Hyphenated LC-ABTS·+ and LC-DAD-HRMS for simultaneous analysis and identification of antioxidant compounds in Astragalus emarginatus Labill. extracts.

The compounds in leaf and stem extracts of Astragalus emarginatus Labill. (AEL), a plant species used in traditional Lebanese medicine, were investigated for antioxidant properties. First, the activity of various extracts was assessed using the Trolox equivalent antioxidant capacity, oxygen radical absorption capacity, and 2,2-diphenyl-1-picryl-hydrazyl-hydrate assays. The extract obtained using 30% ethanol showed the greatest activity. The antioxidant compounds in this extract were screened using a hyphenated high-performance liquid chromatography-2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS·+) system before being separated by ultra-high-performance liquid chromatography and identified using high-resolution mass spectrometry and ultra-violet-visible diode array detection. Approximately 40 compounds were identified. Hydroxycinnamates (caffeic, ferulic, and p-coumaric acid derivatives) and flavonoids (quercetin, luteolin, apigenin, and isorhamnetin derivatives) were the two main categories of the identified compounds. The active compounds were identified as caffeic acid derivatives and quercetin glycosides. In addition, the catechol moiety was shown to be key to antioxidant activity. This study showed that AEL is a source of natural antioxidants, which may explain its medicinal use.

Identification of punicalagin as the bioactive compound behind the antimicrobial activity of pomegranate (Punica granatum L.) peels.

Extracts from 'Zhéri' and 'Hicaznar' varieties of pomegranate, Punica granatum L., were obtained by subjecting powdered peels to extraction using water, water/ethanol (1:1; v/v), ethanol, acetone and heptane. Using the agar diffusion assay, extracts with water and/or ethanol were shown to display significant antimicrobial activity with diameters of inhibition zones up to 20 mm. Ethanolic extracts, which were the most active, were fractionated using SPE, HPLC and UHPLC, and the active compounds they contain were identified by mass spectrometry. Punicalagin, under its α and ß anomeric forms, was identified as the antibacterial compound in pomegranate peel extracts. Both forms were active with MIC values between 0.3 and 1.2 µg.ml-1, and they easily converted from one to the other with an α/ß equilibrium ratio of 3/7. Their spectrum of activity targeted 10 out of 13 Gram positive and two out of three Gram negative bacteria as well as a yeast strain.

Pickering nano-emulsions stabilized by Eudragit RL100 nanoparticles as oral drug delivery system for poorly soluble drugs.

The purpose of this study was to develop Pickering water-in-oil nano-emulsions only stabilized by Eudragit RL100 nanoparticles (NPs), in order to increase the nano-emulsion stability and create a barrier to improve the drug encapsulation and better control the drug release. The first part of this study was dedicated to investigating the nano-emulsion formulation by ultrasonication and understanding the interfacial behavior and role of NPs in the stabilization of the water/oil interface. The focus was on the surface coverage in the function of the formulation parameters (volume fractions) to disclose the extents and limitations of the process. The main physicochemical analysis of the Pickering nano-emulsions was performed by dynamic light scattering and transmission electron microscopy. On the other hand, the second experimental approach was dedicated to understanding the interfacial behavior of the Eudragit RL100 NPs toward a model water/oil interface, using a dynamic tensiometer with axisymmetric drop shape analysis. The study investigated the NPs' adsorption, as well as their rheological behavior. The aim of this part was to reveal the main phenomena that govern the interactions between NPs and the interface in order to understand the origin of Pickering nano-emulsions' stability. The last part of the study was concerned with the stability and in vitro release of a model encapsulated drug (ketoprofen) in a gastric and simulated intestinal environment. The results showed that Pickering nano-emulsions significantly improved the resistance to gastric pH, inducing a significantly slower drug release compared to classical nano-emulsions' stabilized surfactants. These Pickering nano-emulsions appear as a promising technology to modify the delivery of a therapeutic agent, in the function of the pH, and can be, for instance, applied to the oral drug delivery of poorly soluble drugs.

Online acetylcholinesterase inhibition evaluation by high-performance liquid chromatography-mass spectrometry hyphenated with an immobilized enzyme reactor.

