HPLC-Based Automated Synthesis and Purification of Carbohydrates.

Reported herein is a new HPLC-based automated synthesizer (HPLC-A) capable of a temperature-controlled synthesis and purification of carbohydrates. The developed platform allows to perform various protecting group manipulations as well as the synthesis of O- and N-glycosides. A fully automated synthesis and purification was showcased in application to different carbohydrate derivatives including glycosides, oligosaccharides, glycopeptides, glycolipids, and nucleosides.

A review on the upstream production and downstream purification of mannosylerythritol lipids.

Biosurfactants are natural compounds with remarkable surface-active properties that may offer an eco-friendly alternative to conventional surfactants. Among them, mannosylerythritol lipids (MELs) stand out as an intriguing example of a glycolipid biosurfactant. MELs have been used in a variety of sectors for various applications, and are currently commercially produced. Industrially, they are used in the pharmaceutical, cosmetic, food, and agricultural industries, based on their ability to reduce surface tension and enhance emulsification. However, despite their utility, their production is comparatively limited industrially. From a bioprocessing standpoint, two areas of interest to improve the production process are upstream production and downstream (separation and purification) product recovery. The former has seen a significant amount of research, with researchers investigating several production factors: the microbial species or strain employed, the producing media composition, and the production strategy implemented. Improvement and optimization of these are key to scale-up the production of MELs. On the other hand, the latter has seen comparatively limited work presented in the literature. For the most part traditional separation techniques have been employed. This systematic review presents the production and purification methodologies used by researchers by comprehensively analyzing the current state-of-the-art with regards the production, separation, and purification of MELs. By doing so, the review presents different possible approaches, and highlights some potential areas for future work by identifying opportunities for the commercialization of MELs.

Purification and Screening of the Antialgal Activity of Seaweed Extracts and a New Glycolipid Derivative against Two Ichthyotoxic Red Tide Microalgae Amphidinium carterae and Karenia mikimotoi.

Ichthyotoxic red tide is a problem that the world is facing and needs to solve. The use of antialgal compounds from marine macroalgae to suppress ichthyotoxic red tide is considered a promising biological control method. Antialgal substances were screened and isolated from Bangia fusco-purpurea, Gelidium amansii, Gloiopeltis furcate, Hizikia fusifarme, Laminaria japonica, Palmaria palmata, and Sargassum sp. to obtain new materials for the development of algaecides against ichthyotoxic red tide microalgae using bioactivity-guided isolation methods. The fractions of seven macroalgae exhibited selective inhibitory activities against Amphidinium carterae and Karenia mikimotoi, of which the ethyl acetate fractions had the strongest and broadest antialgal activities for the two tested red tide microalgae. Their inhibitory effects on A. carterae and K. mikimotoi were even stronger than that of potassium dichromate, such as ethyl acetate fractions of B. purpurea, H. fusifarme, and Sargassum sp. Thin-layer chromatography and ultraviolet spectroscopy were further carried out to screen the ethyl acetate fraction of Sargassum sp. Finally, a new glycolipid derivative, 2-O-eicosanoyl-3-O-(6-amino-6-deoxy)-ß-D-glucopyranosyl-glycerol, was isolated and identified from Sargassum sp., and it was isolated for the first time from marine macroalgae. The significant antialgal effects of 2-O-eicosanoyl-3-O-(6-amino-6-deoxy)-ß-D-glucopyranosyl-glycerol on A. carterae and K. mikimotoi were determined.

Extraction, Isolation and Biological Activity of Two Glycolipids from Bangia fusco-purpurea.

