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PubMed2002

Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin.

The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS-ML combination system (utilizing lactoperoxidase (LPX) in the range 5-200 mg kg(-1) and ML in the range 50-1,000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS-ML systems over that obtained in comparable LPS only systems was not observed in ground beef The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate.

Animals↗
PubMed1997

Hydrolysis of esters by staphylococci.

The objective of this work was to characterize the hydrolysis of esters by staphylococci in order to understand if they could contribute to the release of free fatty acids in sausage. Cell-free extracts and extracellular concentrates of staphylococci were examined for esterase activities against p-nitrophenyl esters and for lipolytic activities against triolein. Staphylococci showed intracellular and extracellular esterase activities with different esterase electrophoretic patterns. Cell-free extracts of S. xylosus, S. warneri and S. saprophyticus preferentially hydrolysed p-nitrophenyl butyrate whereas their extracellular concentrates were mainly active against p-nitrophenyl butyrate, p-nitrophenyl caproate and p-nitrophenyl caprylate. In addition their extracellular concentrates hydrolysed triolein. The two strains of S. carnosus differed as they did not show pronounced p-nitrophenyl substrate specificity and did not hydrolyse triolein. Staphylococci hydrolysed esters at a high rate between 15 and 25 degrees C; acidic conditions inhibited the hydrolysis. The hydrolysis was also reduced when the water activity was decreased by addition of polyethylene glycol or glycerol.

Esterases↗
PubMed1997

Weak acid inhibition of fermentation by Zygosaccharomyces bailii and Saccharomyces cerevisiae.

The inhibition kinetics of fermentation by Zygosaccharomyces bailii and Saccharomyces cerevisiae were evaluated for weak carboxylic acids. Several regression equations were tried to fit the experimental data, most cases being best fitted to exponential curves. The following parameters were determined: i) acid concentration responsible for 50% inhibition of fermentation (C50%); ii) area under the regression curve up to that concentration (A50%) and iii) exponential inhibition constant (k(i)). These parameters were compared according to their ability to express the inhibitory effect of each acid. In broad terms, the values of k(i) in association with minimum inhibitory concentrations (x(min)), were found best to express the inhibitory effect of the weak acids. However, C50% values were satisfactorily correlated with k(i). The value of A50% more precisely reflected the occasional stimulatory effect of low concentrations of weak acids. Comparison of inhibition parameters for Z. bailii and for S. cerevisiae revealed a higher resistance of the former to acetic, propionic, butyric and benzoic acids and similar resistance to caproic, caprylic and sorbic acids. Previous cultivation in the presence of acetic, propionic and benzoic acids showed a distinct influence on the resistance of both yeasts, although it did not always induce cellular adaptation. Fermentation inhibition showed a good correlation with the lipid solubility of weak acids suggesting that the acids interact with the hydrophobic regions of cell membranes.

Carboxylic Acids↗
PubMed2001

Characterization of the influence of polyol fatty acid esters on the permeation of diclofenac through rat skin.

In this study, we investigated the effect of sefsols on the skin to clarify the mechanism of the sefsol enhancement effect. In vitro percutaneous absorption experiments were performed using Franz diffusion cells. Removal of the stratum corneum and delipidization of the skin increased the permeation of diclofenac from aqueous suspension, with the enhancement effects being similar for both treatments. Further enhancement effects of diclofenac permeation by sefsol through the stripped and delipidized skin were not observed. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was employed to investigate the biophysical changes in the stratum corneum lipids by sefsols. One of the sefsols, propylene glycol mono caprylate (S-218), induced higher and broader absorbance shifts in both asymmetric and symmetric C-H bond stretching regions. However, no significant differences were observed among the sefsols with respect to peak heights and areas for both absorbances when compared with H2O treatment. These results suggest that sefsol may change the lipid-chain fluidity of the stratum corneum without lipid extraction. The accumulated amounts of diclofenac in the skin significantly increased in the presence of sefsol. Also, the amounts of diclofenac in the skin increased with the amount of sefsol in the skin. This sefsol enhancement effect was reversed at 12 h after treatment. Thus, enhancement of the diclofenac flux by sefsols is reversible and may be due to a change in the lipid-chain fluidity of the stratum corneum and improvement in drug partitioning to the stratum corneum.

Animals↗
PubMed2001

Emulsions containing partially water-miscible solvents for the preparation of drug nanosuspensions.

The aim of this study was to investigate the feasibility of partially water-miscible solvents, such as benzyl alcohol, butyl lactate and triacetin, to prepare drug nanosuspensions by a solvent quenching technique. Mitotane, which possesses very poor water solubility and low bioavailability, was used as model drug. Preparation was by emulsifying an organic solution of the drug in an aqueous solution of a stabilising agent followed by rapid displacement of the solvent from the internal into the external phase, provoking solid particle formation. To verify the influence of emulsion droplet size on the drug particle size, 0.1 or 0.2% of different emulsifiers (Tween 80, caprylyl-capryl glucoside or lecithin) and different homogenisation conditions (Ultra Turrax or a high pressure homogenizer at 200 or 1000 bar for three cycles) were used. In general, emulsion droplet size decreased with high pressure homogenization and on increasing the number of cycles. The size of drug particles, obtained after adding water at a constant rate, was dependent on the droplet size in the emulsion. Drug particles of approximately 80 nm were obtained using butyl lactate, supporting the hypothesis that drug particle formation by the emulsification diffusion process involves generating regions of local supersaturation. Because of the increase in available surface area, the dissolution rate of diaultrafiltrated suspensions increased greatly compared to commercial product.

Emulsions↗
PubMed2003

Influence of ion pairing on topical delivery of retinoic acid from microemulsions.

The purpose of the present study was to determinate the significance of ion pairing on the topical permeation of retinoic acid (R.A) using microemulsions as delivery vehicles. Phenylalanine methyl ester, phenylalanine ethylester, histidine methyl ester, tryptophan methyl ester and valine methyl ester were used as counter ions. Results of diffusion studies through polydimethylsiloxane membrane (PDMS) indicate that retinoic acid permeation from ethanol-pH 6.4 buffer mixture significantly increased in the presence of counter ions. A linear relationship was found between apparent partition coefficients and permeation coefficients. The highest values were with valine methyl ester and phenylalanine ethyl ester. In order to develop alternative formulations for topical administration of R.A, microemulsions were evaluated as delivery vehicles. Oil-in-water (O/W) and water-in-oil (W/O) microemulsion formulations were prepared using water, isopropyl myristate, lecithin, caprylyl-capryl glucoside and ethanol or 1,2 hexanediol. Experiments with PDMS membranes showed decreasing permeabilities of R.A from microemulsions in the presence of counter ions. This was related to the increased lipophilicity and different vehicle membrane affinity of the ion pairs The ability of the systems to deliver R.A through the skin was evaluated in vitro using pig-skin. R.A permeabilities were much lower with microemulsions than with solution, while a large increase in R.A skin deposition was observed only from O/W microemulsions in the presence of counter ions. The depth of skin accumulation was below 100 microm after 24 h application. The results suggest that O/W microemulsions containing a counter ion can be used to optimise drug targeting without a concomitant increase in systemic absorption.

Administration, Topical↗
PubMed1999

PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug.