A high-performance liquid chromatography-mass spectrometry technique hyphenated on-line with an immobilized enzyme reactor (IMER) was developed by the use of 3 known acetylcholinesterase (AChE) inhibitors (galanthamine, huperzine A and tacrine). This bioanalytical device allows qualitative comparison of the inhibitory strengths of AChE inhibitors. The AChE inhibitory strengths were evaluated and compared by the corresponding acetylcholine peak areas (mass signal) obtained after a chromatographic separation and the elution through the IMER. Only one injection of the analytes is needed to get this comparative analysis. This bioanalytical device was then applied to the extract of a natural plant, Lycoris radiata, which is known to contain AChE inhibitors such as galanthamine and lycoramine. Aside from the demonstration of the inhibitory activity of the two known AChE inhibitors, the AChE inhibitory activity of another compound (dihydro-latifaliumin C) was revealed. This is the first report describing the AChE inhibitory activity of this compound.

Correction: Pickering nano-emulsions stabilized by solid lipid nanoparticles as a temperature sensitive drug delivery system.

Correction for 'Pickering nano-emulsions stabilized by solid lipid nanoparticles as a temperature sensitive drug delivery system' by Sidy Mouhamed Dieng et al., Soft Matter, 2019, DOI: 10.1039/c9sm01283d.

Treatment of NASH with Antioxidant Therapy: Beneficial Effect of Red Cabbage on Type 2 Diabetic Rats.

AIMS: Oxidative stress (OS) plays a major role in type 2 diabetes and its vascular and hepatic complications, and novel therapeutic approaches include natural antioxidants. Our previous chemical and biological studies demonstrated the antioxidant activities of red cabbage (RC), and here, we aimed to determine the in vivo effects of 2-month long RC consumption using a high-fat/high-fructose model of diabetic rats. RESULTS: This vegetable, associated with lifestyle measurement, was shown to decrease OS and increase vascular endothelial NO synthase expression, ensuring vascular homeostasis. In the liver, RC consumption decreased OS by inhibiting p22phox expression and Nrf2 degradation and increasing catalase activity. It inhibited the activation of SREBP (1c, 2), ChREBP, NF-κB, ERK1/2, PPARγ, and GS and SIRT1 decrease, as observed in diabetic rats. CONCLUSION/INNOVATION: RC consumption led to metabolic profile improvement, together with hepatic function improvements. Although lifestyle changes are not sufficient to prevent diabetic complications, enrichment with RC avoids progression hepatic complications. This antioxidant strategy using RC does not only able to increase antioxidant defense, such as classical antioxidant, but also able to assure a metabolic and energetic balance to reverse complications. Whereas traditional medical therapy failed to reverse NASH in diabetic patients, consumption of RC should be a natural therapy to treat it.

Pickering nano-emulsion as a nanocarrier for pH-triggered drug release.

This study investigates the formulation of surfactant-free Pickering nano-emulsions able to release a drug at specific pH, in order to enhance its oral bioavailability. The stabilizing nanoparticles composed of magnesium hydroxide, were obtained by nano-precipitation method. The oil-in-water Pickering nano-emulsions stabilized with Mg(OH)2 nanoparticles, and encapsulating a model of hydrophobic drug (ibuprofen) were formulated following a high-energy process, using a sonication probe. The experimental approach explored the impact of all formulation parameters, composition and size of Mg(OH)2 nanoparticles, on the physico-chemical properties of the Pickering nano-emulsions. The system was characterized by DLS and transmission electron microscopy. In addition, Mg(OH)2 has the advantage of being solubilized in an acid medium leading to the destabilization of the nano-emulsion and the release of the active ingredient orally. The acid release study (pH = 1.2) showed cumulative release as a function of initial nanodroplet loading and saturation concentration. In basic media (pH = 6.8), we found a significant release of ibuprofen from the nano-emulsions that already had saturation in an acid medium. These nano-emulsions can not only protect patients from the side effects of acid medicines through the basic properties of hydroxides but also can contribute to the increase of the bioavailability of these drugs. In addition, once in the stomach pH is increased by hydroxides and promotes the release of active ingredients such as ibuprofen whose solubility is strongly influenced by pH.

Proteomic analysis of Lactobacillus pentosus for the identification of potential markers of adhesion and other probiotic features.

We analyzed the adhesion capacity to mucus of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives using an immobilized mucin model. On the basis of their adhesive capacity to mucin, three phenotypes were selected for cell-wall protein proteomic analysis to pinpoint proteins involved in the adhesion process: the highly adhesive L. pentosus CF1-43 N (73.49% of adhesion ability), the moderately adhesive L. pentosus CF1-37 N (49.56% of adhesion ability) and the poorly adhesive L. pentosus CF2-20P (32.79% of adhesion ability). The results revealed four moonlighting proteins over-produced in the highly adhesive L. pentosus CF1-43 N, which were under/not produced in the other two L. pentosus strains (CF1-37 N and CF2-20P). These proteins were involved in glycolytic pathway (phosphoglycerate mutase and glucosamine-6-phosphate deaminase), stress response (small heat shock protein) and transcription (transcription elongation factor GreA). Furthermore, the relative fold change in gene expression analysis showed significant up-regulation of the genes coding for these four moonlighting proteins in the highly adhesive L. pentosus CF1-43 N versus the poorly adhesive L. pentosus CF2-20P and also in response to mucin for 20 h which clearly indicate the significant role of these genes in the adhesion capacity of L. pentosus. Thus, these proteins could be used as biomarkers for mucus adhesion in L. pentosus. On the other hand, mucin exposure induced other probiotic effects in L. pentosus strains, enhancing their co-aggregation ability with pathogens and possible inactivation.

Beneficial effects of cherry consumption as a dietary intervention for metabolic, hepatic and vascular complications in type 2 diabetic rats.

BACKGROUND: Oxidative stress (OS) plays an important role in type 2 diabetes (T2D) pathogenesis and its complications. New therapies target natural antioxidants as an alternative and/or supplemental strategy to prevent and control them. Our previous chemical and biological studies highlighted the important antioxidant activities of cherries, among other fruits and vegetables, thus we aimed to determine in vivo effects of 2-month long cherry consumption using a high-fat/high-fructose (HFHF) model of diabetic-rats (Lozano et al. in Nutr Metab 13:15, 2016). METHODS: After 2 months of HFHF, male Wistar rats were divided into: HFHF and HFHF enriched in cherry (nutritional approach) or standard diet ND (lifestyle measures) and ND plus cherry during 2 months. Metabolic, lipidic, oxidative parameters were quantified. Tissues (liver, pancreas and vessels) OS were assessed and hepatic (steatosis, fibrosis, inflammation) and vascular (endothelial dysfunction) complications were characterized. RESULTS: T2D was induced after 2 months of HFHF diet, characterized by systemic hyperglycaemia, hyperinsulinemia, glucose intolerance, dyslipidaemia, hyperleptinemia, and oxidative stress associated with endothelial dysfunction and hepatic complications. Cherry consumption for 2 months, in addition to lifestyle measures, in T2D-rats decreased and normalized the systemic disturbances, including oxidative stress complications. Moreover, in the vessel, cherry consumption decreased oxidative stress and increased endothelial nitric oxide (NO) synthase levels, thus increasing NO bioavailability, ensuring vascular homeostasis. In the liver, cherry consumption decreased oxidative stress by inhibiting NADPH oxidase subunit p22phox expression, nuclear factor erythroid-2 related factor 2 (Nrf2) degradation and the formation of reactive oxygen species. It inhibited the activation of sterol regulatory element-binding proteins (1c and 2) and carbohydrate-responsive element-binding protein, and thus decreased steatosis as observed in T2D rats. This led to the improvement of metabolic profiles, together with endothelial and hepatic function improvements. CONCLUSION: Cherry consumption normalized vascular function and controlled hepatic complications, thus reduced the risk of diabetic metabolic disorders. These results demonstrate that a nutritional intervention with a focus on OS could prevent and/or delay the onset of vascular and hepatic complications related to T2D.

