Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Effect of Long Chain Fatty Acids on Bacterial Respiration and Amino Acid Uptake

S ummary . Long chain fatty acids stimulated oxygen uptake by Gram positive bacteria at bactericidal and protoplast lytic concentrations and produced inhibition at higher levels. The order of activity between individual acids and effects of reversal agents on respiratory activity corresponded to those which produced bactericidal activity. Protoplasts were more susceptible to inhibition than whole cells. Gram negative bacteria were inhibited to a limited extent at high fatty acid concentrations, but spheroplasts were highly sensitive. Fatty acids inhibited amino acid uptake both aerobically and anaerobically at sub-bactericidal levels. The effects were reversed by metal cations, and reflected the activity of dinitrophenol and sodium azide. The susceptibility of organisms to inhibition was of the same order as the sensitivity to other antibacterial effects. The probable mode of action of the fatty acids is discussed in terms of the interference with energy metabolism within the bacterial cell.     

Physicochemical Effects of Long Chain Fatty Acids on Bacterial Cells and their Protoplasts

S ummary . Fatty acids of chain length > C₁₀ induced lysis of protoplasts at pH 7·4 when the concentration was nearly bactericidal. At pH 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysis was produced by the same acids below bactericidal concentrations. The lysis was immediate at pH 8, but at pH 6 the effect was preceded by contraction of protoplasts. At pH 7·4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplasts. Reversal agents antagonized the lysis of protoplasts by fatty acids. A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed.     

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.     

Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters

Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18-C24) unsaturated fatty alcohols and C18-C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.     

Heterologous expression of a fatty acid hydroxylase gene in developing seeds of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Expression of a cDNA encoding the castor bean ( Ricinus communis L.) oleate Delta12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec- cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec- cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos- cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos- cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Delta-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Delta12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Delta12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.     

Effect of Metal Cations and pH on the Antibacterial Activity and Uptake of Long Chain Fatty Acids

S ummary . The bactericidal activity of long chain saturated fatty acids was antagonized by alkaline earth metals. The activity of linoleic acid was less effectively antagonized but was more sensitive to reversal by ferric and stannous ions. With increasing pH value the bactericidal activity of lauric acid decreased but that of the longer chain saturated acids increased. Both Gram positive and Gram negative bacteria reversibly adsorbed fatty acids. Uptake increased with decreasing pH value and increasing chain length. Although adsorbed to a lesser extent, the intrinsic activity of linoleic acid was greater than lauric acid. The uptake appeared to be non-specific and governed by the physicochemical properties of both the acids and the bacterial cell surfaces. Sensitivity to the fatty acids increased with decreasing pH value. Protoplasts of Bacillus megaterium adsorbed fatty acids to a greater extent than whole cells. Resistance of the Gram negative Pseudomonas phaseolicola was not due to non-adsorption of the fatty acids.     

Bactericidal effect of fatty acids on mycobacteria, with particular reference to the suggested mechanism of intracellular killing

The effect of fatty acids on Mycobacterium smegmatis was examined in vitro at pH 5.0 to 7.0 to determine the role of fatty acids in the intracellular killing of mycobacteria. Unsaturated fatty acids showed strong bactericidal activity in low concentrations (0.005 to 0.02 mM), whereas saturated fatty acids, except for lauric and myristic acids, were not very effective even at a concentration of 0.2 mM. Addition of a saturated fatty acid (palmitic or stearic acid) to an unsaturated fatty acid (oleic or linoleic acid) did not strongly interfere with the bactericidal effect of the unsaturated fatty acid at pH 5.0 and 6.0. Ca2+ (3.0 mM), Mg2+ (1.0 mM), and gamma-globulin (0.4%) showed weak reversal effects on the bactericidal activity of unsaturated fatty acids at pH 5.0 and 6.0. Serum albumin and serum showed strong reversal effects. The concentrations of each fatty acid in a mixture (molar ratio, 1:1:1:1) of oleic, linoleic, palmitic, and stearic acids required for the killing of M. smegmatis in the presence of 2% serum (bovine, rabbit, or human) were 0.05 to 0.10 mM at pH 5.0 and 6.0 and 0.05 to 0.20 mM at pH 7.0, depending on the serum used. The susceptibilities of M. kansasii, M. bovis strain BCG, and M. tuberculosis to the mixture of the four fatty acids in the presence of 2% bovine serum were similar to that of M. smegmatis, although M. fortuitum was more resistant.     

Chilling cells enhances the bactericidal action of fatty acids on Escherichia coli.

Preincubation at 0 C considerably increased the bactericidal action of 0.4% nonanoic and decanoic acids on Escherichia coli K-12 154. This lethal effect seemed to be dependent on the media used to grow the bacteria. Stationary-phase cells were more sensitive than those from exponential cultures. A mutant (FA31) resistant to the bactericidal action of "cold shock" and 0.4% deconoic acid was isolated from E. coli FA23 (AN E. coli 154 derivative able to grow on 0.1% decanoic acid) by a recycling selection procedure. Other E. coli strains tested showed behavior similar to that of strain K-12 154. The chilling of cells as a tool to improve the bactericidal action of fatty acids in foods is discussed.     