In order to explore the extraction and activity of macroalge glycolipids, six macroalgae (Bangia fusco-purpurea, Gelidium amansii, Gloiopeltis furcata, Gracilariopsis lemaneiformis, Gracilaria sp. and Pyropia yezoensis) glycolipids were extracted with five different solvents firstly. Considering the yield and glycolipids concentration of extracts, Bangia fusco-purpurea, Gracilaria sp. and Pyropia yezoensis were selected from six species of marine macroalgae as the raw materials for the extraction of glycolipids. The effects of the volume score of methanol, solid-liquid ratio, extraction temperature, extraction time and ultrasonic power on the yield and glycolipids concentration of extracts of the above three macroalgae were analyzed through a series of single-factor experiments. By analyzing the antioxidant activity in vitro, moisture absorption and moisturizing activity, the extraction process of Bangia fusco-purpurea glycolipids was further optimized by response surface method to obtain suitable conditions for glycolipid extraction (solid-liquid ratio of 1:27 g/mL, extraction temperature of 48 °C, extraction time of 98 min and ultrasonic power of 450 W). Bangia fusco-purpurea extracts exhibited a certain scavenging effect on DPPH free radicals, as well as good moisture-absorption and moisture retaining activities. Two glycolipids were isolated from Bangia fusco-purpurea by liquid-liquid extraction, silica gel column chromatography and thin-layer chromatography, and they showed good scavenging activities against DPPH free radicals and total antioxidant capacity. Their scavenging activities against DPPH free radicals were about 60% at 1600 µg/mL, and total antioxidant capacity was better than that of Trolox. Among them, the moisturizing activity of a glycolipid was close to that of sorbierite and sodium alginate. These two glycolipids exhibited big application potential as food humectants and antioxidants.

Exploration of Polysaccharides from Phyllanthus emblica: Isolation, Identification, and Evaluation of Antioxidant and Anti-Glycolipid Metabolism Disorder Activities.

Phyllanthus emblica is a natural medicinal herb with diverse bioactivities. Certain extracts from this herb have been confirmed to possess anti-glycolipid metabolic disorder activity. To further develop its utility value and explore its potential in combating glycolipid metabolic disorders, we designed a series of experiments to investigate the structure, antioxidant activity, and anti-glycolipid metabolic disorder activity of Phyllanthus emblica polysaccharides. In this study, we extracted and purified polysaccharides from Phyllanthus emblica and thoroughly analyzed their structure using various techniques, including NMR, methylation analysis, and surface-enhanced Raman spectroscopy. We investigated the hypolipidemic and anti-glycolipid metabolism disorder activity of Phyllanthus emblica polysaccharides for the first time utilizing oleic acid (OA) and advanced glycation end products (AGEs) as inducers. Additionally, the antioxidant activity of Phyllanthus emblica polysaccharides was assessed in vitro. These findings lay the groundwork for future investigations into the potential application of Phyllanthus emblica polysaccharides as an intervention for preventing and treating diabetes.

Seeking faster, alternative methods for glycolipid biosurfactant characterization and purification.

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

Separation of Glycolipids/Sphingolipids from Glycerophospholipids on TiO2 Coating in Aprotic Solvent for Rapid Comprehensive Lipidomic Analysis with Liquid Microjunction Surface Sampling-Mass Spectrometry.

In lipidomic analysis by direct mass spectrometry (MS), high abundance lipids with high ionizability (such as glycerophospholipids) would cause ion suppression to lipids with poor ionizability and low abundance (such as glycolipids, sphingolipids, or glycerides), which largely limits the detection coverage for lipidomics. In this work, TiO2-based liquid microjunction surface sampling (LMJSS) coupled with MS was used for separation of glycerides, phospholipids and glycolipids/sphingolipids in biological samples and rapid analysis of lipids in different classes with high lipidome coverage. We found that, in nonaqueous aprotic solvents, lipids with a glycosyl or sphingosine group could be selectively separated from lipids with a phosphate group (selectivity >10) after being coenriched on TiO2 by tuning the solvent composition. Accordingly, a selective multistep extraction method was developed by loading the biosamples on TiO2 slides in neutral aprotic solvent, and sequentially eluting glycerides in pure acetonitrile, glycerophospholipids in 6% ammonia-94% acetonitrile (v/v) and glycolipids/sphingolipids in 5% formic acid-95% methanol (v/v) by LMJSS probe from TiO2 slide. Each eluate from TiO2 slide was directly delivered by LMJSS to MS for analysis. The total detection time with three desorption steps would be controlled in 3 min. The method performance for each lipid class was evaluated using lipid standards, including matrix effects (107-128%), RSDs (0.4-16%), linearity (0.98-0.99), detection limits (5-3000 ng/mL), the adsorption equilibrium constants (102-104) and adsorption capacity (1-38 µg/mm2) of TiO2 coated slides to lipids. Finally, the TiO2-based-LMJSS-MS method was applied to lipidomic analysis for blood plasma and brain tissue, and compared with direct infusion MS. Results showed that (2-5)-fold more sphingolipids/glycolipids and 40-50 more glycerophospholipids/glycerides were identified in both plasma and brain extract by the new method comparing with direct infusion MS method. Detected lipids were quantified with standard addition calibration method, and the absolute quantitation results measured by TiO2-based-LMJSS-MS were verified with that by the traditional LC-MS method (correlation coefficient >0.98, slope of correlation line = 0.87-1.05).