Proteomic analysis of Lactobacillus pentosus for the identification of potential markers involved in acid resistance and their influence on other probiotic features.

Acidity often prevents the undesirable microbial colonization both in fermented foods and under gastric conditions. Thus, the acid resistance of Lactobacillus pentosus strains used as starter cultures and/or probiotics requires further understanding. This was investigated by means of comparative proteomic approach using three strains representing the phenotypes: resistant (AP2-15), intermediate (AP2-18) and sensitive (LP-1) to acidic conditions. Proteomic analysis of constitutive phenotypes revealed that the intrinsic resistance of L. pentosus is associated with the over-production of three principal proteins: 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 2 (PGAM-d), elongation factor G and 50S ribosomal protein L10, and additionally on ATP synthase subunit beta and chaperone protein DnaK; they are associated with metabolic pathways of proteins and carbohydrates, energy production and stress responses. Suggested protein biomarkers for acid resistance in L. pentosus include elongation factor G and PGAM-d, both being abundantly found in the constitutive proteome of the resistant phenotype under standard and acidic conditions. Furthermore, L. pentosus strains pre-exposed to acids displayed enhanced probiotic function such as auto-aggregation ability via surface proteins. We conclude that pre-exposure of probiotic L. pentosus strains to acid may strategically enhance their performance as starter cultures and probiotics.

Comparative proteomic analysis of a potentially probiotic Lactobacillus pentosus MP-10 for the identification of key proteins involved in antibiotic resistance and biocide tolerance.

Probiotic bacterial cultures require resistance mechanisms to avoid stress-related responses under challenging environmental conditions; however, understanding these traits is required to discern their utility in fermentative food preparations, versus clinical and agricultural risk. Here, we compared the proteomic responses of Lactobacillus pentosus MP-10, a potentially probiotic lactic acid bacteria isolated from brines of naturally fermented Aloreña green table olives, exposed to sub-lethal concentrations of antibiotics (amoxicillin, chloramphenicol and tetracycline) and biocides (benzalkonium chloride and triclosan). Several genes became differentially expressed depending on antimicrobial exposure, such as the up-regulation of protein synthesis, and the down-regulation of carbohydrate metabolism and energy production. The antimicrobials appeared to have altered Lb. pentosus MP-10 physiology to achieve a gain of cellular energy for survival. For example, biocide-adapted Lb. pentosus MP-10 exhibited a down-regulated phosphocarrier protein HPr and an unexpressed oxidoreductase. However, protein synthesis was over-expressed in antibiotic- and biocide-adapted cells (ribosomal proteins and glutamyl-tRNA synthetase), possibly to compensate for damaged proteins targeted by antimicrobials. Furthermore, stress proteins, such as NADH peroxidase (Npx) and a small heat shock protein, were only over-expressed in antibiotic-adapted Lb. pentosus MP-10. Results showed that adaptation to sub-lethal concentrations of antimicrobials could be a good way to achieve desirable robustness of the probiotic Lb. pentosus MP-10 to various environmental and gastrointestinal conditions (e.g., acid and bile stresses).

Analysis of sitosteryl oleate esters in phytosterols esters enriched foods by HPLC-ESI-MS(2.).

Phytosteryl esters (PE)-enriched spreads are marketed for eating and cooking purposes. Temperature and also light exposure are the major factors leading to the formation of PE oxides in food matrix. In this study a high-speed HPLC-MS(2) method was developed to analyze the major PE present in PE-enriched spreads: sitosteryl oleate (SO) and its oxidation products, by using synthesized compounds as standards. This analytical method was used to quantify seven SO oxides formed in PE-enriched spreads after heating at different temperatures for varying time periods and after prolonged exposure to sunlight. Quantification of remaining native SO was also performed after these different treatments. It was found that under specific heating conditions the decrease of the SO amount was much more important compared to the formation of SO oxides showing that many other products are formed. In contrast to heating, sunlight radiation did not result in the degradation of SO and very few oxides were formed.

TiO2 photocatalysis damages lipids and proteins in Escherichia coli.

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O2·(-)), inhibited this effect by half, showing us that O2·(-) radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO2, respectively, which is accounted for by the cytotoxic effect of TiO2. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO2 increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O2·(-) on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

Comparative proteomic analysis of Listeria monocytogenes exposed to enterocin AS-48 in planktonic and sessile states.

Enterocin AS-48 is a cyclic peptide of great interest for application in food preservation and sanitation. In the present study, the proteome response of Listeria monocytogenes to purified enterocin AS-48 was studied under two different conditions: planktonic cells and sessile cells grown on polystyrene plates. Ten different proteins were differentially expressed in planktonic L. monocytogenes cells treated with 0.1 µg/ml enterocin AS-48 compared to the untreated controls. Overexpressed proteins were related to stress response (DnaK) or carbohydrate transport and metabolism, while underexpressed and unexpressed proteins were related to metabolism (such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate oxidase, glutamate dehydrogenase or glutamate decarboxylase) or stress (GroEL). In the sessile state, L. monocytogenes cells tolerated up to 10 µg/ml bacteriocin, and the treated biofilm cells overexpressed a set of 11 proteins, some of which could be related to stress response (DnaK, GroEL), protein synthesis and carbohydrate metabolism, while glyceraldehyde-3-phosphate dehydrogenase was the only unexpressed protein. Some of the overexpressed proteins (such as elongation factor Tu and GroEL) could also be implicated in cell adhesion. These results suggest different cell responses of L. monocytogenes to enterocin AS-48 in the planktonic and in the sessile state, including stress response and cell metabolism proteins. While in the planktonic state the bacterium may tend to compensate for the cytoplasmic cell permeability changes induced by AS-48 by reinforcing carbohydrate transport and metabolism, sessile cells seem to respond by shifting carbohydrate metabolism and reinforcing protein synthesis. Stress response proteins also seem to be important in the response to AS-48, but the stress response seems to be different in planktonic and in sessile cells.

Effect of heat treatment on the content of individual phospholipids in coffee beans.

In this study, the thermal stability of phospholipids (PLs) extracted from coffee beans was investigated. Chemical analysis was used to obtain information about the effect of heat treatment on the content of PLs in roasted coffee beans. Normal phase high-performance liquid chromatography (HPLC) combined with evaporative light scattering detector (ELSD) was applied to identify and quantify the classes of PLs. The results showed that only two PLs (phosphatidylinositol (PI), and phosphatidylcholine (PC)) were detected in coffee. One gram total lipid of green coffee contained 7.79 mg of PI, 9.6 mg of PC. As the duration of heat treatment was increased, there was a decrease in the content of PI and PC. With increases in temperature at various durations, the content of PI and PC was significantly decreased. Moreover, PI has been found to be more thermo sensitive than PC.

SPE for the simultaneous determination of various isothiocyanates.

Several SPE sorbents were investigated for the extraction of a group of chemically diverse isothiocyanates (ITCs). They included bonded silica, carbon-based, and polymer-based sorbents with various functional groups. Results showed large differences in the ability of these sorbents to simultaneously extract ITCs from standard solutions. Recovery rates were on average the highest with divinylbenzene (DVB) based polymeric sorbents, especially with a DVB/N-vinylpyrrolidone copolymer that had recovery rates ranging between 86.7 and 95.6%. These sorbents achieved the most balanced extraction efficiency between aliphatic and aromatic, polar, and nonpolar ITCs. With graphitized carbon, C(18)-bonded silica, and amide-containing sorbent, recovery levels were higher for the two least polar aromatic ITCs (benzyl ITC and phenylethyl ITC), whereas for the polar aliphatic ITCs levels were the lowest. The least retained one, was methyl ITC that is the most polar with recoveries between 0 and 31.5%. The presence of amide groups, especially in a polyamide sorbent, appeared to be particularly unsuitable for the extraction of aliphatic ITCs. A copolymer made up of DVB and N-vinylpyrrolidone was therefore shown to be the most suited for the extraction of both aliphatic and aromatic ITCs.

Improvement in determination of isothiocyanates using high-temperature reversed-phase HPLC.

