A gram-scale synthesis of very-long chain polyunsaturated fatty acids (VLC-PUFAs).

This manuscript describes our third generation, gram-scale synthesis of very long chain-polyunsaturated fatty acids (VLC-PUFAs), a unique and increasingly important class of lipids. Critical to this work and what makes it different from our previous approach to this family was the avoidance of oxidation sequences. Central to accomplishing this involved the use of a Negishi coupling reaction between the acid chloride derived from DHA and a saturated alkyl zinc reaction. Overall, the general approach required 6 synthetic transformations from DHA and was accomplished with an overall yield of 40%.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Extraction, separation and purification of fatty acid ethyl esters for biodiesel and DHA from Thraustochytrid biomass.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two-step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega-3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single-step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale-up studies in a 4 L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High-performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC-FID analysis.

Metabolic fate of DHA from regio- and stereospecific positions of triacylglycerols in a long-term feeding trial in rats.

This study investigated the impact of regio- and stereospecific position of docosahexaenoic acid (DHA) in dietary triacylglycerols (TAGs) on the fatty acid composition of tissues and organs in rats. Four-week feeding with TAGs containing DHA in sn-1, 2, or 3 position and palmitic acid in the remaining positions at a daily dosage of 500 mg TAG/kg body weight significantly increased the DHA content in all organs and tissues in rats, except in the brain, where the change in DHA level was not statistically significant. The group fed sn-1 DHA showed a significantly higher content of DHA in the plasma TAG than the group fed sn-3 DHA. The sn-3 DHA group had higher levels of DHA in the visceral fat compared to the sn-1, sn-2, as well as all other groups. This is the first study showing that DHA from sn-1 and sn-3 positions of dietary TAGs have differential accumulation in tissues. The new findings improved the current knowledge on the significance of TAG isomeric structure for the bioavailability and metabolic fate of DHA.

Stereoselective Synthesis, Pro-resolution, and Anti-inflammatory Actions of RvD5n-3 DPA.

The methyl ester of resolvin D5n-3 DPA, a lipid mediator biosynthesized from the omega-3 fatty acid n-3 docosapentaenoic acid, was stereoselectively prepared in 8% yield over 12 steps (longest linear sequence). The key steps for the introduction of the two stereogenic secondary alcohols were an organocatalyzed oxyamination and the Midland Alpine borane reduction. For the assembly of the carbon chain, the Sonogashira cross-coupling reaction and the Takai olefination were utilized. The physical properties, including retention time in liquid chromatography and tandem mass spectra, of the synthetic material were matched against material from human peripheral blood and mouse infectious exudates. Synthetic RvD5n-3 DPA, obtained just prior to biological experiments, displayed potent leukocyte-directed activities, upregulating the ability of neutrophils and macrophages to phagocytose bacteria, known as hallmark bioactions of specialized pro-resolving endogenous mediators.

Measuring brain docosahexaenoic acid turnover as a marker of metabolic consumption.

Docosahexaenoic acid (DHA, 22:6n-3) accretion in brain phospholipids is critical for maintaining the structural fluidity that permits proper assembly of protein complexes for signaling. Furthermore, membrane DHA can be released by phospholipase A2 and act as a substrate for the synthesis of bioactive metabolites that regulate synaptogenesis, neurogenesis, inflammation, and oxidative stress. Thus, brain DHA is consumed through multiple pathways including mitochondrial ß-oxidation, autoxidation to neuroprostanes, as well as enzymatic synthesis of bioactive metabolites including oxylipins, synaptamide, fatty-acid amides, and epoxides. By using models developed by Rapoport and colleagues, brain DHA loss has been estimated to be 0.07-0.26 µmol DHA/g brain/d. Since ß-oxidation of DHA in the brain is relatively low, a large portion of brain DHA loss may be attributed to the synthesis of autoxidative and bioactive metabolites. In recent years, we have developed a novel application of compound specific isotope analysis to trace DHA metabolism. By the use of natural abundance in 13C-DHA in the food supply, we are able to trace brain phospholipid DHA loss in free-living mice with estimates ranging from 0.11 to 0.38 µmol DHA/g brain/d, in reasonable agreement with previous methods. This novel fatty acid metabolic tracing methodology should improve our understanding of the factors that regulate brain DHA metabolism.

Differential Behavior of Eicosapentaenoic and Docosahexaenoic Acids on the Organization, Dynamics, and Fusion of Homogeneous and Heterogeneous Membranes.

Membrane fusion is a common course in innumerable biological processes that helps in the survival of eukaryotes. Enveloped viruses utilize this process to enter the host cells. Generally, the membrane lipid compositions play an important role in membrane fusion by modulating the membrane's physical properties and the behavior of membrane proteins in the cellular milieu. In this work, we have demonstrated the role of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, on the organization, dynamics, and fusion of homogeneous and heterogeneous membranes. We have exploited arrays of steady-state and time-resolved fluorescence spectroscopic methods and polyethylene glycol-induced membrane fusion assay to elucidate the behavior of EPA and DHA on dioleoyl phosphatidylcholine (DOPC)/cholesterol (CH) homogeneous and DOPC/sphingomyelin/CH heterogeneous membranes. Our results suggest that EPA and DHA display differential effects on two different membranes. The effects of PUFAs in homogeneous membranes are majorly attributed to their flexible chain dynamics, whereas the ability of PUFA-induced cholesterol transfer from the lo to the ld phase rules their behavior in heterogeneous membranes. Overall, our results provide detailed information on the effect of PUFAs on homogeneous and heterogeneous membranes.

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues.

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (13C/12C ratio or δ13C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ13C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled 13C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the 13C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and 13C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ13C), validated its utility in diet switch studies.

Intestinal absorption of DHA microcapsules with different formulations based on ex vivo rat intestine and in vitro dialysis models.

Intestinal permeability is a key factor affecting the bioavailability and physiological efficacy of docosahexaenoic acid (DHA) encapsulated in microcapsules. However, how the DHA microcapsules are transformed and the components absorbed across the small intestinal membrane has seldom been examined previously. In this study, an ex vivo absorption model based on the permeability of the rat small intestine was established to evaluate the intestinal absorption of DHA microcapsules with five formulations after gastrointestinal digestion in vitro. For pure glucose solutions, the apparent permeability coefficient (Papp) increased from 5.70 ± 0.60 × 10-6 cm s-1 at 5 mg mL-1 to 20.25 ± 0.88 × 10-6 cm s-1 at 30 mg mL-1 and decreased to 15.73 ± 0.91 × 10-6 cm s-1 at 100 mg mL-1. The Papp values obtained using the ex vivo model are comparable to those reported in the human jejunum. For algal oil DHA microcapsules with whey protein as the wall material (A-WP-DHA) after in vitro digestion, the Papp of glucose released was 3.81 × 10-6 cm s-1 with an absorption ratio of 59.55% in the ex vivo model, significantly lower than that from the in vitro porcine casing model. The Papp and absorption ratio varied little among the in vitro dialysis models with different molecular weight cut-off values. A similar trend was observed for the absorption of amino acids. However, the absorption ratio (26.6%) was the highest in the ex vivo model for free fatty acids (FFAs) released from the microcapsules due to the rapid accumulation of compounds on the inner wall of the intestinal sac. In addition, the DHA microcapsules with algal oil as the DHA source (36.40%) exhibited a higher absorption ratio of FFAs than that from tuna oil (14.26%) in the ex vivo model. The wall material compositions seemed to have little effect on FFA absorption. The present study is practically meaningful for the future formulation of DHA microcapsules with enhanced absorption.

Analysis of docosahexaenoic acid hydroperoxide isomers in mackerel using liquid chromatography-mass spectrometry.

Docosahexaenoic acid (DHA) is mostly esterified in food and is easily oxidized by exposure to heat or light. Hydroperoxide positions of DHA mono-hydroperoxide (DHA;OOH) provide information on oxidation mechanisms (e.g., radical- or singlet oxygen oxidation), yet direct identification of esterified DHA;OOH isomers has not been achieved. We previously accomplished the direct analysis of free DHA;OOH isomers with liquid chromatography-mass spectrometry (LC-MS/MS). In this study, we developed an LC-MS/MS method for direct analysis of esterified DHA;OOH based on our previous study. The developed method was capable of distinguishing esterified DHA;OOH isomers in raw- and oxidized mackerel. The result suggested that radical oxidation of esterified DHA can progress even in refrigeration. Different transitions were observed depending on the oxidation mechanism and lipid class. The analytical method and insights obtained in this study would be valuable to further understand and effectively prevent DHA oxidation in food products.

Chain-locked precursor ion scanning based HPLC-MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing ω-3 fatty acyl chains.

Lipase-catalyzed transesterification of structured phospholipids (sPLs) is a hot topic, but the structural variation of the fatty acyl chains in intact phospholipids at the molecular level remains unclear to date. The present study explored the detailed characteristics of synthesized phospholipids through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in precursor ion scan mode. The optimal conditions were in-depth inspected and determined for the reaction system, including phospholipase A1 as catalyst, 15% lipase loading, and 1% water content. The sPLs enriched with EPA/DHA were structurally and quantitatively characterized by focusing on the fragments of m/z 301.6 (eicosapentaenoic acid, EPA) and m/z 327.6 (docosahexaenoic acid, DHA), and the results were statistically analyzed using partial least squares discriminant analysis and clustered heatmap hierarchical clustering analysis. PC 38:6 (18:1/20:5), PC 38:7 (18:2/20:5), PC o-40:6 (o-18:0/22:6), and PE 40:8 (18:2/22:6) etc. were revealed as the main variables that were active in the reaction.

Synaptamide Modulates Astroglial Activity in Mild Traumatic Brain Injury.

At present, the study of the neurotropic activity of polyunsaturated fatty acid ethanolamides (N-acylethanolamines) is becoming increasingly important. N-docosahexaenoylethanolamine (synaptamide, DHEA) is a highly active metabolite of docosahexaenoic acid (DHA) with neuroprotective, synaptogenic, neuritogenic, and anti-inflammatory properties in the nervous system. Synaptamide tested in the present study was obtained using a chemical modification of DHA isolated from squid Berryteuthis magister liver. The results of this study demonstrate the effects of synaptamide on the astroglial response to injury in the acute (1 day) and chronic (7 days) phases of mild traumatic brain injury (mTBI) development. HPLC-MS study revealed several times increase of synaptamide concentration in the cerebral cortex and serum of experimental animals after subcutaneous administration (10 mg/kg/day). Using immunohistochemistry, it was shown that synaptamide regulates the activation of GFAP- and S100ß-positive astroglia, reduce nNOS-positive immunostaining, and stimulates the secretion of neurotrophin BDNF. Dynamics of superoxide dismutase production in synaptamide treatment confirm the antioxidant efficacy of the test compound. We found a decrease in TBI biomarkers such as GFAP, S100ß, and IL-6 in the blood serum of synaptamide-treated experimental animals using Western blot analysis. The results indicate the high therapeutic potential of synaptamide in reducing the severity of the brain damage consequences.

A newly synthesized 17-epi-NeuroProtectin D1/17-epi-Protectin D1: Authentication and functional regulation of Inflammation-Resolution.

The production of specialized pro-resolving mediators (SPMs) during the resolution phase in the inflammatory milieu is key to orchestrating the resolution of the acute inflammatory response. 17-epi-neuroprotectin D1/17-epi-protectin D1 (17-epi-NPD1/17-epi-PD1: 10R,17R-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid) is an SPM of the protectin family, biosynthesized from docosahexaenoic acid (DHA), that exhibits both potent anti-inflammatory and neuroprotective functions. Here, we carried out a new commercial-scale synthesis of 17-epi-NPD1/17-epi-PD1 that enabled the authentication and confirmation of its potent bioactions in vivo and determination of its ability to activate human leukocyte phagocytosis. We provide evidence that this new synthetic 17-epi-NPD1/17-epi-PD1 statistically significantly increases human macrophage uptake of E. coli in vitro and confirm that it limits neutrophilic infiltration in vivo in a murine model of peritonitis. The physical properties of the new synthetic 17-epi-NPD1/17-epi-PD1, namely its ultra-violet absorbance, chromatography, and tandem mass spectrometry fragmentation pattern, matched those of the originally synthesized 17-epi-NPD1/17-epi-PD1. In addition, we verified the structure and complete stereochemical assignment of this new synthetic 17-epi-NPD1/17-epi-PD1 using nuclear magnetic resonance (NMR) spectroscopy. Together, these results authenticate this 17-epi-NPD1/17-epi-PD1 for its structure and potent pro-resolving functions.

Design and Synthesis of Cyclopropane Congeners of Resolvin E3, an Endogenous Pro-Resolving Lipid Mediator, as Its Stable Equivalents.

Resolvins are pro-resolving lipid mediators with highly potent anti-inflammatory effects. Because of their polyunsaturated structures, however, they are unstable to oxygen as a drug prototype. To address this issue, we designed and synthesized CP-RvE3 as oxidatively stable congeners of RvE3 by replacing the cis-olefin with a cis-cyclopropane to avoid the unstable bisallylic structure. Although the oxidative stabilities of CP-RvE3 were not improved, ß-CP-RvE3 was 3.7 times more metabolically stable than RvE3. Thus, we identified ß-CP-RvE3 as a metabolically stable equivalent.

High Lipid Content of Prey Fish and n-3 PUFA Peroxidation Impair the Thiamine Status of Feeding-Migrating Atlantic Salmon (Salmo salar) and Is Reflected in Hepatic Biochemical Indices.

Signs of impaired thiamine (vitamin B1) status in feeding-migrating Atlantic salmon (Salmo salar) were studied in three Baltic Sea areas, which differ in the proportion and nutritional composition of prey fish sprat (Sprattus sprattus) and herring (Clupea harengus). The concentration of n-3 polyunsaturated fatty acids (n-3 PUFAs) increased in salmon with dietary lipids and n-3 PUFAs, and the hepatic peroxidation product malondialdehyde (MDA) concentration increased exponentially with increasing n-3 PUFA and docosahexaenoic acid (DHA, 22:6n-3) concentration, whereas hepatic total thiamine concentration, a sensitive indicator of thiamine status, decreased with the increase in both body lipid and n-3 PUFA or DHA concentration. The hepatic glucose 6-phosphate dehydrogenase activity was suppressed by high dietary lipids. In salmon muscle and in prey fish, the proportion of thiamine pyrophosphate increased, and that of free thiamine decreased, with increasing body lipid content or PUFAs, or merely DHA. The thiamine status of salmon was impaired mainly due to the peroxidation of n-3 PUFAs, whereas lipids as a source of metabolic energy had less effect. Organochlorines or general oxidative stress did not affect the thiamine status. The amount of lipids, and, specifically, their long-chain n-3 PUFAs, are thus responsible for generating thiamine deficiency, and not a prey fish species per se.

Oxidative stability, oxidation pattern and α-tocopherol response of docosahexaenoic acid (DHA, 22:6n-3)-containing triacylglycerols and ethyl esters.

DHA is most often used in supplements either in its triacylglycerol or ethyl ester form. Currently, there is only little published data on the differences in the oxidative stability and α-tocopherol response between the two lipid structures, as well as on the oxidation patterns of pure DHA. This study investigated the oxidative stability, α-tocopherol response and oxidation pattern of DHA incorporated in triacylglycerols and as ethyl esters with an untargeted approach after oxidation at 50 °C in the dark. Liquid and gas chromatographic methods with mass spectrometric detection and nuclear magnetic resonance spectroscopy were applied. DHA was more stable in triacylglycerols than as ethyl esters without α-tocopherol addition. With α-tocopherol added the opposite was observed. The oxidation products formed during triacylglycerol and ethyl ester oil oxidation were mostly similar, but also some structure-related differences were detected in both volatile and non-volatile oxidation products.

Resolvins, Protectins, and Maresins: DHA-Derived Specialized Pro-Resolving Mediators, Biosynthetic Pathways, Synthetic Approaches, and Their Role in Inflammation.

Marine organisms are an important source of natural products with unique and diverse chemical structures that may hold the key for the development of novel drugs. Docosahexaenoic acid (DHA) is an omega-3 fatty acid marine natural product playing a crucial regulatory role in the resolution of inflammation and acting as a precursor for the biosynthesis of the anti-inflammatory specialized pro-resolving mediators (SPMs) resolvins, protectins, and maresins. These metabolites exert many beneficial actions including neuroprotection, anti-hypertension, or anti-tumorigenesis. As dysregulation of SPMs is associated with diseases of prolonged inflammation, the disclosure of their bioactivities may be correlated with anti-inflammatory and pro-resolving capabilities, offering new targets for drug design. The availability of these SPMs from natural resources is very low, but the evaluation of their pharmacological properties requires their access in larger amounts, as achieved by synthetic routes. In this report, the first review of the total organic syntheses carried out for resolvins, protectins, and maresins is presented. Recently, it was proposed that DHA-derived pro-resolving mediators play a key role in the treatment of COVID-19. In this work we also review the current evidence on the structures, biosynthesis, and functional and new-found roles of these novel lipid mediators of disease resolution.

