Extraction Of Phospholipids Research Articles

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Green extraction of marine phospholipids from Conger eel by-product using SC-CO2: Lipidomic profiles and biological activities

This paper presents an eco-friendly approach to recovering phospholipids (PLs) from the Conger myriaster head (CMH) via supercritical carbon dioxide (SC–CO2) extraction using ethanol as a cosolvent. The lipidomic profile and biological activities of the extracted PLs were investigated. Purity assessments, PL composition analysis, and fatty acid profiling were performed to characterize the PLs. Biological activity was determined through antioxidant, antimicrobial, antihypertensive, anti-inflammatory, and anticancer evaluations. The results showed that the yields of the SC–CO2 extracted CMH PLs (SCHP) and organic solvent extracted CMH PLs (OCHP) were 3.26% ± 0.20 % (76.18% ± 0.09% purity) and 9.02% ± 0.21% (purity = 18.29% ± 0.04%), respectively. The 31P NMR analysis revealed a diverse PL composition in both extracts, with the SCHP showing higher PC content and selective extraction characteristics. The SCHP exhibited significantly superior antioxidant, antimicrobial, antihypertensive, and anti-dementia activities compared to the OCHP. Both extracts inhibited nitric oxide production in RAW 264.7 cells, suggesting anti-inflammatory activity. Selective anticancer activity was observed, with high toxicity in SK-N-SH cells but no cytotoxicity in RAW 264.7 and HaCaT cells. The findings highlight the potential of SC-CO2 extraction using ethanol to obtain bioactive PLs from marine by-products, with promising applications in the pharmaceutical, cosmetic, and food industries.

"Turning Waste Into Value: Extraction And Characterization Of Phospholipids From Ghee Residue"

"Turning Waste Into Value: Extraction And Characterization Of Phospholipids From Ghee Residue"

Novel Phytosomal Formulation of Emblica officinalis Extracts with Its In Vivo Nootropic Potential in Rats: Optimization and Development by Box-Behnken Design

Purpose. The present study aimed to improve the aqueous solubility, permeability, bioavailability, and nootropic potential of standardized Emblica officinalis extract (EOE) by developing a novel phytosomal formulation. Method. Emblica officinalis extract-loaded phytosomes (EOPs) were prepared using solvent evaporation. The EOP was prepared at different molar ratios of extract and phospholipid. Herein, the effects of phospholipid extract ratio (A), temperature (B), and reaction time (C) were systematically investigated on entrapment efficiency using Box-Behnken design. In vitro and in vivo characterizations of the optimized formulation were performed. Results. Optimized EOP formulation (89.90 ± 0.24 μg/ml) exhibited improved aqueous solubility than plain EOE (11.85 ± 0.25 μg/ml). The optimized formulation’s particle size and Zeta potential were 198.4 ± 0.20 nm and −39.0 ± 0.40 mv. DSC and XRD studies confirmed the partial amorphization of EOE in phytosomes. Optimized formulation exhibited 69.82 ± 0.17% of EOE release at 12 h and followed zero-order release kinetics. Moreover, the phytosomal formulation of EOE exhibited its rationality with an improvement of bioavailability by 2.7 folds compared with pure EOE. Compared to EOE, EOP showed significantly (p<0.05 lower escape and transfer latencies on both days in MWMT and EPMT, indicating more effective memory-enhancing activity. Furthermore, EOP-treated rats exhibited improved acetylcholine (Ach) levels than EOE. Brain tissue concentrations measured following EOP oral administration (1.06 ± 0.04 μg/ml) were substantially greater (p<0.05) than those following EOE (0.32 ± 0.07 μg/ml). The brain dopamine and serotonin concentration were found to be higher (16.27 ± 1.209 and 43.28 ± 1.550 ng/ml) in the EOP-treated group as compared to the pure extract-treated group (10.40 ± 1.185 and 32.79 ± 1.738 ng/ml). Conclusion. Improvement of aqueous solubility, permeability, dissolution, bioavailability, and narrower particle size distribution could facilitate enhancement in the nootropic potential of EOE phytosomal formulation.

