Polar Lipids of Marine Microalgae Nannochloropsis oceanica and Chlorococcum amblystomatis Mitigate the LPS-Induced Pro-Inflammatory Response in Macrophages.

Microalgae are recognized as a relevant source of bioactive compounds. Among these bioactive products, lipids, mainly glycolipids, have been shown to present immunomodulatory properties with the potential to mitigate chronic inflammation. This study aimed to evaluate the anti-inflammatory effect of polar lipids isolated from Nannochloropsis oceanica and Chlorococcum amblystomatis. Three fractions enriched in (1) digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG), (2) monogalactosyldiacylglycerol (MGDG), and (3) diacylglyceryl-trimethylhomoserine (DGTS) and phospholipids (PL) were obtained from the total lipid extracts (TE) of N. oceanica and C. amblystomatis, and their anti-inflammatory effect was assessed by analyzing their capacity to counteract nitric oxide (NO) production and transcription of pro-inflammatory genes Nos2, Ptgs2, Tnfa, and Il1b in lipopolysaccharide (LPS)-activated macrophages. For both microalgae, TE and Fractions 1 and 3 strongly inhibited NO production, although to different extents. A strong reduction in the LPS-induced transcription of Nos2, Ptgs2, Tnfa, and Il1b was observed for N. oceanica and C. amblystomatis lipids. The most active fractions were the DGTS-and-PL-enriched fraction from N. oceanica and the DGDG-and-SQDG-enriched fraction from C. amblystomatis. Our results reveal that microalgae lipids have strong anti-inflammatory capacity and may be explored as functional ingredients or nutraceuticals, offering a natural solution to tackle chronic inflammation-associated diseases.

Chlorella vulgaris Extracts as Modulators of the Health Status and the Inflammatory Response of Gilthead Seabream Juveniles (Sparus aurata).

This study aimed to evaluate the effects of short-term supplementation, with 2% Chlorella vulgaris (C. vulgaris) biomass and two 0.1% C. vulgaris extracts, on the health status (experiment one) and on the inflammatory response (experiment two) of gilthead seabream (Sparus aurata). The trial comprised four isoproteic (50% crude protein) and isolipidic (17% crude fat) diets. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of C. vulgaris biomass (Diet D1); 0.1% inclusion of C. vulgaris peptide-enriched extract (Diet D2) and finally a 0.1% inclusion of C. vulgaris insoluble fraction (Diet D3). Diets were randomly assigned to quadruplicate groups of 97 fish/tank (IBW: 33.4 ± 4.1 g), fed to satiation three times a day in a recirculation seawater system. In experiment one, seabream juveniles were fed for 2 weeks and sampled for tissues at 1 week and at the end of the feeding period. Afterwards, randomly selected fish from each group were subjected to an inflammatory insult (experiment two) by intraperitoneal injection of inactivated gram-negative bacteria, following 24 and 48 h fish were sampled for tissues. Blood was withdrawn for haematological procedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters. The anterior gut was also collected for gene expression measurements. After 1 and 2 weeks of feeding, fish fed D2 showed higher circulating neutrophils than seabream fed CTR. In contrast, dietary treatments induced mild effects on the innate immune and antioxidant functions of gilthead seabream juveniles fed for 2 weeks. In the inflammatory response following the inflammatory insult, mild effects could be attributed to C. vulgaris supplementation either in biomass form or extract. However, the C. vulgaris soluble peptide-enriched extract seems to confer a protective, anti-stress effect in the gut at the molecular level, which should be further explored in future studies.

Bioactive Hydrolysates from Chlorella vulgaris: Optimal Process and Bioactive Properties.