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Isolation and antimicrobial evaluation of isomeric hydroxy ketones in leaf cuticular waxes of Annona squamosa

A novel natural compound, 11-hydroxy-16-hentriacontanone, has been isolated from the leaf cuticular wax of Annona squamosa along with its known isomer 10-hydroxy-16-hentriacontanone in a ratio of 67:33. This isomeric mixture of hydroxy ketones constituted together 16.5% of the total cuticular waxes. The new compound was characterised using spectral and chromatographic techniques. The major component was found to be 16-hentriacontanone (palmitone), which constituted up to 48% of the total cuticular wax, together with a homologous series of hydrocarbons, fatty aldehydes, fatty alcohols, fatty acids and sterols as minor components. The antimicrobial activity of the isomeric hydroxy ketones was tested against selected Gram-positive and Gram-negative bacterial strains, and also some selected fungal strains, and compared with palmitone. The antibacterial activity of palmitone was significantly higher than that of the isomeric hydroxy ketones, but their antifungal activities were comparable.     

Occurrence of lipoxygenase products in membranes of rabbit reticulocytes. Evidence for a role of the reticulocyte lipoxygenase in the maturation of red cells

A lipoxygenase has been found in the reticulocytes of all mammalian species tested so far (rabbit, rat, mouse, monkey, and humans); evidence from in vitro studies suggests that the lipid-peroxidizing effects of this enzyme could render the mitochondrion and other intracellular organelles prone to the proteolytic degradation which is a natural step in development of the reticulocyte to the mature red cell. In this study we sought evidence of an active lipoxygenase in vivo. A bleeding anemia was induced in rabbits, and in the course of the subsequent reticulocytosis the red cell membranes were examined for the presence of the characteristic lipoxygenase products of linoleic and arachidonic acids. Erythrocyte membranes from control collections contained only small amounts of hydroxy fatty acids (0.03-0.08% of the polyenoic fatty acids). In contrast, reticulocyte-enriched red cells contained up to 3.3% of the polyenoic acids as hydroxylated derivatives. The main hydroxy fatty acid in reticulocyte membranes was identified as 13-L(S)-hydroxy-9Z,11E-octadecadienoic acid. Small amounts of other hydroxy derivatives including 15-hydroxy-5,8,11,13-(Z,Z,Z,E)eicosatetraenoic acid were also detected. These products appeared about 3 days after development of reticulocytosis. The precise structures of the hydroxylated polyenoic fatty acids and the time course of their appearance strongly suggest that their formation is due to the intracellular action of the cell-specific reticulocyte lipoxygenase. These findings are the first evidence for an activity of this enzyme in vivo, and the results support the hypothesis that enzymic peroxidation of reticulocyte intracellular membranes is a step in preparation of the intracellular organelles for proteolytic degradation.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Measurement of Hydroxy and Hydroperoxy Fatty Acids by a High Pressure Liquid Chromatography with a Column-switching System

Hydroxy and hydroperoxy fatty acids were labeled with 9-bromomethylacridine at room temperature. They were separated from the degradation products and less polar fatty acid derivatives on an octyl silicagel column, and put on an octadecyl silicagel column by on-line column switching. By this method, picomolar levels of the derivatives were measured with good reproducibility. The detection limit of 16-hydroxy-hexadecanoic acid as a representative was 0.9 pmol (S/N =3) and the relative standard deviation of its peak areas was 2.5% (18.5 pmol, n = 7). The method was used for the measurement of hydroxy fatty acids derived from hydroperoxy fatty acids and phosphatidylcholine (PC) hydroperoxides spiked in human plasma. By incubation at 37°C for 4h with human plasma, the hydroperoxy fatty acid was reduced to the corresponding hydroxy fatty acid. In this condition, the PC hydroperoxides showed a considerable decrease, however, a small portion (2.5–3%) of PC hydroperoxides decomposed gave the corresponding hydroxy fatty acids.     

Dihydroxy and monohydroxy fatty acids in Legionella pneumophila

Five strains of Legionella pneumophila were examined for the presence of hydroxy fatty acid. The cellular distribution of the fatty acids was also determined, as was the variation of hydroxy acid production on five growth media. The strains tested all produced approximately 5 mol% of hydroxy fatty acid, most of which was found in the nonextractable, alkali-stable, acid-labile (wall-associated, amide-linked) fraction. Three major hydroxy acids were found, along with several minor components. The major hydroxy acids were analyzed by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and infrared spectrophotometry. These compounds were tentatively identified as 3-hydroxy-12-methyltridecanoate, 3-hydroxy-n-eicosanoate, and a novel dihydroxy acid, 2,3-dihydroxy-12-methyltridecanoate. The total amount of hydroxy acid produced, as well as the profile of the hydroxy acids, remained relatively unchanged with respect to strain and growth medium.     

Spectrophotometric assay of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid

A spectrophotometric method has been developed for the determination of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid. The assay is based on the absorbance produced in the 290 to 300-nm range from their reaction with sulfuric acid at 100°C. α,β-Unsaturated aliphatic acids give absorption bands at 235–240 nm and thus can be easily differentiated from unsaturated fatty acids having the double bond(s) at positions not conjugated with the carboxyl group. A certain minimum chain length is required for full development of the absorption band at 300 nm. Position and intensity of the so-formed absorption band is independent on the position and number of the double bonds or hydroxyl groups. Carboxyl groups are not essential, as unsaturated hydrocarbons and higher alcohols likewise react with sulfuric acid to produce the absorbing species at 300 nm, providing a minimum chain length of 5 carbon atoms is present. The nature of the absorbing species at 300 nm is discussed.     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.