Mesoporous nanoperforators as membranolytic agents via nano- and molecular-scale multi-patterning

Plasma membrane lysis is an effective anticancer strategy, which mostly relying on soluble molecular membranolytic agents. However, nanomaterial-based membranolytic agents has been largely unexplored. Herein, we introduce a mesoporous membranolytic nanoperforators (MLNPs) via a nano- and molecular-scale multi-patterning strategy, featuring a spiky surface topography (nanoscale patterning) and molecular-level periodicity in the spikes with a benzene-bridged organosilica composition (molecular-scale patterning), which cooperatively endow an intrinsic membranolytic activity. Computational modelling reveals a nanospike-mediated multivalent perforation behaviour, i.e., multiple spikes induce nonlinearly enlarged membrane pores compared to a single spike, and that benzene groups aligned parallelly to a phospholipid molecule show considerably higher binding energy than other alignments, underpinning the importance of molecular ordering in phospholipid extraction for membranolysis. Finally, the antitumour activity of MLNPs is demonstrated in female Balb/c mouse models. This work demonstrates assembly of organosilica based bioactive nanostructures, enabling new understandings on nano-/molecular patterns co-governed nano-bio interaction.

Extraction of neutral lipids and phospholipids from marine biomasses using subcritical and supercritical fluids

Supercritical carbon dioxide (scCO2) and subcritical dimethylether (DME) were used to extract lipids from partially to fully dry marine biomass - whole jack mackerel (WJM), Hoki frames and heads (HFH), and Greenshell mussel meat (GSM). scCO2 gave a lower extraction yield of total lipids from GSM (42%) compared to WFH and HFH (86 - 88% yield) due to the higher concentration of phospholipids present in GSM. DME gave a higher extraction yield of total lipids and phospholipids than scCO2 for all marine biomass types. Phospholipid yield from GSM using DME was dependent on moisture content of the feed marine biomass and decreased with decreasing moisture content. Our results suggested that phospholipid extraction yield can be optimised by partly drying biomass to 30 - 50% moisture prior to extraction. DME extracts contained good levels of omega-3 fatty acids with minimal hydrolytic phospholipid degradation, thus providing a potential processing technology option to recover them from marine biomass.

Effects of phospholipids from embryonated egg yolk on lipopolysaccharide-induced acute lung injury by regulating the serum metabolism

Acute lung injury is a severe and intricate condition characterized by the disruptions in the alveolar barrier, pulmonary edema, enhanced inflammation, and alveolar apoptosis. Existing therapeutic approaches often exhibit limited efficacy and undesirable side effects. Egg yolk phospholipids have shown some potential as nutritional supplements due to their anti-apoptotic and anti-inflammatory properties under various conditions. This study aimed to explore whether phospholipid supplementation, particularly those rich in phosphatidylcholine, could alleviate lipopolysaccharide-induced lung injury in mice. Mice received daily oral administrations of phospholipid extracts (800 mg/kg) from different sources (embryonated egg yolk on day 18 and unfertilized fresh egg yolk) for 7 days or a single dose of dexamethasone (positive control). Subsequently, lipopolysaccharide (10 mg/kg) was intraperitoneally injected, and the mice were euthanized after 6 h. Various samples were collected for histological examination, assessment of inflammation and oxidative stress, serum metabolome analysis, and identification of gut flora. Results demonstrated that pretreatment with phospholipids effectively mitigated damages to the alveolar-capillary barrier and reduced lung edema by suppressing the release of oxidative and inflammatory cytokines, with embryonated egg extracts exhibiting superior efficacy. Additionally, the protective mechanism of phospholipids seemed associated with the metabolic distribution and absorption, helping to maintain the right lipid metabolism and restore protein synthesis, thus mitigating the impact of lung injury. In conclusion, supplementation with phosphatidylcholine-enriched phospholipids may serve as an innovative nutritional strategy to alleviate the adverse effects of bacterial infection on alveolar function.