Diagnostic strategy for females suspected of Fabry disease.

A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso-GL-3 (lyso-Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso-GL-3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.

Neutral Lipids, Glycolipids, and Phospholipids, Isolated from Sandfish (Arctoscopus japonicus) Eggs, Exhibit Anti-Inflammatory Activity in LPS-Stimulated RAW264.7 Cells through NF-κB and MAPKs Pathways.

Total lipids were extracted from sandfish (Arctoscopus japonicus), and then they were separated into the following three lipid fractions: neutral lipids, glycolipids, and phospholipids. In this study, we analyzed the lipid fractions of A. japonicus eggs and we determined their anti-inflammatory activity in RAW264.7 macrophage cells. In these three lipid-fractions, the main fatty acids were as follows: palmitic acid (16:0), oleic acid (18:1n-9), docosahexaenoic acid (DHA, 22:6n-3), and eicosapentaenoic acid (EPA, 20:5n-3). Among the lipid fractions, phospholipids showed the highest concentration of DHA and EPA (21.70 ± 1.92 and 18.96 ± 1.27, respectively). The three lipid fractions of A. japonicus significantly suppressed the production of NO in macrophages. Moreover, they also significantly inhibited the expression of iNOS, COX-2, IL-6, IL-1ß, and TNF-α, in a dose-dependent manner. Furthermore, the lipid fractions of A. japonicus suppressed the nuclear translocation of NF-κB p65 subunits in a dose-dependent manner. In addition, they attenuated the activation of MAPKs (p38, ERK1/2, and JNK) phosphorylation in LPS-stimulated RAW264.7 cells. These results indicate that all the lipid fractions of A. japonicus exert anti-inflammatory activity by suppressing the activation of NF-κB and MAPK pathways. Therefore, the lipid fractions of A. japonicus might be potentially used as anti-inflammatory agents.

Selective enrichment of milk fat globules using functionalized polyvinylidene fluoride membrane.

We report on the development of a functionalized membrane-based technology for selective enrichment of milk fat globules from raw bovine milk. Functionalization was conducted by in situ polymerization of acrylic acid within a polyvinylidene fluoride membrane, followed by the electrostatic attachment of a cationic polymer to impart a net positive charge. The functionalized membrane-based technology enabled a one-step method of selective separation of globules directly from milk-based on size and charge. The presence of globules in the eluate was confirmed by fluorescence microscopy. Quantification of the extracted phospholipids from globules in the eluant revealed a significantly higher amount of polar lipids than the permeate. Our study describes a comprehensive analysis of selective enrichment of fat globules using a functionalized membrane and demonstrates the beneficial effect of extracted phospholipids from enriched fat globules.

Antibacterial Efficacy and Mechanism of Mannosylerythritol Lipids-A on Listeria monocytogenes.

Mannosylerythritol lipids-A (MEL-A) is a novel biosurfactant with excellent surface activity and potential biomedical applications. In this study, we explored the antibacterial activity and the underlying mechanisms of MEL-A against the important food-borne pathogen Listeria monocytogenes. The bacterial growth and survival assays revealed a remarkable antibacterial activity of MEL-A. Since MEL-A is a biosurfactant, we examined the cell membrane integrity and morphological changes of MEL-A-treated bacteria by biochemical assays and flow cytometry analysis and electron microscopes. The results showed obvious damaging effects of MEL-A on the cell membrane and morphology. To further explore the antibacterial mechanism of MEL-A, a transcriptome analysis was performed, which identified 528 differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed that the gene categories of membrane, localization and transport were enriched among the DEGs, and the analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways demonstrated significant changes in the maltodextrin ABC transporter system and stress response system. Furthermore, the growth of L. monocytogenes could also be significantly inhibited by MEL-A in milk, a model of a real food system, suggesting that MEL-A could be potentially applied as an natural antimicrobial agent to control food-borne pathogens in the food industry.

Production and investigation of the physico-chemical properties of MEL-A from glycerol and coconut water.

