The Impact of Beeswax and Glycerol Monolaurate on Camellia Oil Oleogel's Formulation and Application in Food Products.

This study assessed the nutritional profile of camellia oil through its fatty acid composition, highlighting its high oleic acid content (81.4%), followed by linoleic (7.99%) and palmitic acids (7.74%), demonstrating its excellence as an edible oil source. The impact of beeswax (BW) and glycerol monolaurate (GML) on camellia oil oleogels was investigated, revealing that increasing BW or GML concentrations enhanced hardness and springiness, with 10% BW oleogel exhibiting the highest hardness and springiness. FTIR results suggested that the structure of the oleogels was formed by interactions between molecules without altering the chemical composition. In biscuits, 10% BW oleogel provided superior crispness, expansion ratio, texture, and taste, whereas GML imparted a distinct odor. In sausages, no significant differences were observed in color, water retention, and pH between the control and replacement groups; however, the BW group scored higher than the GML group in the sensory evaluation. The findings suggest that the BW oleogel is an effective fat substitute in biscuits and sausages, promoting the application of camellia oil in food products.

The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid.

By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

Fabrication and characterization of fast dissolving glycerol monolaurate microemulsion encapsulated gelatin nanofibers with antimicrobial activity.

BACKGROUND: Electrospun fibers are a good candidate for the delivery of bioactive compounds in the food industry because of their advantages that include a tunable diameter, high porosity and a high specific surface area. In the present study, we fabricated gelatin/glycerol monolaurate (GML) microemulsion nanofibers by solubilizing GML in Tween-80 followed by mixing with gelatin solution for electrospinning. We hypothesized that the addition of GML microemulsions affects the properties of the gelatin solution and modifies the physical and antimicrobial properties of the resulting nanofibers. RESULTS: Both pure gelatin solution and gelatin/GML microemulsions showed shear-thinning behavior. However, electrospinnability was not affected by the addition of GML microemulsions. A significantly higher average diameter of nanofibers (1147 nm) with 5% GML was observed compared to the gelatin fiber diameter of 560 nm. Fourier transform infrared spectroscopy showed hydrogen bonding between gelatin molecules and GML microemulsions. Thermal analysis and X-ray diffraction indicated an amorphous structure of gelatin/GML microemulsion nanofibers, although a small amount of crystalline GML existed in the nanofibers with high GML content. Gelatin/GML microemulsion nanofibers showed high thermal stability and improved hydrophilicity. Nanofibers with 5% GML (weight with respect to nanofiber) (D64 nanofibers) showed effective antimicrobial activity against Escherichia coli and Staphylococcus aureus. CONCLUSION: Gelatin/GML microemulsion nanofibrous films demonstrate superhydrophilicity and fast dissolution properties as a result of the high surface-to-volume ratio, amorphous structure and improved hydrophilicity of the nanofiber surface. The results indicate the potential application of gelatin/GML microemulsion nanofibrous films as edible antimicrobial food packaging. © 2021 Society of Chemical Industry.

Redesigning Solvatochromic Probe Laurdan for Imaging Lipid Order Selectively in Cell Plasma Membranes.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

Nanodisc self-assembly is thermodynamically reversible and controllable.

Many highly ordered complex systems form by the spontaneous self-assembly of simpler subunits. An important biophysical tool that relies on self-assembly is the Nanodisc system, which finds extensive use as native-like environments for studying membrane proteins. Nanodiscs are self-assembled from detergent-solubilized mixtures of phospholipids and engineered helical proteins called membrane scaffold proteins (MSPs). Detergent removal results in the formation of nanoscale bilayers stabilized by two MSP "belts." Despite their numerous applications in biology, and contributions from many laboratories world-wide, little is known about the self-assembly process such as when the bilayer forms or when the MSP associates with lipids. We use fluorescence and optical spectroscopy to probe self-assembly at various equilibria defined by the detergent concentration. We show that the bilayer begins forming below the critical micellar concentration of the detergent (10 mM), and the association of MSP and lipids begins at lower detergent levels, showing a dependence on the concentrations of MSP and lipids. Following the dissolution process by adding detergent to purified Nanodiscs demonstrates that the self-assembly is reversible. Our data demonstrate that Nanodisc self-assembly is experimentally accessible, and that controlling the detergent concentration allows exquisite control over the self-assembly reaction. This improved understanding of self-assembly could lead to better functional incorporation of hitherto intractable membrane target proteins.

Pursuing Specific Chemotherapy of Orthotopic Breast Cancer with Lung Metastasis from Docking Nanoparticles Driven by Bioinspired Exosomes.

Breast cancer develops from local tissue but is characterized by a distinct metastatic pattern involving regional lymph nodes and distant organs, which is the primary cause of high mortality in breast cancer patients. Herein, optimal docking nanoparticles (NPs) composed of a laurate-functionalized Pt(IV) prodrug (Pt(lau)), human serum albumin (HSA), and lecithin were predicted by computational modeling, prepared by nanoprecipitation, and validated by fluorescence spectroscopy. As macrophages have been reported to be preferentially recruited by breast cancer, Rex, the exosome spontaneously secreted by murine RAW 264.7 cells, was isolated to encapsulate the NPs. This high-performance delivery system, called NPs/Rex, possessed the desired physicochemical properties, enhanced colloidal stability, and redox-triggered release profile. Investigations of cytodynamics proved that NPs/Rex was internalized through multiple pathways, avoided entrapment by bilayers, and successfully platinized nucleic acids after bioreduction in the cytosol. Intracellular activation of Pt(lau) was confirmed by observing the characteristic effects of cisplatin on cell proliferation and the cell cycle following treatment with NPs/Rex. During in vivo application, the bioinspired Rex coating endowed docking NPs with prolonged blood circulation, smart organ tropism, and enhanced biocompatibility, as well as robust platinum (Pt) chemotherapy for breast cancer cells in orthotopic tumors of fat pads and metastatic nodules of lungs. Therefore, this favorable nanoplatform might provide valuable insight into the derivatization and development of Pt anticancer drugs used currently in the clinic.

Structure and Rheological Properties of Glycerol Monolaurate-Induced Organogels: Influence of Hydrocolloids with Different Surface Charge.

Organogel (OG) is a class of semi-solid gel, entrapping organic solvent within a three-dimensional network, which is formed via the self-assembly of organogelators. In the present study, OG was produced by glycerol monolaurate (GML) as organogelator. The influence of hydrocolloids with different surface charges (chitosan (CS), konjac glucomannan (KGM) and sodium alginate (SA)) on the physiochemical properties of OG was investigated. Rheological studies demonstrated that OG and pure hydrocolloid solution showed shear-thinning behavior. After incorporation of the hydrocolloid, the initial viscosity of OG was lowered from ~100 Pa·s to <10 Pa·s, and then the viscosity increased to more than 100 Pa·s at a low shear rate of 0.1-0.2 s-1, which subsequently decreased with a higher shear rate. OGs in the presence of hydrocolloids still kept the thermo-sensitivity, while the melting point of the OG decreased with the incorporation of hydrocolloids. Hydrocolloid addition greatly shortened the gelling time of the OG from 21 min to less than 2 min. The presence of hydrocolloids increased the particle size of oil droplets in the molten OG. Some aggregation and coalescence of oil droplets occurred in the presence of positive-charged CS and negative-charged SA, respectively. After gelling, the gel structure converted into a biphasic-like network. Hydrocolloids improved the hardness, stickiness and the oil-holding stability of OGs by 18.8~33.9%. Overall, hydrocolloid incorporation could modulate the properties of OGs through their different surface charge properties. These novel OGs have potential as nutrient carriers or low-fat margarine alternatives and avoid the trans-fatty acid intake.

Preparation and Compatibilization of PBS/Whey Protein Isolate Based Blends.

In this paper the production of biopolymeric blends of poly(butylene succinate) PBS and plasticized whey protein (PWP), obtained from a natural by-product from cheese manufacturing, has been investigated for the production of films and/or sheets. In order to add the highest possible whey protein content, different formulations (from 30 to 50 wt.%) were studied. It was found that by increasing the amount of PWP added to PBS, the mechanical properties were worsened accordingly. This trend was attributed to the low compatibility between PWP and PBS. Consequently, the effect of the addition of soy lecithin and glycerol monostearate (GMS) as compatibilizers was investigated and compared to the use of whey protein modified with oleate and laurate groups obtained by Schotten-Baumann reaction. Soy lecithin and the Schotten-Baumann modified whey were effective in compatibilizing the PWP/PBS blend. In fact, a significant increase in elastic modulus, tensile strength and elongation at break with respect to the not compatibilized blend was observed and the length of aliphatic chains as well as the degree of modification of the Schotten-Baumann proteins affected the results. Moreover, thanks to DSC investigations, these compatibilizers were also found effective in increasing the PBS crystallinity.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Solvatochromic Modeling of Laurdan for Multiple Polarity Analysis of Dihydrosphingomyelin Bilayer.

The hydration properties of the interface between lipid bilayers and bulk water are important for determining membrane characteristics. Here, the emission properties of a solvent-sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan), were evaluated in lipid bilayer systems composed of the sphingolipids D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM). The glycerophospholipids 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine were used as controls. The fluorescence properties of Laurdan in sphingolipid bilayers indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center-of-mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components based on the solvent model enabled us to identify the varieties of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Sphingolipids showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to PSM, even though DHPSM has a higher Tm than PSM. Because DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups), the interfacial region of the DHPSM bilayer was expected to be in a highly polar environment. The careful analysis of Laurdan emission spectra through the four-component deconvolution in this study provides important insights for understanding the multiple polarity in the lipid membrane.

Characterizing the Membrane-Disruptive Behavior of Dodecylglycerol Using Supported Lipid Bilayers.

Monoglycerides are esterified adducts of fatty acid and glycerol molecules that disrupt phospholipid membranes, leading to a wide range of biological functions such as antimicrobial activity. Among monoglycerides, glycerol monolaurate (GML) exhibits particularly high antimicrobial activity, although enzymatic hydrolysis of its ester group can diminish potency. Consequently, there have been efforts to identify more chemically stable versions of GML, most notably its alkylglycerol ether equivalent called dodecylglycerol (DDG). However, despite high structural similarity, biological studies indicate that DDG and GML are not functionally equivalent and it has been speculated that the two compounds might have different interaction profiles with phospholipid membranes. To address this outstanding question, herein, we employed supported lipid bilayer (SLB) platforms to experimentally characterize the interactions of DDG with phospholipid membranes. Quartz crystal microbalance-dissipation experiments identified that DDG causes concentration-dependent membrane morphological changes in SLBs and the overall extent of membrane remodeling events was greater than that caused by GML. In addition, time-lapsed fluorescence microscopy imaging experiments revealed that DDG causes extensive membrane tubulation that is distinct from how GML induces membrane budding. We discuss how differences in the head group properties of DDG and GML contribute to distinct membrane interaction profiles, offering insight into how the molecular design of DDG not only improves chemical stability but also enhances membrane-disruptive activity.

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing.

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).

Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections.

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 µg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.

In Vitro Antimicrobial Activities of Organic Acids and Their Derivatives on Several Species of Gram-Negative and Gram-Positive Bacteria.

The objective of this study was to determine the in vitro antimicrobial activity of several organic acids and their derivatives against Gram-positive (G+) and Gram-negative (G-) bacteria. Butyric acid, valeric acid, monopropionin, monobutyrin, monovalerin, monolaurin, sodium formate, and ProPhorce-a mixture of sodium formate and formic acid (40:60 w/v)-were tested at 8 to 16 concentrations from 10 to 50,000 mg/L. The tested bacteria included G- bacteria (Escherichia coli, Salmonella enterica Typhimurium, and Campylobacter jejuni) and G+ bacteria (Enterococcus faecalis, Clostridium perfringens, Streptococcus pneumoniae, and Streptococcus suis). Antimicrobial activity was expressed as minimum inhibitory concentration (MIC) of tested compounds that prevented growth of tested bacteria in treated culture broth. The MICs of butyric acid, valeric acid, and ProPhorce varied among bacterial strains with the lowest MIC of 500-1000 mg/L on two strains of Campylobacter. Sodium formate at highest tested concentrations (20,000 mg/L) did not inhibit the growth of Escherichia coli, Salmonella Typhimurium, and Enterococcus faecalis, but sodium formate inhibited the growth of other tested bacteria with MIC values from 2000 to 18,800 mg/L. The MIC values of monovalerin, monolaurin, and monobutyrin ranged from 2500 to 15,000 mg/L in the majority of bacterial strains. Monopropionin did not inhibit the growth of all tested bacteria, with the exception that the MIC of monopropionin was 11,300 mg/L on Clostridia perfringens. Monolaurin strongly inhibited G+ bacteria, with the MIC value of 10 mg/L against Streptococcus pneumoniae. The MIC tests indicated that organic acids and their derivatives exhibit promising antimicrobial effects in vitro against G- and G+ bacteria that are resistant to antimicrobial drugs. The acid forms had stronger in vitro antimicrobial activities than ester forms, except that the medium chain fatty acid ester monolaurin exhibited strong inhibitory effects on G+ bacteria.

Clarithromycin laurate salt: physicochemical properties and pharmacokinetics after oral administration in humans.

OBJECTIVE: To prepare and characterize the physicochemical and pharmacokinetic properties of clarithromycin laurate (CLM-L), a fatty acid salt of clarithromycin (CLM). METHODS: CLM-L was prepared by a simple co-melting process. The formation of CLM-L was confirmed using FTIR, 1H NMR, and 13C NMR. Solubility, intrinsic dissolution rate (IDR), and partitioning properties of CLM-L were determined and compared to those of CLM. Bioavailability of CLM from CLM-L tablets was evaluated in healthy volunteers and compared to immediate release CLM tablets. RESULTS: CLM-L showed lower aqueous solubility, higher partitioning coefficient, and slower dissolution rate. Tablets of CLM-L also showed a significantly slower in vitro release in comparison to CLM tablets. Cmax, Tmax and AUC0→∞ of CLM-L tablets and immediate release CLM tablets did not show a significant difference. However, the AUC0→∞ for the CLM-L tablets tended to be higher than that of CLM tablets at all-time points. CONCLUSION: CLM-L was successfully prepared and its formation was confirmed. CLM-L was more hydrophobic than CLM. It exhibited a slight in vivo absorption enhancement in comparison to CLM. However, its pharmacokinetic behavior was comparable to that of CLM.

Time-Resolved Laurdan Fluorescence Reveals Insights into Membrane Viscosity and Hydration Levels.

Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here, we first developed a method for a time-resolved general polarization (GP) analysis with fluorescence-lifetime imaging microscopy that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity, whereas curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a supercritical angle fluorescence objective, to our knowledge the first time fluorescence-lifetime imaging microscopy images were generated with supercritical angle fluorescence. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes.