A method using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode was developed for the analysis of fatty acids as methyl esters (FAMEs) in order to determine their percentage contribution to the fatty acid profile in food. In the GC/EI-MS-SIM mode, saturated fatty acids were determined with m/z 87, monoenoic fatty acids were determined with m/z 74, and polyenoic fatty acids were determined via the sum of m/z 79 and m/z 81. The ratios of these fragment ions and the GC retention data provided additional information for tentative structural assignments. The 28 FAME standards tested provided similar results for the novel GC/EI-MS-SIM method and GC/EI-MS in the full scan mode, both of which were slightly worse than GC/flame ionization detection (FID). Analysis of sunflower oil, suet, and cod liver oil verified that both major and minor fatty acids (20-60% and down to 0.001% contribution to the fatty acid pattern) were determined with sufficient quality that justifies application of the GC/EI-MS-SIM method for the analysis of food samples. Furthermore, the method was approximately 20- or approximately 10-fold more sensitive than GC/EI-MS in the full scan mode or GC/FID, respectively. The method is suited for both quantitative purposes and fatty acid identification in samples where only low amounts of lipids are available.

A Gas Chromatography/Electron Ionization−Mass Spectrometry−Selected Ion Monitoring Method for Determining the Fatty Acid Pattern in Food after Formation of Fatty Acid Methyl Esters

The main function of cuticular lipids in insects is the restriction of water transpiration through the surface. Lipids are involved in various types of chemical communication between species and reduce the penetration of insecticides, chemicals, and toxins and they also provide protection from attack by microorganisms, parasitic insects, and predators. Hydrocarbons, which include straight-chain saturated, unsaturated, and methyl-branched hydrocarbons, predominate in the cuticular lipids of most insect species; fatty acids, alcohols, esters, ketones, aldehydes, as well as trace amounts of epoxides, ethers, oxoaldehydes, diols, and triacylglycerols have also been identified. Analyses of cuticular lipids are chemically relatively straightforward, and methods for their extraction should be simple. Classically, extraction has relied mainly on application of apolar solvents to the entire insect body. Recently, several alternative methods have been employed to overcome some of the shortcomings of solvent extraction. These include the use of solid-phase microextraction (SPME) fibers to extract hydrocarbons from the headspace of heated samples, SPME to sample live individuals, and a less expensive method (utilized for social wasps), which consists of the collection of cuticular lipids by means of small pieces of cotton rubbed on the body of the insect. Both classical and recently developed extraction methods are reviewed in this work. The separation and analysis of the insect cuticular lipids were performed by column chromatography, thin-layer chromatography (TLC), high performance liquid chromatography with a laser light scattering detector (HPLC-LLSD), gas chromatography (GC), and GC-mass spectrometry (MS). The strategy of lipid analysis with the use of chromatographic techniques was as follows: extraction of analytes from biological material, lipid class separation by TLC, column chromatography, HPLC-LLSD, derivatization, and final determination by GC, GC-MS, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS, and liquid chromatography-mass spectrometry (LC-MS).

Cuticular lipids of insects as potential biofungicides: methods of lipid composition analysis.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

Fatty Acids Regulate Stress Resistance and Virulence Factor Production for Listeria monocytogenes

Characterisation of capillary ionic liquid columns for gas chromatography–mass spectrometry analysis of fatty acid methyl esters

A Gram-positive-staining, aerobic, non-motile, coccoid shaped, halotolerant bacterium (strain JG 06(T)) was isolated from the roots of Salicornia brachiata, an extreme halophyte. Phylogenetic analysis based on 16S rRNA gene sequence showed that the novel strain had sequence similarities of 99.2% to Brachybacterium paraconglomeratum JCM 11608(T), 99.0% to Brachybacterium conglomeratum DSM 10241 and 98.2% to Brachybacterium faecium DSM 4810(T). DNA-DNA hybridization with B. paraconglomeratum DSM 46341(T), B. conglomeratum DSM 10241(T), B. faecium DSM 4810(T), Brachybacterium tyrofermentans DSM 10673(T), Brachybacterium alimentarium DSM 10672(T), Brachybacterium fresconsis DSM 14564(T), Brachybacterium sacelli DSM 14566(T) and Brachybacterium muris DSM 15460(T) resulted in reassociation values of 36.2%, 36.5%, 35.8%, 27.6%, 27.9%, 28.2%, 28.7% and 11.2%, respectively. The peptidoglycan type of strain JG 06(T) was variant A4γ. The menaquinone content was MK7 (100%). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, monogalactosyl diglyceride, three unidentified phospholipids and three glycolipids. The predominant fatty acid was anteiso-C(15:0) (52.07%); significant amounts of iso-C(16:0)(12.38%), iso-C(15:0 )(8.59%) and anteiso-C(17:0)(10.03%) were also present. The G+C content of the DNA was 73.0 mol%. The strain formed a growth pellicle in nitrogen-free semisolid NFb medium containing NaCl at levels of up to 4% (w/v) and reduced acetylene to ethylene, a result indicative of N(2) fixation. In nutrient broth medium the novel strain grew at NaCl concentrations up to 15% (w/v). It also had the ability to produce indole-3-acetic acid (IAA) and siderophores, utilized 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen and possessed the ACC deaminase enzyme. On the basis of physiological, biochemical data and phylogenetic analyses, strain JG 06(T) should be placed in the genus Brachybacterium. Strain JG 06(T) represents a novel species of the genus Brachybacterium for which the name Brachybacterium saurashtrense sp. nov. is proposed (type strain JG 06(T)=DSM 23186(T)=IMCC 252(T)).

Brachybacterium saurashtrense sp. nov., a halotolerant root-associated bacterium with plant growth-promoting potential.

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 ω...

and M. Pete Yurawecz

Papers

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli.