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.     

Ethanol Extraction of Polar Lipids from Nannochloropsis oceanica for Food,Feed, and Biotechnology Applications Evaluated Using Lipidomic Approaches

Nannochloropsis oceanica can accumulate lipids and is a good source of polar lipids, which are emerging as new value-added compounds with high commercial value for the food, nutraceutical, and pharmaceutical industries. Some applications may limit the extraction solvents, such as food applications that require safe food-grade solvents, such as ethanol. However, the effect of using ethanol as an extraction solvent on the quality of the extracted polar lipidome, compared to other more traditional methods, is not yet well established. In this study, the polar lipid profile of N. oceanica extracts was obtained using different solvents, including chloroform/methanol (CM), dichloromethane/methanol (DM), dichloromethane/ethanol (DE), and ethanol (E), and evaluated by modern lipidomic methods using LC-MS/MS. Ultrasonic bath (E + USB)- and ultrasonic probe (E + USP)-assisted methodologies were implemented to increase the lipid extraction yields using ethanol. The polar lipid signature and antioxidant activity of DM, E + USB, and E + USP resemble conventional CM, demonstrating a similar extraction efficiency, while the DE and ethanol extracts were significantly different. Our results showed the impact of different extraction solvents in the polar lipid composition of the final extracts and demonstrated the feasibility of E + USB and E + USP as safe and food-grade sources of polar lipids, with the potential for high-added-value biotechnological applications.     

Potential Anti-Obesity,Anti-Steatosis,and Anti-Inflammatory Properties of Extracts from the Microalgae Chlorella vulgaris and Chlorococcum amblystomatis under Different Growth Conditions

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.     

Composition and Quantitation of Microalgal Lipids by ERETIC 1H NMR Method

Accurate characterization of biomass constituents is a crucial aspect of research in the biotechnological application of natural products. Here we report an efficient, fast and reproducible method for the identification and quantitation of fatty acids and complex lipids (triacylglycerols, glycolipids, phospholipids) in microalgae under investigation for the development of functional health products (probiotics, food ingredients, drugs, etc.) or third generation biofuels. The procedure consists of extraction of the biological matrix by modified Folch method and direct analysis of the resulting material by proton nuclear magnetic resonance (¹H NMR). The protocol uses a reference electronic signal as external standard (ERETIC method) and allows assessment of total lipid content, saturation degree and class distribution in both high throughput screening of algal collection and metabolic analysis during genetic or culturing studies. As proof of concept, the methodology was applied to the analysis of three microalgal species (Thalassiosira weissflogii, Cyclotella cryptica and Nannochloropsis salina) which drastically differ for the qualitative and quantitative composition of their fatty acid-based lipids.     

Valorization of the Red Algae Gelidium sesquipedale by Extracting a Broad Spectrum of Minor Compounds Using Green Approaches

Until now, the red algae Gelidium sesquipedale has been primarily exploited for agar production, leaving an undervalued biomass. In this work, the use of eco-friendly approaches employing ultrasound-assisted extraction (UAE) and green solvents was investigated to valorize the algal minor compounds. The green methods used herein showed an attractive alternative to efficiently extract a broad spectrum of bioactive compounds in short extraction times (15 to 30 min vs. 8 h of the conventional method). Using the best UAE conditions, red seaweed extracts were characterized in terms of total phenolics (189.3 ± 11.7 mg GAE/100 g dw), flavonoids (310.7 ± 9.7 mg QE/100 g dw), mycosporine-like amino acids (MAAs) (Σ MAAs = 1271 mg/100 g dw), and phycobiliproteins (72.4 ± 0.5 mg/100 g dw). Additionally, produced algal extracts exhibited interesting antioxidant and anti-enzymatic activities for potential applications in medical and/or cosmetic products. Thus, this study provides the basis to reach a superior valorization of algal biomass by using alternative methods to extract biologically active compounds following eco-friendly approaches. Moreover, the strategies developed not only open new possibilities for the commercial use of Gelidium sesquipedale, but also for the valorization of different algae species since the techniques established can be easily adapted.     

豆科植物异黄酮提取技术研究进展

豆和豆制品,红三叶草,葛藤中都含有异黄酮,包括染料木苷(Genistin),黄豆苷(Daidzin),黄豆黄苷(Glycitin)以及它们的衍生物如丙二酰(malonyl),乙酰(acetyl),苷元(aglucone )等.由于不同的组分在原料中的含量差别很大,寻找最优的异黄酮提取法是很困难的.传统的提取法利用甲醇、乙醇、乙腈、丙酮、n-丁醇、二甲基亚砜、Genapol等有机溶剂提取异黄酮.有机溶剂的大量使用对环境和人体都造成较大损害.近几年来,研究人员已发现几种高效的、环境友好型的提取法:超临界流体萃取法(SFE),超声波辅助萃取法(UAE),高压液体萃取法(PLE),固相萃取法(SPE),微波辅助萃取法(MAE)等.文章综述了从豆科植物中提取异黄酮的溶剂提取法和新近发展的提取技术.     

Evaluation of Ultrasound,Microwave, Ultrasound–Microwave,Hydrothermal and High Pressure Assisted Extraction Technologies for the Recovery of Phytochemicals and Antioxidants from Brown Macroalgae

This study aims to explore novel extraction technologies (ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), ultrasound–microwave-assisted extraction (UMAE), hydrothermal-assisted extraction (HAE) and high-pressure-assisted extraction (HPAE)) and extraction time post-treatment (0 and 24 h) for the recovery of phytochemicals and associated antioxidant properties from Fucus vesiculosus and Pelvetia canaliculata. When using fixed extraction conditions (solvent: 50% ethanol; extraction time: 10 min; algae/solvent ratio: 1/10) for all the novel technologies, UAE generated extracts with the highest phytochemical contents from both macroalgae. The highest yields of compounds extracted from F. vesiculosus using UAE were: total phenolic content (445.0 ± 4.6 mg gallic acid equivalents/g), total phlorotannin content (362.9 ± 3.7 mg phloroglucinol equivalents/g), total flavonoid content (286.3 ± 7.8 mg quercetin equivalents/g) and total tannin content (189.1 ± 4.4 mg catechin equivalents/g). In the case of the antioxidant activities, the highest DPPH activities were achieved by UAE and UMAE from both macroalgae, while no clear pattern was recorded in the case of FRAP activities. The highest DPPH scavenging activities (112.5 ± 0.7 mg trolox equivalents/g) and FRAP activities (284.8 ± 2.2 mg trolox equivalents/g) were achieved from F. vesiculosus. Following the extraction treatment, an additional storage post-extraction (24 h) did not improve the yields of phytochemicals or antioxidant properties of the extracts.     

A Critical Comparison of the Advanced Extraction Techniques Applied to Obtain Health-Promoting Compounds from Seaweeds

Marine macroalgae are rich in bioactive compounds that can be applied in several fields, mainly food, cosmetics, and medicine. The health-promoting effects of bioactive compounds, such as polyphenols, polysaccharides, carotenoids, proteins, and fatty acids, have been increasingly explored, especially regarding their antioxidant activity and improvement in human health. To extract these valuable compounds, advanced technologies that include Supercritical-Fluid Extraction (SFE), Pressurised-Liquid Extraction (PLE), Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), Enzyme-Assisted Extraction (EAE), Ultrasound-Microwave-Assisted Extraction (UMAE) and Liquefied Gas Extraction (LGE) have been assessed due to their notable advantages over the conventional methods (Solid–Liquid and Soxhlet extraction). These advanced techniques are considerably influenced by different extraction parameters such as temperature, pressure, type of solvent, extraction time, solvent:solid material ratio, power (MAE, UAE, and UMAE), enzymes used (EAE), and factors related to the macroalgae matrix itself. Optimizing these process parameters for each method is critical to obtain better efficiency results for the targeted bioactive compounds. Macroalgae are natural sources with undeniable beneficial effects on human health. In this context, optimising the extraction techniques discussed in this review should prioritise exploiting these valuable resources’ wide range of bioactive properties.     

In Vivo Nutritional Assessment of the Microalga Nannochloropsis gaditana and Evaluation of the Antioxidant and Antiproliferative Capacity of Its Functional Extracts

Nannochloropsis gaditana is a microalga with interesting nutritional and functional value due to its high content of protein, polyunsaturated fatty acids, and bioactive compounds. However, the hardness of its cell wall prevents accessibility to these components. This work aimed to study the effect of a treatment to increase the fragility of the cell wall on the bioavailability of its nutrients and functional compounds. The antioxidant and antiproliferative capacity of functional extracts from treated and untreated N. gaditana was assessed, and the profile of bioactive compounds was characterized. Furthermore, to study the effect of treatment on its nutrient availability and functional capacity, an in vivo experiment was carried out using a rat experimental model and a 20% dietary inclusion level of microalgae. Functional extracts from treated N. gaditana exhibited higher antioxidant activity than the untreated control. Furthermore, the treated microalga induced hypoglycemic action, higher nitrogen digestibility, and increased hepatic antioxidant activity. In conclusion, N. gaditana has interesting hepatoprotective, antioxidant, and anti-inflammatory potential, thus proving itself an ideal functional food candidate, especially if the microalga is treated to increase the fragility of its cell wall before consumption.     

Microalgae as Sustainable Bio-Factories of Healthy Lipids: Evaluating Fatty Acid Content and Antioxidant Activity

The demand for sustainable and environmentally friendly food sources and food ingredients is increasing, and microalgae are promoted as a sustainable source of essential and bioactive lipids, with high levels of omega-3 fatty acids (ω-3 FA), comparable to those of fish. However, most FA screening studies on algae are scattered or use different methodologies, preventing a true comparison of its content between microalgae. In this work, we used gas-chromatography mass-spectrometry (GC-MS) to characterize the FA profile of seven different commercial microalgae with biotechnological applications (Chlorella vulgaris, Chlorococcum amblystomatis, Scenedesmus obliquus, Tetraselmis chui, Phaeodactylum tricornutum, Spirulina sp., and Nannochloropsis oceanica). Screening for antioxidant activity was also performed to understand the relationship between FA profile and bioactivity. Microalgae exhibited specific FA profiles with a different composition, namely in the ω-3 FA profile, but with species of the same phylum showing similar tendencies. The different lipid extracts showed similar antioxidant activities, but with a low activity of the extracts of Nannochloropsis oceanica. Overall, this study provides a direct comparison of FA profiles between microalgae species, supporting the role of these species as alternative, sustainable, and healthy sources of essential lipids.     

Development of the “Applied Proteomics” Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the “omics” approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana.     

Effects of Structural and Compositional Changes of Nanochloropsis oceania after Enzyme Treatment on EPA-Rich Lipids Extraction

Improved methods for the extraction of eicosapentaenoic acid (EPA), an essential and economically important polyunsaturated fatty acid, are urgently required. However, lipid extraction rates using food-grade solvents such as ethanol are usually low. To improve the ethanol-based extraction rate, and to elucidate the relevant mechanisms, we used cellulase and laccase to treat powdered Nannochloropsis, one of the most promising microalgal sources of EPA. Cellulase and laccase synergistically increased lipid yields by 69.31% and lipid EPA content by 42.63%, by degrading the amorphous hemicellulose and cellulose, improving crystallinity, and promoting the release and extraction of lysodiacylglyceryltrimethylhomoserine. Scanning electron microscopy showed that cell morphology was substantially altered, with cell-wall rupture, loss of cell boundaries, and the release of intracellular substances. In conclusion, Nannochloropsis lipid yields may be directly linked to cell-wall hemicellulose structure, and enzymatic treatment to alter this may improve lipid yields.     

Advances in Technologies for Highly Active Omega-3 Fatty Acids from Krill Oil: Clinical Applications

Euphausia superba, commonly known as krill, is a small marine crustacean from the Antarctic Ocean that plays an important role in the marine ecosystem, serving as feed for most fish. It is a known source of highly bioavailable omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In preclinical studies, krill oil showed metabolic, anti-inflammatory, neuroprotective and chemo preventive effects, while in clinical trials it showed significant metabolic, vascular and ergogenic actions. Solvent extraction is the most conventional method to obtain krill oil. However, different solvents must be used to extract all lipids from krill because of the diversity of the polarities of the lipid compounds in the biomass. This review aims to provide an overview of the chemical composition, bioavailability and bioaccessibility of krill oil, as well as the mechanisms of action, classic and non-conventional extraction techniques, health benefits and current applications of this marine crustacean.     

The effect of different extraction techniques on extraction yield, total phenolic, and anti-radical capacity of extracts from Pinus radiata Bark

The performance of four techniques, conventional maceration, Soxhlet extraction, microwave assisted extraction (MAE), and ultrasound assisted extraction (UAE), for extraction of Pinus radiata bark, in one and several stages, were evaluated. For each technique, the mass extracted (g extract/g bark), total phenols (by Folin-Cicalteau), and tannin (by precipitation) concentration and anti-radical capacity (diphenyl-picrylhydrazyl, DPPH) were quantified. In one stage, the extracted mass increased in the following order: maceration < UAE < MAE < Soxhlet (p < 0.05). The total phenols and tannin levels were also higher with the Soxhlet technique. With additional extraction stages, only the samples produced with MAE and UAE techniques improved their parameters. Additionally, MAE extracts presented a higher anti-radical capacity than does Soxhlet and Pycnogenol^® extracts. Therefore, MAE was a simple and rapid method that was useful for extraction of P. radiata bark. Scanning electron micrographs (SEM) provided evidence of the mechanical effects on cell walls, mainly evidenced by cell destruction produced by Soxhlet, MAE, and UAE on the bark. In contrast, maceration only results in slightly ruptured cell pores, which could explain its low extraction yield.     

Optimization of the ultrasound-assisted extraction of tryptophan and its derivatives from rice (Oryza sativa) grains through a response surface methodology

An analytical ultrasound-assisted extraction (UAE) technique has been optimized and validated for the extraction of tryptophan and its derivatives from rice grains. A Box–Behnken design in conjunction with a response surface methodology based on six factors and three levels was used to evaluate the effects of the studied factors prior to optimizing the UAE conditions. The significant (p < 0.05) response surface models with high coefficients of determination were fitted to the experimental data. The most significant (p < 0.0001) effect is the solvent-to-sample ratio while quadratic effects caused by temperature and solvent-to-sample ratio were of moderate importance (p < 0.05). The optimal UAE conditions were as follows: extraction time of 5 min, ultrasound amplitude of 30%, cycle of 0.7 s⁻¹, extraction temperature of 30 °C, 8% methanol in water as the extraction solvent at pH 3 and a solvent/solid ratio 5:1. The method validation ensured that appropriate values were obtained for the LOD, LOQ, precision and recovery. Furthermore, the method was successfully applied to the analysis of a number of rice samples of different varieties. It was demonstrated that this particular UAE method is an interesting tool for the determination of tryptophan and tryptophan derivatives in rice grain samples.     

Carotenoids,Phenolic Compounds and Tocopherols Contribute to the Antioxidative Properties of Some Microalgae Species Grown on Industrial Wastewater

This study aimed at investigating the potential of microalgae species grown on industrial waste water as a new source of natural antioxidants. Six microalgae from different classes, including Phaeodactylum sp. (Bacillariophyceae), Nannochloropsis sp. (Eustigmatophyceae), Chlorella sp., Dunaniella sp., and Desmodesmus sp. (Chlorophyta), were screened for their antioxidant properties using different in vitro assays. Natural antioxidants, including pigments, phenolics, and tocopherols, were measured in methanolic extracts of microalgae biomass. Highest and lowest concentrations of pigments, phenolic compounds, and tocopherols were found in Desmodesmus sp. and Phaeodactylum tricornuotom microalgae species, respectively. The results of each assay were correlated to the content of natural antioxidants in microalgae biomass. Phenolic compounds were found as major contributors to the antioxidant activity in all antioxidant tests while carotenoids were found to contribute to the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity, ferrous reduction power (FRAP), and ABTS-radical scavenging capacity activity. Desmodesmus sp. biomass represented a potentially rich source of natural antioxidants, such as carotenoids (lutein), tocopherols, and phenolic compounds when cultivated on industrial waste water as the main nutrient source.     

A Saponification Method for Chlorophyll Removal from Microalgae Biomass as Oil Feedstock

Microalgae oil is an optimal feedstock for nutraceutical, pharmaceutical and biodiesel production, but its high levels of chlorophyll limit its large-scale application. To date, few effective approaches have been developed to remove chlorophyll from microalgae oil. The main purpose of this study was to present a preprocessing method of algae oil feedstock (Scenedesmus) to remove chlorophyll by saponification. The results showed that 96% of chlorophyll in biomass was removed. High quality orange transparent oil could be extracted from the chlorophyll reduced biomass. Specifically, the proportion of neutral lipids and saturation levels of fatty acids increased, and the pigments composition became carotenoids-based. The critical parameters of chlorophyll reduced biodiesel conformed to the standards of the USA, China and EU. Sodium copper chlorophyllin could be prepared from the bleaching effluent. The results presented herein offer a useful pathway to improve the quality of microalgae oil and reduce the cost of microalgae biodiesel.     

Ultrasound-assisted extraction and profile characteristics of seed oil from Isatis indigotica Fort

In order to further exploit the by-products of Isatis indigotica Fort., the seed oil was studied for its extraction and physicochemical properties. Ultrasound-assisted extraction (UAE) was used, and the parameters affecting seed oil recovery were optimized through response surface methodology (RSM). The optimum conditions were as follows: solvent-to-sample, 24:1; particle size, 110 meshes; extraction temperature, 49 °C; and extraction time, 44 min. Which resulted in a maximum oil recovery of 81.20 ± 0.21% (n = 3). Furthermore, the effects of UAE on the yield, fatty acid compositions, physicochemical properties, and microstructure of the seed powder were also investigated by calculating the recovery rate, utilizing a gas chromatograph fitted with a mass spectrometer (GC-MS), and performing scanning electron microscopy, respectively. The results show that UAE was an effective method for the seed oil extraction and the high content of unsaturated fatty acids (93.81%) demonstrates the oil has potential benefits for the cosmetics, edible products, or pharmaceutical industries.     

Year-Round Cultivation of Tetraselmis sp. for Essential Lipid Production in a Semi-Open Raceway System

There is increasing demand for essential fatty acids (EFAs) from non-fish sources such as microalgae, which are considered a renewable and sustainable biomass. The open raceway system (ORS) is an affordable system for microalgae biomass cultivation for industrial applications. However, seasonal variations in weather can affect biomass productivity and the quality of microalgal biomass. The aim of this study was to determine the feasibility of year-round Tetraselmis sp. cultivation in a semi-ORS in Korea for biomass and bioactive lipid production. To maximize biomass productivity of Tetraselmis sp., f medium was selected because it resulted in a significantly higher biomass productivity (1.64 ± 0.03 g/L) and lower omega-6/omega-3 ratio (0.52/1) under laboratory conditions than f/2 medium (0.70/1). Then, we used climatic data-based building information modeling technology to construct a pilot plant of six semi-ORSs for controlling culture conditions, each with a culture volume of 40,000 L. Over 1 year, there were no significant variations in monthly biomass productivity, fatty acid composition, or the omega-6/omega-3 ratio; however, the lipid content correlated significantly with photosynthetic photon flux density. During year-round cultivation from November 2014 to October 2017, areal productivity was gradually increased by increasing medium salinity and injecting CO₂ gas into the culture medium. Productivity peaked at 44.01 g/m²/d in October 2017. Throughout the trials, there were no significant differences in average lipid content, which was 14.88 ± 1.26%, 14.73 ± 2.44%, 12.81 ± 2.82%, and 13.63 ± 3.42% in 2014, 2015, 2016, and 2017, respectively. Our results demonstrated that high biomass productivity and constant lipid content can be sustainably maintained under Korean climate conditions.     

Crypthecodinium cohnii Growth and Omega Fatty Acid Production in Mediums Supplemented with Extract from Recycled Biomass

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h⁻¹, while the biomass yield from substrate reached 0.601 g·g⁻¹. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h⁻¹) and yield from the substrate (0.234 g·g⁻¹). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.