This work reports the production of MEL-A using coconut water as the carbon source. Proximate analysis of coconut water indicated the presence of nutrients necessary for growth of the organism and production of desired metabolite. The amount of MEL produced using coconut water was 3.85 g/L (± 0.35) with 74% of it being MEL-A when compared to 2.58 g/L (± 0.15) with 60% being MEL-A using glycerol, a conventional carbon source. MEL-A from coconut water consisted of 38.1% long-chain saturated fatty acids (C16:0 and C18:0) whereas with glycerol it was 9.6%. The critical micellar concentration of the biosurfactant from coconut water was 2.32 ± 0.21 µM when compared to 4.41 ± 0.25 µM from glycerol. The stability of O/W emulsion was reduced by 50% and 90% after incubation for 8 h in the case of MEL-A from coconut water and glycerol respectively when compared to synthetic surfactant, Tween-20. MEL-A from both the sources exhibited free radical scavenging activity (DPPH assay) in a dose-dependent manner wherein MEL-A from coconut water showed two fold higher activity than the other. The interaction of coconut water MEL-A with DPPC for drug encapsulation applications was also studied. The DSC measurements showed the differences in the interaction of drugs with DPPC/MEL-A liposome. The differences were also observed in the solubility of drugs after encapsulation with DPPC/MEL-A liposome.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2013-2014.

This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation?

The membrane (Milk Fat Globule Membrane - MFGM) surrounding the milk fat globule is becoming increasingly studied for its use in food applications due to proven nutritional and technological properties. This review focuses first on current researches which have been led on the MFGM structure and composition and also on laboratory and industrial purification and isolation methods developed in the last few years. The nutritional, health benefits and techno-functional properties of the MFGM are then discussed. Finally, new techno-functional opportunities of MFGM glycoproteins as a possible ingredient for Lactic Acid Bacteria (LAB) encapsulation are detailed. The ability of MFGM to form liposomes entrapping bioactive compounds has been already demonstrated. One drawback is that liposomes are too small to be used for bacteria encapsulation. For the first time, this review points out the numerous advantages to use MFGM glycoproteins as a protecting, encapsulating matrix for bacteria and especially for LAB.

Dokdolipids A-C, Hydroxylated Rhamnolipids from the Marine-Derived Actinomycete Actinoalloteichus hymeniacidonis.

Three new hydroxylated rhamnolipids, dokdolipids A-C (1-3) were obtained from the marine actinomycete Actinoalloteichus hymeniacidonis, which was isolated from a sediment sample collected off the coasts of Dokdo island, Republic of Korea. The structures of the isolated compounds were elucidated on the basis of 1D and 2D NMR and mass spectrometric data analyses. Their absolute configurations were assigned using the modified Mosher's method and specific rotation values, as well as acid hydrolysis, chemical derivatizations and subsequent HPLC analysis to determine the configuration of the sugar moieties. All new compounds were evaluated for their cytotoxicity against six cancer cell lines, HCT-15, NUGC-3, NCI-H23, ACHN, PC-3 and MDA-MB-231. Compounds 1-3 displayed moderate cytotoxicity against all the cell lines tested with IC50 values ranging from 13.7-41.5 µM.

Immunostimulatory Phosphatidylmonogalactosyldiacylglycerols (PGDG) from the Marine Diatom Thalassiosira weissflogii: Inspiration for a Novel Synthetic Toll-Like Receptor 4 Agonist.

An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG, 1) rich in polyunsaturated fatty acids was isolated from the marine diatoms Thalassiosira weissflogii. Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.

Recovery of milk fat globule membrane (MFGM) from buttermilk: effect of Ca-binding salts.

In this Research Communication we present a study of the effect of Ca-binding salts on the recovery of milk fat globule membrane (MFGM) from buttermilk. Sodium phosphate buffer was used for the purpose of MFGM recovery from buttermilk for the first time and we showed that 0.1 M buffer at pH 7.2 was the most effective for the recovery of MFGM. The fact of high efficacy of sodium phosphate buffer in recovery of MFGM from buttermilk allowed us to suggest that MFGM in buttermilk is present in association with casein through Ca- bridges formed between phospholipids of MFGM and phosphate groups of casein, primarily with k-casein as the peripheral protein of casein micelles.

Dirhamnolipid Produced by the Pathogenic Fungus Colletotrichum gloeosporioides BWH-1 and Its Herbicidal Activity.