Microalgae have been described as a source of bioactive compounds, such as peptides. Microalgae are easy to produce, making them a sustainable resource for extracting active ingredients for industrial applications. Several microalgae species have interesting protein content, such as Chlorella vulgaris with around 52.2% of protein, making it promising for peptide hydrolysate production. Therefore, this work focused on the production of water-soluble hydrolysates rich in proteins/peptides from the microalgae C. vulgaris and studied bioactive properties. For that, a design of experiments (DOE) was performed to establish the optimal conditions to produce hydrolysates with higher levels of protein, as well as antioxidant and antihypertensive properties. Four experimental factors were considered (cellulase percentage, protease percentage, hydrolysis temperature, and hydrolysis duration) for three responses (protein content, antioxidant activity, and antihypertensive activity). The optimal conditions determined by the DOE allowed producing a scaled-up hydrolysate with 45% protein, with antioxidant activity, measured by oxygen radical absorbance capacity assay, of 1035 µmol TE/g protein, IC50 for angiotensin-converting enzyme inhibition activity of 286 µg protein/mL, and α-glucosidase inhibition of 31% (30 mg hydrolysate/mL). The obtained hydrolysates can be used as functional ingredients for food and nutraceuticals due to their antioxidant, antihypertensive, and antidiabetic potential. Moreover, the antioxidant potential of the extracts may be relevant for the cosmetic industry, especially in antiaging formulations.

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.

Enzymatic hydrolysis allows an integral valorization of Nannochloropsis oceanica resulting in the production of bioactive peptide extracts and an eicosapentaenoic acid enriched fraction.

Nannochloropsis oceanica is a microalga with relevant protein content, making it a potential source of bioactive peptides. Furthermore, it is also rich in fatty acids, with a special focus on eicosapentaenoic acid (EPA), an omega-3 fatty acid mainly obtained from marine animal sources, with high importance for human health. N. oceanica has a rigid cell wall constraining protein extraction, thus hydrolyzing it may help increase its components' extractability. Therefore, a Box-Behnken experimental design was carried out to optimize the hydrolysis. The hydrolysate A showed 67% ± 0.7% of protein, antioxidant activity of 1166 ± 63.7 µmol TE g-1 of protein and an ACE inhibition with an IC50 of 379 µg protein mL-1 . The hydrolysate B showed 60% ± 1.8% of protein, antioxidant activity of 775 ± 13.0 µmol TE g-1 of protein and an ACE inhibition with an IC50 of 239 µg protein mL-1 . The by-product showed higher yields of total fatty acids when compared to "raw" microalgae, being 5.22% and 1%, respectively. The sustainable developed methodology led to the production of one fraction rich in bioactive peptides and another with interesting EPA content, both with value-added properties with potential to be commercialized as ingredients for different industrial applications, such as functional food, supplements, or cosmetic formulations.

Evaluation of the fertilizer potential of Chlorella vulgaris and Scenedesmus obliquus grown in agricultural drainage water from maize fields.

Producing microalgae with agricultural drainage water (ADW) allows recycling water and nutrients, with the production of a biofertilizer, avoiding receiving waters' contamination. Chlorella vulgaris and Scenedesmus obliquus were cultivated using ADW and standard media supplementation and presented higher productivities, relatively to the control industrial growth medium (using freshwater). Selected strains were grown outdoors in pilot flat panel photobioreactors, reaching 2.20 g L-1 for S. obliquus and 1.15 g L-1 for C. vulgaris, and degrading herbicides in the ADW to non-quantifiable concentrations. The potential of the C. vulgaris and S. obliquus suspensions to replace 50% of nitrogen (N) mineral fertilization of lettuce (0.5 g pot-1) was evaluated through a pot trial, also using a 2-times (1.0 g pot-1) and 5-times (2.5 g pot-1) higher dose, applied 31 days before lettuce transplanting. Even the lower dose of N, applied via C. vulgaris or S. obliquus suspensions, was able to provide significantly higher lettuce fresh matter yield, relatively to the mineral fertilized control. Soil enzymatic activities were improved, with significantly higher dehydrogenase, ß-glucosidase, and acid phosphatase activities for the 2.5 g pot-1 dose, more marked for S. obliquus, which was also able to increase soil organic matter content. Both the non-fertilized control and microalgae fertilized pots led to similar soil electrical conductivities, 3-fold lower than in the N-mineral fertilized pots, evidencing the capacity of microalgae fertilizers to avoid soil secondary salinization. Results suggest benefits from using ADW from maize cultivation to produce C. vulgaris or S. obliquus suspensions, that can be further used as liquid organic slow-release fertilizer.