The nanoprecipitation technique for preparation of nanoparticles suffers the drawback of poor incorporation of water soluble drugs. The aim of this study was therefore to assess various formulation parameters to enhance the incorporation of a water soluble drug (procaine hydrochloride) into poly(dl-lactide-co-glycolide) (PLGA) nanoparticles prepared by this technique. Approaches investigated for drug incorporation efficiency enhancement included the influence of aqueous phase pH, replacement of procaine hydrochloride with procaine dihydrate and the inclusion of excipients: poly(dl-lactide) (PLA) oligomers, poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA) or fatty acids into the formulation. The nanoparticles produced were submicron size (<210 nm) and of low polydispersity. It was found that an aqueous phase pH of 9.3, replacement of procaine hydrochloride with procaine dihydrate and the incorporation of PMMA-MA, lauric and caprylic acid into the formulation could enhance drug incorporation efficiency without the size, morphology and nanoparticle recovery being adversely influenced. For instance changing the aqueous phase pH from 5.8 to 9.3 increased nanoparticle recovery from 65.1 to 93.4%, drug content from 0.3 to 1.3% w/w and drug entrapment from 11.0 to 58.2%. However, the presence of high ratios of lauric acid and procaine dihydrate in the formulation adversely affected the morphology and size of the nanoparticles. Also, PLA oligomers were not considered a feasible approach since it decreased drug entrapment from 11.0 to 8.4% and nanoparticle recovery from 65.1 to 19.6%. Drug release from nanoparticles appears to consist of two components with an initial rapid release followed by a slower exponential stage. This study has demonstrated that formulation variables can be exploited in order to enhance the incorporation of a water soluble drug into PLGA nanoparticles by the nanoprecipitation technique.

Buffers↗
PubMed1977

[Properties of and information about a new preparation of immunoglobulins (IgGAM) enriched in IgA and IgM].

A new immunoglobulin preparation, called IgGAM, has been obtained from fraction III. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III; the immunoglobulin concentrate is finally obtained by ethanol precipitation of the supernatant. The IgGAM preparation contains IgG,IgA and IgM in various proportions according to the modifications of the initial technique, and we can obtain fractions more specially enriched in IgA or in IgM. The clinical results obtained with IgGAM preparation are summarized; different types of antibody deficiency syndromes have been successfully treated. It can be injected once à week IM, at a dose of 0.5 ml/kg. No side effects, nor appearance of anti IgA antibodies have been observed.

Female↗
PubMed2000

Application of atmospheric pressure chemical ionization liquid chromatography-mass spectrometry in identification of lymph triacylglycerols.

Atmospheric pressure chemical ionization liquid chromatography-mass spectrometry was used in the identification of triacylglycerol molecular species in lymph samples from rats given either a structured lipid or safflower oil. The structured lipid was MLM-type (M, medium-chain fatty acid; L, long-chain fatty acid) and manufactured from caprylic acid (8:0) and the oil (safflower oil or high-oleic sunflower oil). The triacylglycerol composition of lymph varied significantly between structured triacylglycerols and safflower oil. Diacylglycerol fragment ions were found for all triacylglycerols and we could also observe the ammonium adduct molecular ion [M+NH4]+ for all the triacylglycerols at the selected conditions. Protonated molecular ions were formed from triacylglycerols containing unsaturated fatty acids, and fatty acid fragment ions were also observed in the case of strong fragmentation. The lymph triacylglycerols were identified from their ammonium adduct molecular ions and diacylglycerol fragment ions. In addition to the intact MLM-type structured triacylglycerols, the MLL- and LLL-type triacylglycerols were also identified. The absorption pathway of MLM-type structured triacylglycerols is most likely the same as that of conventional long-chain triacylglycerols, i.e. they were hydrolyzed into 2-monoacylglycerol and medium-chain fatty acids, which were then used for resynthesis of triacylglycerols. The present study thereby also demonstrates the possibility to study the absorption pathway of triacylglycerol via identification of triacylglycerol species in biological samples.

Animals↗
PubMed2001

Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products, by HPTLC coupled with in situ densitometric analysis.

The development of new and simple high performance thin layer chromatography (HPTLC) assays for the quantification of medium chain triglycerides (MCT, tricaprylin) and long chain triglycerides (LCT, triolein) and their lipolytic products, bile salts (BS) and phospholipids (PL) are described. Different classes of lipids (PL, BS, fatty acids, monoglycerides, diglycerides, and triglycerides) were separated on a single silica gel 60 HPTLC plate by Automated Multiple Development (AMD) methods using a Camag AMD 2. Post-chromatographic staining of long chain lipids (triolein, diolein, monoolein, and oleic acid), PL and BS with a solution of copper sulphate-phosphoric acid and medium chain lipids (tricaprylin, dicaprylin, monocaprylin, and caprylic acid) with a solution of ammonium molybdate-perchloric acid allowed visualisation of the lipids. Lipids were quantified by in situ spectrodensitometric measurements using a Camag TLC scanner 3. The intra- and inter-assay accuracy was between 83 and 115% and the assay was precise to within a CV of less than 20% over a range of 0.1-1 and 5-50 microg for long chain lipids and medium chain lipids, respectively. The methods have been employed to study the kinetics of triolein and tricaprylin lipolysis in an in vitro lipid digestion model commonly used to assess the digestibility of novel oral lipid-based formulations.

Chromatography, Thin Layer↗
PubMed2002

Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis.

The fatty acids have an important role in the control of leukocyte metabolism and function. Higher concentrations of certain fatty acids, particularly polyunsaturated fatty acids (PUFAs) and volatile fatty acids, can cause cell death via apoptosis or, when concentrations are greater, necrosis. In this study, we determined the highest concentrations of various fatty acids that are non-toxic to two human leukemic cell lines, Jurkat (T-lymphocyte) and Raji (B-lymphocyte). Toxicity was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. There were no remarkable differences for the toxicity of the fatty acids between B and T cell lines. The cytotoxicity of the fatty acids was related to the carbon chain length and number of double bonds: docosahexaenoic acid=eicosapentaenoic acid=arachidonic acid=gamma-linolenic acid=stearic acid=palmitic acid > linoleic acid=palmitoleic acid > vacenic acid=lauric acid > oleic acid > elaidic acid > capric acid > butyric acid > caprylic acid=caproic acid=propionic acid. The proportion of cells undergoing apoptosis or necrosis, induced by the fatty acids tested, remains to be investigated.

Apoptosis↗
PubMed2001

Dilatational rheology and foaming properties of sucrose monoesters in the presence of beta-lactoglobulin.

The foaming properties and the dilatational rheology of systems containing purified sucrose caprylate (SM800), caprate (SM1000), laurate (SM1200) and palmitate (SM1600) have been studied. Addition of beta-lactoglobulin (beta-lg) at a low concentration (0.050 wt.%) can aid the foam formation in the cases, where the surfactant concentration is insufficient to support foam formation. However, foams where both species co-existed exhibited poor stability. beta-lg was found to affect the dilatational properties of surfactant films even at low concentrations. It is thought that this could be related to the effect of the protein on the adsorption-desorption relaxation mechanism, or to the possible formation of a protein-surfactant complex in the bulk. The age of the protein film was also found to affect the kinetics of protein displacement by SM1000, as monitored by the change in the dilatational properties (Langmuir trough technique) and the relative reflectivity of the interface (Brewster angle microscopy) with time. An insoluble monolayer of sucrose stearate (Suc18) and beta-lg was also studied and it was found that the presence of small amounts of Suc18 in the protein film lead to a reduction of the interfacial elasticity. This is believed to be due to the disruption of the protein network. A possible mechanism could involve the obstruction of the hydrogen-bond intermolecular protein association by the strongly hydrated sucrose headgroup or the obstruction of the protein-protein hydrophobic interactions by the formation of an interfacial protein-surfactant complex.

Journal Article↗
PubMed1999

Purification and characterization of lipase from Rhizopus chinensis cells.

Lipase from Rhizopus chinensis cells was purified and characterized. The molecular mass of purified lipase was 28.4 kDa and the optimal temperature and pH for its activity were 37 degrees C and 5.5, respectively. Purified lipase exhibited high hydrolytic activity against fatty acid methyl esters such as methyl caprylate, methyl laurate, and methyl palmitate. Freeze-dried lipase catalyzed the transesterification between olive oil and methyl laurate in n-hexane.