Fungal phytotoxins used as ecofriendly bioherbicides are becoming efficient alternatives to chemical herbicides for sustainable weed management. Previous study found that cultures of the pathogenic fungus Colletotrichum gloeosporioides BWH-1 showed phytotoxic activity. This study further isolated the major phytotoxin from cultures of the strain BWH-1 using bioactivity-guided isolation, by puncturing its host plant for an activity test and analyzing on the HPLC-DAD-3D mode for a purity check. Then, the active and pure phytotoxin was characterized as a dirhamnolipid (Rha-Rha-C10-C10) using the NMR, ESIMS, IR and UV methods. The herbicidal activity of dirhamnolipid was evaluated by the inhibition rate on the primary root length and the fresh plant weight of nine test plants, and the synergistic effect when combining with commercial herbicides. Dirhamnolipid exhibited broad herbicidal activity against eight weed species with IC50 values ranging from 28.91 to 217.71 mg L-1 and no toxicity on Oryza sativa, and the herbicidal activity could be synergistically improved combining dirhamnolipid with commercial herbicides. Thus, dirhamnolipid that originated from C. gloeosporioides BWH-1 displayed the potential to be used as a bioherbicide alone, or as an adjuvant added into commercial herbicides, leading to a decrease in herbicides concentration and increased control efficiency.

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis.

Glycolipids (GLs), involved in biological processes and pathologies, such as viral, neurodegenerative and oncogenic transformations are in the focus of research related to method development for structural analysis. This review highlights modern separation techniques coupled to mass spectrometry (MS) for the investigation of GLs from various biological matrices. First section is dedicated to methods, which, although provide the separation in a non-liquid phase, are able to supply important data on the composition of complex mixtures. While classical thin layer chromatography (TLC) is useful for MS analyses of the fractionated samples, ultramodern ion mobility (IMS) characterized by high reproducibility facilitates to discover minor species and to apply low sample amounts, in addition to providing conformational separation with isomer discrimination. Second section highlights the advantages, applications and limitations of liquid-based separation techniques such as high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography (HILIC) in direct or indirect coupling to MS for glycolipidomics surveys. The on- and off-line capillary electrophoresis (CE) MS, offering a remarkable separation efficiency of GLs is also presented and critically assessed from the technical and application perspective in the final part of the review.

From lab to market: An integrated bioprocess design approach for new-to-nature biosurfactants produced by Starmerella bombicola.

Glycolipid microbial biosurfactants, such as sophorolipids (SLs), generate high industrial interest as 100% biobased alternatives for traditional surfactants. A well-known success story is the efficient SL producer Starmerella bombicola, which reaches titers well above 200 g/L. Recent engineering attempts have enabled the production of completely new types of molecules by S. bombicola, e.g. the bolaform SLs. Scale-up of bolaform SL production was performed at 150 L scale. The purified product was evaluated in detergent applications, as classic SLs are mostly applied in eco-friendly detergents. In this paper, we show that they can be used as green and non-irritant surfactants in for example (automatic) dishwashing applications. However, due to the presence of an ester function in the biosurfactant molecule a limited chemical stability at higher pH values (>6.5) was noticed, (therefore called 'non-symmetrical' (nsBola)) which, is a major drawback that will most likely inhibit market introduction. An integrated bioprocess design (IBPD) strategy was thus applied to resolve this issue. The strategy was to replace the fed fatty acids with fatty alcohols, to generate so-called "symmetrical bolaform (sBola) sophorosides (SSs)," containing two instead of one glycosidic bond. Next to a change in feeding strategy, the blocking of the fatty alcohols from metabolizing/oxidizing through the suggested ω-oxidation pathway was necessary. For the latter, two putative fatty alcohol oxidase genes (fao1 and fao2) were identified in the S. bombicola genome and deleted in the bolaform SL producing strain (ΔatΔsble). Shake flask experiments for these new strains (ΔatΔsbleΔfao1 and ΔatΔsbleΔfao2) were performed to evaluate if the fed fatty alcohols were directly implemented into the SL biosynthesis pathway. Indeed, sBola sophorosides (SSs) production up to 20 g/L was observed for the ΔatΔsbleΔfao1 strain. Unexpectedly, the ΔatΔsbleΔfao2 strain only produced minor amounts of sBola sophorosides (SSs), and mainly nsBola SLs (alike the parental ΔatΔsble strain). The sBola sophorosides (SSs) were purified and their symmetrical structure was confirmed by NMR. They were found to be significantly more stable at higher pH, opening up the application potential of the biosurfactant by enhancing its stability properties.