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.

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.

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.

Structural identities of four glycosylated lipids in the oral bacterium Streptococcus mutans UA159.

The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have alpha-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.

Lipid Composition of Liposomal Membrane Largely Affects Its Transport and Uptake through Small Intestinal Epithelial Cell Models.

Lipid composition of liposomal bilayer should alter the cell response for permeability, transport, and uptake in small intestine. This work was done to investigate the transport and uptake of liposomes composed of docosahexaenoic acid-enriched phosphatidylcholine (PtdCho), phosphatidylserine (PtdSer), and sulfoquinovosyl diacylglycerol (SQDG) derived from marine products on multilamellar vesicles (MLV) in small intestinal epithelial cell models. The results showed that addition of PtdSer and SQDG as liposomal bilayer could improve the efficiency entrapment of liposomes. The liposomes containing PtdSer showed higher transport and uptake through both Caco-2 cell and M cell monolayers as compared to PtdCho-MLV. SQDG-containing liposomes exhibited only higher transport through M cell monolayer, while its uptake effect was higher both in Caco-2 cell and M cell monolayers. The results of experiments done with endocytosis inhibitors indicated that PtdCho-MLV must be transported via macropinocytosis and uptaken by phagocytosis in M cell monolayer model. PtdCho/PtdSer-MLV and PtdCho/SQDG-MLV might be transported and uptaken through M cell monolayer by phagocytosis. The result also indicated that PtdCho/SQDG-MLV could open the tight junction of small intestinal epithelial cell monolayers. Furthermore, our findings demonstrated that the surface status of cholesterol-containing liposomes were smooth, but they did not affect their transport and uptake through Caco-2 cell and M cell monolayers.

Protective mechanism of beta-SQAG9 liposome, a sulfonoglycolipid extracted from sea urchin intestines, against hepatic ischemia reperfusion injury.

We previously reported that beta-SQAG9 liposome, a sulfonoglycolipid extracted from sea urchin intestines, had a protective effect against hepatic ischemia reperfusion (I/R) injury. In this study, we made a detailed investigation of this protective effect and its mechanism. Rats were pretreated either with beta-SQAG9 liposome (treated group) or with phosphate-buffered saline solution (control group). Thereafter, they were subjected to partial hepatic I/R. The serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and histological damage was evaluated with hematoxylin and eosin staining. To investigate the protective mechanism of beta-SQAG9 liposome on I/R injury, the serum levels and the tissue messenger RNA levels of TNF-alpha and IL-1beta were measured, and polymorphonuclear neutrophil (PMN) infiltration was histologically evaluated by immunohistochemistry. Moreover, to investigate an interaction between beta-SQAG9 liposome and L-selectin on PMNs, flow cytometric analysis and immunofluorescence were performed. beta-SQAG9 liposome reduced the hepatic I/R injury. The pretreatment with beta-SQAG9 liposome reduced the PMN infiltration into the liver parenchyma. On the other hand, there was no apparent difference in the serum levels and the tissue messenger RNA levels of the proinflammatory cytokines between the two groups. Thus, beta-SQAG9 liposome might reduce the hepatic I/R injury by inhibition of the PMN infiltration into the liver parenchyma, which was independent of the regulation of cytokine production. Moreover, we demonstrated that beta-SQAG9 liposome specifically bound to L-selectin on PMN cell surface, which mediated the PMN infiltration. beta-SQAG9 liposome might competitively antagonize L-selectin on PMNs and suppress the subsequent PMN infiltration, resulting in the reduction in I/R injury.

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis.

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac2SGL) and the phosphatidyl-myo-inositol dimannosides (PIM2) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6'-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines.

Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant for induction of cell-mediated immunity.

Archaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

Microfiltration of butter serum upon casein micelle destabilization.

The gross composition of butter serum, the aqueous phase of butter, is comparable to that of buttermilk, except that it has a higher content of material derived from the milk fat globule membrane (MFGM). As such, butter serum is a good source for further purification of MFGM material. The purified fraction could be of interest for its emulsifying and nutritional properties. The effect of sodium citrate and ethanol on the dissociation of butter serum casein micelles, and their effect on casein retention upon tangential microfiltration were investigated. Optimal conditions of casein micelle dissociation were assessed by using an experimental design (response surface full central composite orthogonal design) with temperature and ethanol or sodium citrate concentration as design variables and the Hunter L* value as response variable. For both dissociating agents, a highly significant reduced quadratic model was fit to the data. Microfiltration tests were performed on pure butter serum, and on butter serum in the presence of sodium citrate, under optimal dissociation conditions (50 degrees C, 80 mM). A cellulose acetate membrane with a pore size of 0.15 microm was used. From the filtration curves and fouling coefficients it was clear that the addition of sodium citrate improved the permeation flux, and minimized fouling. All fractions were analyzed for dry matter, protein, lactose, lipid, and polar lipid contents. The protein fraction was further characterized by sodium dodecyl sulfate-PAGE. It was shown that sodium citrate greatly enhanced casein transmission through the membrane, but at the expense of substantial losses of polar lipids.

Treatment with beta-SQAG9 prevents rat hepatic ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) injury occurs in various situations, including transplantation, trauma, and shock. We previously reported that the synthetic beta-SQDG (18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessed immunosuppressive effects, such as inhibition of T-cell responses in human allogenic human mixed lymphocyte reactions (MLR) and skin allograft survival in rats. beta-SQAG9 was synthesized from beta-SQDG (18:0) to improve structural stability in aqueous solution with the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin) in vitro. We hypothesized that beta-SQAG9 might attenuate leukocyte rolling on the endothelium and neutrophil infiltration in which L-selectin and P-selectin are key molecules. We investigated the protective effect of beta-SQAG9 against hepatic I/R injury. METHODS: Male Lewis rats were divided into 6 groups: sham, control, and treatment. Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30 minutes. They were injected with PBS or beta-SQAG9 at doses of 5, 10, 25, and 50 mg/kg into the penile vein immediately before reperfusion. To assess the damage to the hepatic parenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured and histological evaluation was performed at 6 hours after reperfusion. RESULTS: In the group treated with beta-SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDH were significantly reduced, and the amount of neutrophil infiltration also was significantly reduced. CONCLUSIONS: Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R injury.

Mycobacterial glycopeptidolipid interactions with membranes: a monolayer study.

Mycobacterial glycopeptidolipid (GPL) interactions with membranes were analysed with monolayer experiment, using GPLs bearing 3, 1, or 0 carbohydrate residues (GPL3, GPL1, GPL0). Compression isotherms and surface potential determinations suggested that the glycopeptidic moiety of GPL3 permanently dipped in water, while those of GPL1 and GPL0 can lay in the interface. Insertion of GPL molecules into a preformed phospholipid monolayer was observed using GPL3 or GPL1 dispersions, but not from GPL0. It is postulated that the activity of GPL0 is low due to its failure to become inserted into membranes, as is that of GPL3 owing to its insertion only by its acyl chain. GPL1 is likely to disturb membranes by inserting its glycopeptidic moiety into the interface.

Interaction of hopanoids with phosphatidylcholines containing oleic and omega-cyclohexyldodecanoic acid in lipid bilayer membranes.

Lipid bilayer membranes were made from hopanoid phosphatidylcholine mixtures dissolved in decane. The specific capacity of the mixed membranes was found to increase with increasing hopanoid content. This indicates an interaction between hopanoids and lipids which leads to a reduction of the chemical potential of the solvent in the membranes. The structural properties of mixtures of hopanoids and phosphatidylcholines were investigated using charged probe molecules, the negatively charged lipophilic ions dipicrylamine (DPA) and tetraphenylborate (TphiB) and the positively charged potassium complex PV-K+ (PV, cyclo (D-Val-L-Pro-L-Val-D-Pro)3). The transport properties of the lipophilic ions in the mixed membranes indicate that the electrical properties like dipolar potential and surface potentials of phosphatidylcholine membranes are not changed by the insertion of the hopanoids. The translocation rate constant K of the PV-K+ complex is drastically reduced in the hopanoid phosphatidylcholine membranes with increasing hopanoid content. This effect is discussed on the basis of an alteration of the microviscosity in the mixed membranes. There exists a close analogy between the action of cholesterol and hopanoids in bilayer membranes from phosphatidylcholines. A bilayer membrane composed of di-omega-cyclohexyldodecanoyl-phosphatidylcholine (DCPC) was found to possess a higher specific capacity as compared to other phosphatidylcholines. Also a lower translocation rate constant for PV-K+ was observed which may be caused by the relative high microviscosity of this lipid even above the phase transition temperature.

Lipase-mediated deacetylation and oligomerization of lactonic sophorolipids.

The direct enzymatic polymerization of lactonic sophorolipids (SLs) was investigated with four lipases, including porcine pancreatic lipase (PPL), immobilized Mucor miehei lipase (MML), lyophilized Candida antarctica lipase (Fraction B, CAL-B), and lyophilized Pseudomonas sp. lipase (PSL). Several organic solvents, covering a wide range of polarity, were compared for suitability as the reaction medium. Isopropyl ether and toluene were found most effective. According to the quantification and structure identification by HPLC and LC-MS, the reaction proceeded with the formation of monoacetylated lactonic SLs and the subsequent conversion of the intermediates to oligomers and polymers, presumably through ring-opening polymerization. Temperature was found to have significant effects on the reaction. Both the conversion of reactant SLs and the subsequent formation of oligomers and polymers from the intermediates were faster at 60 degrees C than at 50 degrees C. The substrate selectivity among the three dominant reactant SLs also differed with the temperature. The conversion rate increased with the ring size of the lactones at 60 degrees C, but it decreased with the size at 50 degrees C.

Galactose-specific lectin from Viscum album as a mediator of aggregation and priming of human platelets.