Journal Article↗
PubMed2003

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae.

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Journal Article↗
PubMed2003

Evidence of a relationship between autolysis of starter bacteria and lipolysis in cheddar cheese during ripening.

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 degrees C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese 'juice' extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4:0) to linolenic acid (C18:3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4:0) to linolenic (C18:3) acids increased significantly (P<0.05) during ripening in cheeses made with either starter culture. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids were significantly higher (P<0.05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.

Bacteriolysis↗
PubMed1998

Rumen-protected methionine or lysine supplementation of Comisana ewes' diets: effects on milk fatty acid composition.

The effect of graded levels of dietary rumen-protected methionine (rp-Met) or lysine (rp-Lys) on the fatty acid composition of Comisana ewes' milk was investigated. The animals lambed in autumn and were separated from their lambs 7 weeks after parturition; they were divided into five groups of nine which were given different rations. The control group was fed on a mixture of vetch and oat hay with a pelleted concentrate; the other groups were given the control ration supplemented with rp-Met (3.5 or 7.0 g/kg) or rp-Lys (10.5 or 21.0 g/kg). Milk fatty acid composition was determined by gas chromatography, and these values were subjected to principal components analysis. A correlation was found between fatty acid composition and diet. We demonstrated that giving the experimental diets significantly affected milk fat, to an extent that was independent of the levels of the supplementary amino acids. The milk fat of the control group contained largely caprylic, capric and lauric acids, whereas that of the groups given the supplementary amino acids contained largely palmitic, palmitoleic, stearic and oleic acids. Thus supplementation resulted in an increase in the proportion of 16:0-18:3 (primarily palmitic and stearic) and a reduction in the proportion of 4:0-12:0 fatty acids. There was also a modest increase in the unsaturated:saturated fatty acid ratio.

Animals↗
PubMed1994

Characterization of the effector mechanisms of a transmission-blocking antibody upon differentiation of Plasmodium berghei gametocytes into ookinetes in vitro.

The transmission-blocking monoclonal antibody 13.1, which recognizes the ookinete surface antigen Pbs21 of Plasmodium berghei, and an IgG2a isotype control antibody 26.37 were purified by caprylic acid and ammonium sulphate precipitation. Fab fragments were prepared by papain digestion. IgG but not Fab from antibody 13.1 reduced ookinete formation by P. berghei in culture by as much as 94% at a concentration of 100 micrograms/ml. There was little difference in antibody efficacy in the range 6.25-400 micrograms/ml in this assay. The parasite was most sensitive to antibody activity in the first 6-9 h of culture, i.e. the gamete/zygote and early retort stages. Peripheral blood leucocytes (PBL) were essential to achieve maximal inhibition by mAb 13.1 (activity was abrogated totally if PBL were removed). Together the data suggest that one of the mechanisms of action of this antibody is antibody-mediated PBL killing. Phagocytosis of parasites was noted in these experiments in all cultures. We have not attempted in this study to distinguish between Fc-mediated opsonization, as opposed to antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1994

Novel membraneless amperometric peroxide biosensor based on a tetrathiafulvalene-p-tetracyanoquinodimethane electrode.

Amperometric biosensors for the detection of hydrogen peroxide are prepared by adsorbing peroxidase (POD, EC 1.11.1.7, lipophilized with caprylic aldehyde) to TTF-TCNQ/silicone oil paste electrodes. This is the first time a reductase is coupled to an organic conducting salt electrode. At -50 mV vs Ag/AgCl and pH 6.0, the current vs concentration function can be described by the enzyme kinetic Michaelis-Menten formalism. Stable signals are obtained within 10 s. The detection limit is typically in the low nanomolar range for H2O2. The enzyme stability under storage, standby, and various operation conditions is discussed.

Biosensing Techniques↗