One-Step Preparative Separation of Fucoxanthin from Three Edible Brown Algae by Elution-Extrusion Countercurrent Chromatography

A method for batch preparation of fucoxanthin from brown algae was established, which possessed the advantages of high yield and high purity. The ultrasonic-assisted extraction method was used to obtain a crude extract from Sargassum fusiforme as the separation sample. Then the crude extract was separated by elution-extrusion countercurrent chromatography. The optimum preparation conditions of fucoxanthin were determined as follows: n-hexane-ethanol-water (20:9:11, v:v:v) as a two-phase solvent system, the mobile phase flow rate was 5 mL min⁻¹, the revolution speed was 800 r min⁻¹, the loading capacity was 60 mg 10 mL⁻¹ and the temperature was 25 °C. By this method, 12.8 mg fucoxanthin with a purity of 94.72% was obtained from the crude extract of Sargassum fusiforme. In addition, when the loading capacity was 50 mg 10 mL⁻¹, the purity of fucoxanthin reached 96.01%. Two types of by-products, chlorophyll and pheophytin, could also be obtained during the process of separation. This optimal method was further applied to separate fucoxanthin from Laminaria japonica and Undaria pinnatifida, and 6.0 mg and 9.7 mg fucoxanthin with a purity of 96.24% and 92.62% were acquired, respectively. Therefore, it was demonstrated that the preparation method of fucoxanthin established in this study had an applicability to brown algae, which improved the utilization value of raw materials.     

Protein extraction from Atriplex lampa leaves: Potential use as forage for animals used for human diets

The purpose of this study was to evaluate the effectof pH on the extraction of protein nitrogen from Atriplex lampa leaves (Moquin) Dietrich. Thechemical characterization of the dry matter indicated thefollowing (g/100 g): protein, 26.93; ash, 21.80; etherextract, 4.65; dry matter, 37.30; sodium, 6.05; and calcium, 0.41.Non-critical values were obtained for saponins andnitrates. The high concentration of oxalic acid (8.52 g/100 g),together with elevated salt content accountfor the low palatability of the studied species.In order to determine the parameters needed to improvethe extraction in protein nitrogen from leaves, freshmaterial was macerated with 2% sodium metasulfite,followed by pulping with a hand-driven grinder.Extractions were performed at different pH values(2–12) adjusting the value with 5N HCL or NaOH, withagitation followed by centrifugation and pressing.Supernatants were collected and kept. The lastextraction was performed with Tween 20 in order toobtain maximum nitrogen recovery from the residuecake. Highest extraction (41.23%) was obtained at pH10 with a 1:5 ratio (leaf : deionized water, w/v). Itis proposed that this regional natural resource may beused to elaborate a protein concentrate, which can bemade more palatable by decreasing potassium and sodiumsalt content with the use of membrane technology.