Dual effect of TAT functionalized DHAH lipid nanoparticles with neurotrophic factors in human BBB and microglia cultures.

BACKGROUND: Neurodegenerative diseases (NDs) are an accelerating global health problem. Nevertheless, the stronghold of the brain- the blood-brain barrier (BBB) prevents drug penetrance and dwindles effective treatments. Therefore, it is crucial to identify Trojan horse-like drug carriers that can effectively cross the blood-brain barrier and reach the brain tissue. We have previously developed polyunsaturated fatty acids (PUFA)-based nanostructured lipid carriers (NLC), namely DHAH-NLC. These carriers are modulated with BBB-permeating compounds such as chitosan (CS) and trans-activating transcriptional activator (TAT) from HIV-1 that can entrap neurotrophic factors (NTF) serving as nanocarriers for NDs treatment. Moreover, microglia are suggested as a key causative factor of the undergoing neuroinflammation of NDs. In this work, we used in vitro models to investigate whether DHAH-NLCs can enter the brain via the BBB and investigate the therapeutic effect of NTF-containing DHAH-NLC and DHAH-NLC itself on lipopolysaccharide-challenged microglia. METHODS: We employed human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) to capitalize on the in vivo-like TEER of this BBB model and quantitatively assessed the permeability of DHAH-NLCs. We also used the HMC3 microglia cell line to assess the therapeutic effect of NTF-containing DHAH-NLC upon LPS challenge. RESULTS: TAT-functionalized DHAH-NLCs successfully crossed the in vitro BBB model, which exhibited high transendothelial electrical resistance (TEER) values (≈3000 Ω*cm2). Specifically, the TAT-functionalized DHAH-NLCs showed a permeability of up to 0.4% of the dose. Furthermore, using human microglia (HMC3), we demonstrate that DHAH-NLCs successfully counteracted the inflammatory response in our cultures after LPS challenge. Moreover, the encapsulation of glial cell-derived neurotrophic factor (GNDF)-containing DHAH-NLCs (DHAH-NLC-GNDF) activated the Nrf2/HO-1 pathway, suggesting the triggering of the endogenous anti-oxidative system present in microglia. CONCLUSIONS: Overall, this work shows that the TAT-functionalized DHAH-NLCs can cross the BBB, modulate immune responses, and serve as cargo carriers for growth factors; thus, constituting an attractive and promising novel drug delivery approach for the transport of therapeutics through the BBB into the brain.

Dynamics of the Acinetobacter baumannii inner membrane under exogenous polyunsaturated fatty acid stress.

Exogenous polyunsaturated fatty acids (PUFAs) are readily incorporated into the synthesis pathways of A. baumannii membrane phospholipids, where they contribute to reduced bacterial fitness and increased antimicrobial susceptibility. Here we examine the impact of PUFA membrane modification on membrane organisation and biophysical properties using coarse grained MARTINI simulations of chemically representative membrane models developed from mass-spectrometry datasets of an untreated, arachidonic acid (AA) treated and docosahexaenoic acid (DHA) treated A. baumannii membranes. Enzymatic integration of AA or DHA into phospholipids of the A. baumannii membrane resulted in modulation of membrane biophysical properties. Membrane thickness decreased slightly following PUFA treatment, concomitant with changes in the lateral area per lipid of each lipid headgroup class. PUFA treatment resulted in a decrease in membrane ordering and an increase in lipid lateral diffusion. Changes in lateral membrane organisation were observed in the PUFA treated membranes, with a concurrent increase in ordered cardiolipin domains and disordered PUFA-containing domains. Notably, separation between ordered and disordered domains was enhanced and was more pronounced for DHA relative to AA, providing a possible mechanism for greater antimicrobial action of DHA relative to AA observed experimentally. Furthermore, the membrane active antimicrobial, pentamidine, preferentially adsorbs to cardiolipin domains of the A. baumannii model membranes. This interaction, and membrane penetration of pentamidine, was enhanced following PUFA treatment. Cumulatively, this work explores the wide-ranging effects of PUFA incorporation on the A. baumannii membrane and provides a molecular basis for bacterial inner membrane disruption by PUFAs.

Innovative method to grow the probiotic Lactobacillus reuteri in the omega3-rich microalga Isochrysis galbana.

Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.