Galactose-specific lectin from Viscum album (VAA) was found to induce aggregation of human platelets in a dose- and sugar-dependent manner. Small nonaggregating concentrations of VAA primed the response of platelets to known aggregants (ADP, arachidonic acid, thrombin, ristocetin, and A23187). VAA-induced platelet aggregation was completely reversible by addition of the sugar inhibitor lactose and the platelets from disrupted aggregates maintained the response to other aggregants. The lectin-induced aggregation of washed platelets was more resistant to metabolic inhibitors than thrombin- or arachidonic acid-dependent cell interaction. In contrast to the related galactose-specific lectin from Ricinus communis and the soy bean agglutinin, the lectin did not aggregate liposomes prepared from total platelet lipids, indicating different affinities of aggregation-mediating lectins to platelet glycolipids.

A new screening method for antimitotic substances and isolation of glycolipids as stimulators of tubulin polymerization from Okinawan sponge Pseudoceratina sp.

A new screening method to detect antimitotic substances utilizing purified porcine brain microtubule proteins was developed. This method observes the inhibitory and stimulatory activities on microtubule polymerization and inhibitory activity on depolymerization in sequence. Two glycolipids, 1-O-beta-D-galactopyranosyl-2,3-di-O-acylglycerol and 1-O-tetrahydroxycyclopentyl-2-O-acyl-3-O-alkylglycerol were isolated from Okinawan marine sponge Pseudoceratina sp. by this screening method. These compounds stimulated the microtubule polymerization at 10 degrees C.

[In vitro activation of immune cells by a mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate, derived from Gordona aurantiaca].

A mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate (GaGM), derived from Gordona aurantiaca, an acid-fast bacteria closely related taxonomically to Mycobacterium, was investigated for its immune adjuvant activity in vitro. The liposomes containing GaGM showed strong mitogenic effects on murine spleen cells at the doses used (25-100 micrograms/ml), but not on T-cell-depleted spleen cells or macrophage-depleted spleen cells. These results suggest that the mitogenic property of liposomes containing GaGM differs from that of such as lipopolysaccharide, a B-cell mitogen and that its mitogenic effects depend on the presence of macrophages. In addition, liposomes containing GaGM augmented the mixed lymphocyte reaction (MLR) and in vitro induction of cytotoxic T-lymphocytes (CTLs) against allogeneic tumor cells. These results suggest that liposomes containing GaGM have immune adjuvant properties in vitro and the adjuvant activity may be related to such cytokines as interleukin-1 and -2.

Oral peptide delivery by tetraether lipid liposomes.

The aim of this study is to improve of oral peptide delivery by a novel type of liposomes containing tetraether lipids (TELs) derived from archaea bacteria. Liposomes were used for the oral delivery of the somatostatin analogue octreotide. TELs were extracted from Sulfolobus acidocaldarius and subsequently purified to single compounds. Liposomes were prepared by the film method followed by extrusion. Vesicles in size between 130 and 207 nm were obtained as confirmed by photon correlation spectroscopy. The pharmacokinetics of radiolabeled TELs in liposomes was investigated after oral administration to rats. 1.6% of the applied radioactivity in fed and 1.5% in fasted rats was recovered in the blood and inner organs after 2h, while most of the radioactivity remained in the gastro-intestinal tract. After 24h the percentage of radioactivity in inner organs was reduced to 0.6% in fed rats, respectively 1.0% in fasted animals. Several liposomal formulations containing dipalmitoyl phosphatidylcholine (DPPC) and TELs in different ratios were loaded with octreotide and orally administered. Liposomes with 25% TEL could improve the oral bioavailability of octreotide 4.1-fold and one formulation with a cationic TEL derivative 4.6-fold. TEL-liposomes probably act by protecting the peptide in the gastro-intestinal tract.

Glycolipid composition of Hevea brasiliensis latex.

Glycolipids of fresh latex from three clones of Hevea brasiliensis were characterized and quantified by HPLC/ESI-MS. Their fatty acyl and sterol components were further confirmed by GC/MS after saponification. The four detected glycolipid classes were steryl glucosides (SG), esterified steryl glucosides (ESG), monogalactosyl diacylglycerols (MGDG) and digalactosyl diacylglycerols (DGDG). Sterols in SG, ESG and total latex unsaponifiable were stigmasterol, ß-sitosterol and Δ5-avenasterol. The latter was found instead of fucosterol formerly described. Galactolipids were mainly DGDG and had a fatty acid composition different from that of plant leaves as they contained less than 5% C18:3. Glycolipids, which represented 27-37% of total lipids, displayed important clonal variations in the proportions of the different fatty acids. ESG, MGDG and DGDG from clone PB235 differed notably by their higher content in furan fatty acid, which accounted for more than 40% of total fatty acids. Clonal variation was also observed in the relative proportions of glycolipid classes except MGDG (8%), with 43-51% DGDG, 30-34% SG and 7-19% ESG. When compared with other plant cell content, the unusual glycolipid composition of H. brasiliensis latex may be linked to the peculiar nature of this specialized cytoplasm expelled from laticiferous system, especially in terms of functional and structural properties.

Archaebacterial tetraetherlipid liposomes.

Liposomes are widely investigated for their applicability as drug delivery systems. However, the unstable liposomal constitution is one of the greatest limitations, because the liposomes undergo fast elimination after application to the human body. In the presented study, novel archeal lipids were used to prepare liposomal formulations which were tested for their stability at elevated temperatures, at different pH-values and after heat sterilization.