Toxicity of hexagonal boron nitride nanosheets to freshwater algae: Phospholipid membrane damage and carbon assimilation inhibition

Hexagonal boron nitride (h-BN) nanomaterials have attracted numerous attentions for application in various fields, including environmental governance. Understanding the environmental implications of h-BN is a prerequisite for its safe and sustainable use; nevertheless, information on the negative effect of h-BN on aquatic organisms and the underlying toxicity mechanisms is scarce. The present study found that low exposure doses (0.1–1 μg/mL) of micron-sized h-BN lamella apparently suppressed (maximally 45.3%) the growth of Chlorella vulgaris (a freshwater alga) via membrane damages and metabolic reprogramming. Experimental and simulation results verified that h-BN can penetrate into and then extract phospholipids from the cell membrane of algae due to the strong hydrophobic interactions between h-BN nanosheets and lipids, resulting in membrane permeabilization and integrity reduction. Oxidative stress-triggered lipid peroxidation also contributes to membrane destruction of algae. Metabolomics assay demonstrated that h-BN down-regulated the CO2-fixation associated Calvin cycle and glycolysis/gluconeogenesis pathways in algae, thereby inhibiting energy synthesis and antioxidation process. Despite releasing soluble B inside cells, the B species exhibited negligible toxicity. These findings highlight the phenomena and mechanisms of h-BN toxicity in photosynthetic algae, which have great implications for guiding their safe use under the scenarios of global carbon neutrality.

Optimised extraction of phospholipids and omega‐3 fatty acids from Patagonian squid waste with low‐toxicity solvents

SummaryExtracting conditions of lipid compounds (phospholipids, PLs; ω3 fatty acids, FAs) from Patagonian squid (Doriteuthis gahi) waste were optimised by employment of a Simplex‐Lattice design. For it, different concentrations of low‐toxicity solvents (ethanol, acetone and ethyl acetate; single and binary and ternary mixtures) were tested. As a result, the multiple regression anova proved that the quadratic model was significant (P < 0.05) for PL and eicosapentaenoic acid (EPA) contents and for total polyunsaturated/total saturated and total ω3/total ω6 FA ratios. Thus, the anova application of the fitting model led to the following optimised values for such parameters: 477.42 g kg−1 lipids, 21.48 g 100 g−1 total FAs, 3.48 and 13.03, respectively. The combination of process variable levels which maximised the desirability function (0.49 value) was ethanol/ethyl acetate (0.50:0.50, v/v) and led to values of 412.50 (g kg−1 lipids), 19.05 (g 100 g−1 total FAs), 2.59 and 12.86, respectively, for the above‐mentioned lipid parameters. Results were compared to those obtained by the conventional chloroform/methanol procedure. By including an advanced mathematical design, a novel eco‐friendly strategy based on the employment of low‐toxicity solvents is proposed for the extraction from a marine waste substrate of highly valuable lipid compounds with healthy and nutritional properties.

Selective extraction of phospholipids from human milk using glass fabric modified with zirconium-based metal organic framework

Phospholipids (PLs) are important and complex trace lipids in milk, which have positive effects on the infants' nervous and immune system development. Herein, a new method for selective extraction of PLs using glass fabric @ MOF-808 was proposed. Based on Lewis acid-base interaction, MOF-808 containing abundant Zr-OH groups was selected as the adsorption body, and glass fabric was used as a substrate to make the adsorbent easy to remove and reuse. The influencing factors such as loading solution, extraction time, eluent and elution time were further investigated. The adsorbent showed high adsorption capacity (3.31–6.54 mg/g for PLs) and good reusability (reused at least five times). The method showed low detection limits (1.61 μg/L - 10.24 μg/L) and quantification limits (5.24 μg/L–51.21 μg/L) for eight classes of PLs. The analysis of PLs in human milk at different lactation stages by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry could obtain up to 206 PLs, indicating that the method has extremely high extraction and anti-interference capabilities. This work is the first time to introduce MOF materials to selectively extract PLs and use glass fabric as a substrate for MOF-808, which has the advantages of easy recovery and high sensitivity. It provides technical support for the discovery of more PL species and has potential applications in phospholipidomics.

Modified styrene-maleic anhydride copolymer-based chromatographic stationary phase for phospholipid separation and analysis

Phospholipids are important signaling molecules, and their metabolism is closely related to various diseases, such as neurodegenerative diseases and cancers. Phospholipids are typically characterized with extreme complexity and structural diversity. For example, phospholipids present in many different forms, such as sn position isomers, double-bond position isomers, double-bond stereochemical isomers, and enantiomers. Therefore, further research on novel separation and analytical techniques for phospholipids is of great importance. As an amphiphilic alternating copolymer, styrene-maleic anhydride copolymer (SMA) can be inserted into the phospholipid bilayer of biofilms to form lipid nanodisks with membrane proteins as the centers, thereby solubilizing membrane proteins and phospholipids. Thus, the introduction of SMA into a chromatographic stationary phase can potentially improve the separation and analysis of phospholipids. In this paper, SMA was successfully grafted onto the surface of silica gel via the "click" reaction and free radical polymerization. After further ring-opening modification of SMA with methyl methionine hydrochloride (MME·HCl), a novel SMA-modified stationary phase material (Sil-SMA-MME) was fabricated. The Sil-SMA-MME stationary phase was characterized using thermogravimetric analysis and Fourier transform infrared spectroscopy (FT-IR), and the results indicated the successful fabrication of the target material. The retention mechanism of the packed Sil-SMA-MME chromatographic column was investigated using hydrophilic nucleosides and nucleic acid bases via high performance liquid chromatography (HPLC) and UV detection. According to the retention characteristics of the nucleosides and nucleic acid bases in different mobile phases, the Sil-SMA-MME chromatographic column exhibited a typical hydrophilic-interaction-based retention mechanism, similar to that of a commercially available amino (SiO2-NH2) column. The separation performance of the Sil-SMA-MME column was evaluated using three types of small-molecule substances, including amides, nucleoside/nucleic acid bases, and phenols. Cyanoacetamide, 2-iodoacetamide, benzamide, p-aminobenzamide, and nicotinamide were used to evaluate the chromatographic performance of the developed Sil-SMA-MME column. When acetonitrile-H2O (96∶4, v/v) was used as the mobile phase, the five compounds exhibited good peak shapes and could be baseline-separated within 8 min. The highest column efficiency achieved was 90900 N/m. By contrast, under the same chromatographic conditions, the test substances were not separated effectively on the SiO2-NH2 column. Regardless of the mobile phase ratio, the peaks of benzamide and 2-iodoacetamide overlapped. These results demonstrate that the developed Sil-SMA-MME column has good separation selectivity. The separation performance of the Sil-SMA-MME column for phospholipid samples was also investigated by HPLC and evaporative light scattering detection (ELSD) to explore its feasibility for phospholipid separation and analysis. Different phospholipid standards were used to evaluate the separation performance of the column. Under certain mobile phase conditions, baseline separation could be achieved for dipalmityl phosphatidyl serine sodium (DPPS), diolyl phosphatidyl choline (DOPC), and dipalmityl phosphatidyl ethanolamine (DPPE), as well as four phosphatidyl choline (PC) standards, namely, lysophosphatidylcholine (LysoPC), dimyristoyl phosphatidyl choline (DMPC), distearyl phosphatidyl choline (DSPC), and dipalmitoyl phosphatidyl choline (DPPC). The separation potential of the developed Sil-SMA-MME column was further evaluated by separating and analyzing phospholipid extracts from Antarctic krill oil and human serum. The results showed that the developed Sil-SMA-MME column has good potential for phospholipid separation and analysis.

Development of a new online SPE-HPLC-MS/MS method for the profiling and quantification of sphingolipids and phospholipids in red blood cells – Application to the study of Gaucher's disease

Red blood cells (RBCs) are the subject of clinical attention due to their biological importance. Recently, it has been shown that certain erythrocyte pathologies could be linked to an abnormal lipid composition. In this work, we have developed a simple and fast method using online sample preparation with liquid chromatography coupled to mass spectrometry (SPE-HPLC-MS/MS), to identify a large number of sphingolipids (SL) and phospholipids (PL). The use of online sample preparation considerably reduces analysis times (15 min including extraction and separation of lipids + 2 min for system re-equilibration) and facilitates experimentation while ensuring very good extraction yields. This method was then successfully applied to the quantification of 30 sphingolipids and phospholipids in plasma and erythrocyte extracts from a cohort of individuals with Gaucher disease, treated or not by enzymotherapy. Our results for the study of this disease, led us to establish the lipid profile of the healthy red blood cells, still not very well-known to date. For this, we adopted a semi-targeted approach, based on the use of a triple-quadrupole analyzer and identified more than two hundred different lipid species. These promising results will hopefully enable us to enrich our knowledge of the normal red blood cells lipidome.

Green extraction of phospholipids from ghee residue using pulsed electric field processing: Process optimisation and analysis of structural and compositional parameters

Process parameters were optimised using Taguchi orthogonal array design for extraction of phospholipids (PLs) from ghee residue (GR) with pulsed electric field (PEF) assistance. For higher PL yield and antioxidant activity, process voltage and time were observed to be significant factors. Optimised conditions were determined as 60 kV/cm voltage at 7.5 (v/w) solvent (water)‐to‐solid ratio for 5 min. Physical disruption of GR matrix due to PEF treatment was established through scanning electron micrographs. Lipidomic profile of the extract ascertained through liquid chromatography‐mass spectrophotometry (LC–MS) revealed multiple species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine in the resultant extract.

Bifunctional Ti4+-modified paper for selective extraction or removal of phospholipids and paper spray mass spectrometry for bioanalysis in urine and plasma

BackgroundPhospholipids (PLs) are major constituents of cell membranes, play important roles in cell proliferation and death, as well as in signal transduction, and therefore are relevant biomarkers for different pathologies. On the other hand, when the analysis of small compounds, such as therapeutics in blood is desired, then phospholipids are part of the matrix and cause serious interference during analysis. Currently, both the analysis and removal of PLs from biological samples are limited by extensive sample preparation and instrumental separation. ResultsA fast and simple quantitative Ti4+-modified paper spray tandem mass spectrometric (TiPS-MS/MS) method was established in urine, where the enrichment of phospholipids was achieved, as well as reduction of matrix effects (primarily caused by high salt content) that ultimately led to improved sensitivity and selectivity. The method could achieve a physiologically relevant limit of detection (0.01–0.03 μg mL−1). Also, the usefulness of the Ti4+-modified paper was investigated in the opposite mode, namely for the selective removal of phospholipids from matrices such as plasma. Clonidine is used as model compound, as the detection of this compound is known to suffer from ion suppression by phospholipids. Compared with blank paper spray tandem mass spectrometry, the limit of detection could be improved from 0.3 μg mL−1 to 0.03 μg mL−1 by employing a Ti4+-modified paper on top of the spray tip to capture phospholipids from the sample. Significance and noveltyA novel Ti4+-modified paper was developed to allow for rapid solid-phase extraction of phospholipids from urine or selective removal from plasma, followed by direct paper spray mass spectrometric detection as a fast and convenient sample preparation and analysis combination. The paper properties are based on the Ti4+ metal ion, which can selectively bind phosphate-containing compounds under acidic conditions, and its applicability was demonstrated in relevant biological matrices.

Comparison of the Effect of Phospholipid Extracts from Salmon and Silver Carp Heads on High-Fat-Diet-Induced Metabolic Syndrome in C57BL/6J Mice.

Metabolic syndrome (MetS) is a global health problem, and EPA/DHA-enriched phospholipids (EPA/DHA-PLs) have been found to have positive effects on MetS improvement. Currently, research on EPA/DHA-PL mainly focuses on special and rare seafood, such as phospholipids derived from krill, sea cucumber, squid, and fish roe. However, it has been recently demonstrated that abundant EPA/DHA-PL can also be found in bulk fish and its by-products. Nonetheless, there is still limited research on the biological activities of EPA/DHA-PL derived from these sources. The aim of this study was to investigate the effect of phospholipid extracts from the heads of salmon and silver carp (S-PLE and SC-PLE) on the high-fat-diet-induced MetS in C57/BL mice. After an 8-week intervention, both SC-PLE and S-PLE had a significant ameliorating effect on MetS. Moreover, SC-PLE was more effective than S-PLE in reducing liver inflammation and fasting glucose. Both of the PL extracts were able to regulate the expression of key genes in lipid synthesis, fatty acid β-oxidation, and insulin signaling pathways. Compared with S-PLE, dietary SC-PLE had a greater influence on liver metabolomics. Pathway enrichment analysis showed that the differential metabolites of SC-PLE were mainly involved in arachidonic acid metabolism and glutathione metabolism. The results indicated that the different metabolic regulation methods of S-PLE and SC-PLE could be related to their variant molecular composition in EPA/DHA-PL.