The largely adopted reversed-phase HPLC analysis of the molecular species of isothiocyanates (ITCs) was performed and showed losses during the chromatographic run with eight ITCs. These losses, which obviously impact the accuracy of quantitative determinations, were due to precipitation in the chromatographic system. At 22°C, they ranged from 5.4% for sulforaphane (SFN) to 11.0% for benzyl-ITC when ITCs were injected at 80 µg mL(-1) , but they were up to three times higher at 1 mg mL(-1) reaching 31.9% for benzyl-ITC. The water solubility of the ITCs was a key determinant of the extent of the measured loss. When the column was heated at 60°C, losses in injected ITCs were reduced, in comparison with 22, 40, and 50°C, by two to ten times depending on the ITC considered. A reversed-phase HPLC method based on column heating was suggested and its quantitative performance was determined. It was then applied to the separation of methylene chloride extracts of various cruciferous vegetables. Ally-ITC, SFN, and iberin in cabbage; SFN and iberin in cauliflower; and allyl-ITC and phenylethyl-ITC in horseradish could be identified and quantified. The obtained results cast doubt on quantitative determinations of ITCs that are carried out at room temperature using reversed-phase HPLC.

Determination of phosphatidylethanolamine molecular species in various food matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS2).

A liquid chromatographic-electrospray ionization-tandem mass spectrometric (LC-ESI-MS(2)) method has been developed for determination of the molecular species of phosphatidylethanolamine (PE) in four food matrices (soy, egg yolk, ox liver, and krill oil). The extraction and purification method consisted of a pressurized liquid extraction procedure for total lipid (TL) extraction, purification of phospholipids (PLs) by adsorption on a silica gel column, and separation of PL classes by semi-preparative normal-phase HPLC. Separation and identification of PE molecular species were performed by reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry (ESI-MS(2)). Methanol containing 5 mmol L(-1) ammonium formate was used as the mobile phase. A variety of PE molecular species were detected in the four food matrices. (C16:0-C18:2)PE, (C18:2-C18:2)PE, and (C16:0-C18:1)PE were the major PE molecular species in soy. Egg yolk PE contained (C16:0-C18:1)PE, (C18:0-C18:1)PE, (C18:0-C18:2)PE, and (C16:0-C18:2)PE as the major molecular species. Ox liver PE was rich in the species (C18:0-C18:1)PE, (C18:0-C20:4)PE, and (C18:0-C18:2)PE. Finally, krill oil which was particularly rich in (C16:0(alkyl)-C22:6(acyl))plasmanylethanolamine (PakE), (C16:0-C22:6)PE, and (C16:0-C20:5)PE, seemed to be an interesting potential source for supplementation of food with eicosapentaenoic acid and docosahexaenoic acid.

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics.

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.

Liquid chromatography-tandem mass spectrometry for the determination of sphingomyelin species from calf brain, ox liver, egg yolk, and krill oil.

In this study, molecular species of sphingomyelin (SM) in egg yolk, calf brain, ox liver, and krill oil were investigated. Classes of phospholipids (PLs) were purified, identified, and quantified by normal phase semipreparative high-performance liquid chromatography (HPLC) combined with evaporative light scattering detectors (ELSD). For SM molecular species identification, pure SM collected through a flow splitter was loaded to HPLC-electrospray ionization-tandem mass spectrometry (LC-ESI-MS(2)), with 100% methanol containing 5 mM ammonium formate as mobile phase. In addition to classes of PLs, the used approach allowed the determination of profiles of SM species in egg yolk, ox liver, and calf brain, whereas krill oil turned out not to contain any SM. It also allowed the separation and identification of SM subclasses, as well as tentative identification of species with the same molecular mass, including isomers. The results showed that egg yolk contained the highest proportion of (d18:1-16:0)SM (94.1%). The major SM molecular species in ox liver were (d18:1-16:0)SM (25.5%), (d18:1-23:0)SM (19.7%), (d18:1-24:0)SM (13.2%), and (d18:1-22:0)SM (12.5%). Calf brain SM was rich in species such as (d18:1-18:0)SM (40.7%), (d18:1-24:1)SM (17.1%), and (d18:1-20:0)SM (10.8%).