Extraction of phospholipids from beta‐serum using switchable solvents and solid‐phase extraction

AbstractWe report the extraction of milk phospholipids (MPLs) from beta‐serum with a tertiary amine (N‐N dimethylcyclohexylamine, CyNMe2). The extraction time (3, 10, and 18 h), temperature (25, 40, and 60°C), and sample‐to‐solvent ratio (1/3, 1/10, and 1/18 v/v) were evaluated according to a factorial design. The extracted lipids were fractionated with solid‐phase extraction and quantified by HPLC‐CAD. Extraction conducted at 60°C yielded values of 14.86 ± 2.95%, 69.79 ± 3.19%, 79.39 ± 5.55% after 3, 10, and 18 h and a ratio of 1/18 (w/w). Microstructure images showed significant damage to the solid matrix that released the inner MPLs into the aqueous medium. The highest extraction yields of MPLs (79.39 ± 5.55%) were obtained after 18 h of extraction at 60°C and a sample‐to‐solvent ratio of 18 (w/w). The recovered fraction was made of phosphatidylinositol (~41%), phosphatidylserine (~6%), phosphatidylethanolamine (~34%), phosphatidylcholine (~9%), and sphingomyelin (~8%). This work outlines different extraction conditions for recovering MPLs from beta‐serum, a dairy byproduct.

Kinetics of Egg-Yolk Protein Hydrolysis and Properties of Hydrolysates.

Lecithin-free egg yolk (LFEY) is a byproduct of the extraction of egg-yolk phospholipids, which contain approximately 46% egg yolk proteins (EYPs) and 48% lipids. The enzymatic proteolysis is the alternative to increase the commercial value of LFEY. The kinetics of proteolysis in full-fat and defatted LFEY with Alcalase 2.4 L was analyzed in terms of the Weibull and Michaelis-Menten models. A product inhibition effect was also studied in the full-fat and defatted substrate hydrolysis. The molecular weight profile of hydrolysates was analyzed by gel filtration chromatography. Results pointed out that the defatting process did not importantly affect the maximum degree of hydrolysis (DHmax) in the reaction but rather the time at which DHmax is attained. The maximum rate of hydrolysis (Vmax) and the Michaelis-Menten constant KM were higher in the hydrolysis of the defatted LFEY. The defatting process might have induced conformational changes in the EYP molecules, and this affected their interaction with the enzyme. Consequently, the enzymatic reaction mechanism of hydrolysis and the molecular weight profile of peptides were influenced by defatting. A product inhibition effect was observed when adding 1% hydrolysates containing peptides lower than 3 kDa at the beginning of the reaction with both substrates.

Wrapping-Trapping versus Extraction Mechanism of Bactericidal Activity of MoS2 Nanosheets against Staphylococcus aureus Bacterial Membrane.

The promising broad-spectrum antibacterial activity of two-dimensional molybdenum disulfide (2D MoS2) has been widely recognized in the past decade. However, a comprehensive understanding of how the antibacterial pathways opted by the MoS2 nanosheets varies with change in lipid compositions of different bacterial strains is imperative to harness their full antibacterial potential and remains unexplored thus far. Herein, we present an atomistic molecular dynamics (MD) study to investigate the distinct modes of antibacterial action of MoS2 nanosheets against Staphylococcus aureus (S. aureus) under varying conditions. We observed that the freely dispersed nanosheets readily adhered to the bacterial membrane outer surface and opted for an unconventional surface directed "wrapping-trapping" mechanism at physiological temperature (i.e., 310 K). The adsorbed nanosheets mildly influenced the membrane structure by originating a compact packing of the lipid molecules present in its direct contact. Interestingly, these surface adsorbed nanosheets exhibited extensive phospholipid extraction to their surface, thereby inducing transmembrane water passage analogous to the cellular leakage, even at a slight increment of 20 K in the temperature. The strong van der Waals interactions between lipid fatty acyl tails and MoS2 basal planes were primarily responsible for this destructive phospholipid extraction. In addition, the MoS2 nanosheets bound to an imaginary substrate, controlling their vertical alignment, demonstrated a "nano-knives" action by spontaneously piercing inside the membrane core through their sharp corner, subsequently causing localized lipid ordering in their vicinity. The larger nanosheet produced a more profound deteriorating impact in all of the observed mechanisms. Keeping the existing knowledge about the bactericidal activity of 2D MoS2 in view, our study concludes that their antibacterial activity is strongly governed by the lipid composition of the bacterial membrane and can be intensified either by controlling the nanosheet vertical alignment or by moderately warming up the systems.

Antibacterial pathway of MoS2 nanosheets against Escherichia coli membrane via phospholipid extraction and localized phase transition.

The rapid emergence of antimicrobial resistance among pathogens have stimulated the quest for new generation antibacterials with minimal resistance inducing capabilities such as nanomaterials. Two-dimensional molybdenum disulfide (MoS2), a transition metal chalcogenide, has recently been recognized to exhibit excellent broad-spectrum bactericidal efficiency. However, a detailed assessment of the molecular level interactions responsible for their antibacterial action is imperative to modulate their efficacy. Through an atomistic molecular dynamics study, the present study delineates the interactions happening at the nano-bio interface responsible for their antibacterial action against Escherichia coli (E. coli) membrane [Soft Matter, 2022, 18,7159-7170]. The study conveys a two-step pathway of MoS2 nanosheet interactions with the bacterial membrane; (i) spontaneous phospholipid extraction by the nanosheet via van der Waals interactions with lipid acyl tails followed by (ii) complete ingestion of the nanosheet in the bilayer hydrophobic core through a synergic effect of MoS2-lipid interactions and lipid-lipid self-interactions. The phospholipid extraction to the MoS2 surface originates cavity formation in the extracellular leaflet which in turn creates a water passage across the membrane, signifying the possibility of cytoplasmic leakage in realistic membranes. Further, on complete insertion in the bilayer, the nanosheet induces the formation of densely packed lipid layers in its vicinity and causes localized phase transition in the membrane. These structural deteriorations in the bacterial membrane can be escalated by increasing the size of the nanosheet as well as by increasing the nanosheet concentrations. Thus, the study outlines both phospholipid extraction as well as localized alterations in membrane morphology responsible for the antibacterial activity of MoS2 nanosheets against E. coli bacterial membrane.

Lipidomic Profiling of Rice Bran after Green Solid-Liquid Extractions for the Development of Circular Economy Approaches.

Rice bran is a rather underutilized by-product of the rice industry that nowadays is far from being valorized. In this study, the lipidomic profile of bran of the Italian rice variety, Roma, has been evaluated through ultra performance liquid chromatography-tandem mass spectrometry. Crude lipid extracts were obtained from rice bran treated with different green solvents (1-butanol, ethanol and methyl tert-butyl ether/methanol mixture) in combination with an ultrasonic pre-treatment, and then compared with extracts obtained with standard solvents (chloroform/methanol mixture). Lipid yield, number and type of lipids and composition of prevalent lipid classes extracted were evaluated in order to provide an exhaustive lipid profile of the rice bran and to identify the most efficient green solvent for solid-liquid extractions. Twelve different lipid classes and a maximum of 276 lipids were identified. Ethanol and methyl tert-butyl ether/methanol solvents provided higher lipid extraction yields, the former being the most effective solvent for the extraction of triglycerides and N-acylethanolamines and the latter the most effective for the extraction of diglycerides, phospholipids and ceramides at 4 °C. Moreover, extraction with ethanol at 20 °C gave similar results as at 4 °C in terms of lipid yield and for most of the classes of lipids extracted. Taken together, our results indicate ethanol and methyl tert-butyl ether/methanol as excellent solvents for lipid extraction from rice bran, with the aim to further valorize this food by-product in the perspective of a circular economy.