Non-volatile Fatty Acids Research Articles

Analytical Quality by Design: Achieving Robustness of an LC-CAD Method for the Analysis of Non-Volatile Fatty Acids.

An alternative to the time-consuming and error-prone pharmacopoeial gas chromatography method for the analysis of fatty acids (FAs) is urgently needed. The objective was therefore to propose a robust liquid chromatography method with charged aerosol detection for the analysis of polysorbate 80 (PS80) and magnesium stearate. FAs with different numbers of carbon atoms in the chain necessitated the use of a gradient method with a Hypersil Gold C18 column and acetonitrile as organic modifier. The risk-based Analytical Quality by Design approach was applied to define the Method Operable Design Region (MODR). Formic acid concentration, initial and final percentages of acetonitrile, gradient elution time, column temperature, and mobile phase flow rate were identified as critical method parameters (CMPs). The initial and final percentages of acetonitrile were fixed while the remaining CMPs were fine-tuned using response surface methodology. Critical method attributes included the baseline separation of adjacent peaks (α-linolenic and myristic acid, and oleic and petroselinic acid) and the retention factor of the last compound eluted, stearic acid. The MODR was calculated by Monte Carlo simulations with a probability equal or greater than 90%. Finally, the column temperature was set at 33 °C, the flow rate was 0.575 mL/min, and acetonitrile linearly increased from 70 to 80% (v/v) within 14.2 min.

Reducing the effect of non-volatile fatty acids (non-VFAs) on polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge

Thermal-hydrolyzed sludge (THS) can be fermented to produce volatile fatty acids (VFAs) rich liquids. These fermentative liquids are considered as a potential feedstock for polyhydroxyalkanoates (PHA) production. However, the presence of high levels of non-VFAs organics supporting the growth response instead of PHA accumulation hindered an efficient culture selection in a feast and famine regime. Lowering the non-VFAs content can compromise activities of microorganisms to take up these external carbon sources and improve the selective pressure; thus two enhanced strategies were tested to optimize the selection process: 1) increasing the proportion of VFAs in the original substrate or 2) removing most of the non-VFAs at the end of the feast phase. Results showed that the strategies resulted in PHA yields on VFAs respectively of 0.62 and 0.54 Cmol/Cmol, significantly higher than that in original SBR (0.16–0.35 Cmol/Cmol), confirming that reducing the adverse effect of non-VFAs can impose effective internal limitation and induce high PHA storage responses. In PHA accumulation tests, cultures selected with synthetic substrates accumulated a maximum PHA content of 61.4 wt%, which is the highest as ever reported among PHA production from THS. In summary, the study provided valuable references for improving PHA production from complex substrates.

Solid Phase Micro-extraction (SPME) with In Situ Transesterification: An Easy Method for the Detection of Non-volatile Fatty Acid Derivatives on the Insect Cuticle.

Triacylglycerides (TAGs) and other non-volatile fatty acid derivatives (NFADs) occur in large amounts in the internal tissues of insects, but their presence on the insect cuticle is controversially discussed. Most studies investigating cuticular lipids of insects involve solvent extraction, which implies the risk of extracting lipids from internal tissues. Here, we present a new method that overcomes this problem. The method employs solid phase micro-extraction (SPME) to sample NFADs by rubbing the SPME fiber over the insect cuticle. Subsequently, the sampled NFADs are transesterified in situ with trimethyl sulfonium hydroxide (TMSH) into more volatile fatty acid methyl esters (FAMEs), which can be analyzed by standard GC/MS. We performed two types of control experiments to enable significant conclusions: (1) to rule out contamination of the GC/MS system with NFADs, and (2) to exclude the presence of free fatty acids on the insect cuticle, which would also furnish FAMEs after TMSH treatment, and thus might simulate the presence of NFADs. In combination with these two essential control experiments, the described SPME technique can be used to detect TAGs and/or other NFADs on the insect cuticle. We analyzed six insect species from four insect orders with our method and compared the results with conventional solvent extraction followed by ex situ transesterification. Several fatty acids typically found as constituents of TAGs were detected by the SPME method on the cuticle of all species analyzed. A comparison of the two methods revealed differences in the fatty acid compositions of the samples. Saturated fatty acids showed by trend higher relative abundances when sampled with the SPME method, while several minor FAMEs were detected only in the solvent extracts. Our study suggests that TAGs and maybe other NFADs are far more common on the insect cuticle than usually thought.

Crude glycerol as feedstock for polyhydroxyalkanoates production by mixed microbial cultures

The increase in global biodiesel production makes imperative the development of sustainable processes for the use of its main by-product, crude glycerol. In this study the feasibility of polyhydroxyalkanoates (PHA) production by a mixed microbial community using crude glycerol as feedstock was investigated. The selected culture had the ability to consume both glycerol and methanol fraction present in the crude. However, glycerol seemed to be the only carbon source contributing for the two biopolymers stored: poly-3-hydroxybutyrate (PHB) and glucose biopolymer (GB). In this work the culture reached a maximum PHB content of 47% (cdw) and a productivity of 0.27 g X/L.d, with an aerobic mixed cultures and a real waste substrate with non-volatile fatty acids (VFA) organic matter. The overall PHA yield on total substrate obtained was in the middle range of those reported in literature. The fact that crude glycerol can be used to produce PHA without any pre-treatment step, makes the overall production process economically more competitive, reducing polymer final cost.

Emission modeling of styrene from vinyl ester resins with low hazardous air pollutant contents

Styrene is a commonly used co-monomer in vinyl ester (VE) resins, which acts as a reactive diluent and is required in most liquid molding fabrication methods to reduce viscosity and improve overall resin performance. Resins containing low hazardous air pollutant contents have been developed to reduce the styrene emissions during composite fabrication. VE monomers with a bimodal molecular weight distribution have been used to effectively decrease the amount of styrene in the system while maintaining low resin viscosities. Fatty acid vinyl ester (FAVE) resins partially replace styrene with non-volatile fatty acid monomers to reduce styrene emissions. The emissions from bimodal and FAVE resins were measured as a function of time and various parameters, including styrene content, VE molecular weight, and fatty acid monomer content and chain length. The initial emission rate from VE resins is only dependent on styrene content for constant evaporation geometry. Furthermore, the evaporation rate constant was the same regardless of VE molecular weight, styrene content, or the use of co-reactive diluent (MFA monomers). The diffusivity was not dependent on the styrene content in the resin, but decreased linearly as the VE molecular weight increased because of a corresponding increase in the resin viscosity. The diffusivity also increased as the content of MFA increased because of a decrease in the resin viscosity with high MFA content at high emission time. Furthermore, the emission profiles were accurately modeled using a modified version of 1D diffusion through a planar sheet that accounts for the depth change as a function of styrene evaporation. Overall, the model predicted emission profiles similar to the experimentally measured profiles as a function of time for various styrene contents, VE molecular weights, and fatty acid monomer contents.

Volatile fatty acids, metabolic by-products of periodontopathic bacteria, inhibit lymphocyte proliferation and cytokine production.

Short-chain fatty acids are a major by-product of anaerobic metabolism and can be detected in gingival fluid from periodontal pockets. Since most T cells are present subjacent to the pocket epithelium in conjunction with the plasma cells, it is important to know how these T cells are affected by short-chain fatty acids produced by subgingival plaque. The purpose of this study is to examine the effects of extracellular metabolites from periodontopathic bacteria on the proliferation and cytokine production of mouse splenic cells as a potential mechanism of imbalance among host-microbial interactions. A low-molecular-weight, heat-stable agent present in the two-day culture filtrate of Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum significantly depressed Con A- and LPS- induced cell proliferation. To determine whether short-chain fatty acids present in the filtrate could account for the depression, we tested extracted volatile and non-volatile fatty acids for their effects on mitogenic activity. The volatile fatty acids extracted from immunosuppressive supernatants greatly inhibited T- and B- cell proliferation. Among these volatile fatty acids, butyric, propionic, valeric, and isovaleric acids impaired cell proliferation dose-dependently. From gas-liquid chromatographic analysis data, it is suggested that immuno-inhibitory activities in culture filtrates are mainly attributable to butyric and isovaleric acids in P. gingivalis, to propionic, butyric, and isovaleric acids in P. loescheii, and to butyric acid in F. nucleatum. Furthermore, these fatty acids significantly depressed interleukin 2 (IL-2), IL-4, IL-5, IL-6, and IL-10 production by Con A-stimulated splenic-T cells dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of CDC group 1 and group 1-like coryneform bacteria isolated from clinical specimens.

Fifteen strains of CDC group 1 coryneform and biochemically similar bacteria were isolated from clinical specimens. Of the 15 strains isolated, 11 were derived from abscesses and purulent lesions, mostly from the upper part of the body, and 3 were grown from blood cultures. Nine strains were associated with mixed anaerobic but no other aerobic flora. Seven strains exhibited the classical biochemical profile of CDC coryneform group 1; however, eight strains were unable to reduce nitrate and were called "group 1-like." Other reactions to differentiate CDC group 1 and group 1-like coryneform rods include alpha-hemolysis on human blood agar, fermentation of adonitol, and the presence of alkaline phosphatase. Fifteen strains showed marked CAMP reactions on different erythrocyte agars. Gas-liquid chromatography of volatile and nonvolatile fatty acids as well as cellular fatty acid patterns and the composition of cell wall components suggest that CDC group 1 and group 1-like coryneform bacteria do not belong to the genus Corynebacterium but possibly to the genus Actinomyces or Arcanobacterium. DNA-DNA hybridization studies revealed that group 1 and group 1-like strains represent different species.

An Identification Scheme for Oral Non-pigmentedPrevotella(Bacteroides) Species

Three approaches were used to characterise 13 reference strains of non-pigmented Prevotella ( Bacteroides ) spp. and 91 clinical isolates from 21 adult patients with periodontal diseases. Strains were examined in tests for: (1) biochemical and morphological characters comprising microscopic and colonial appearance, biochemical and fermentation activity, tolerance of bile salts and dyes, susceptibility to antibiotic disks and fluorescence under U.V. light; (2) production of volatile and non-volatile fatty acids by gas-liquid chromatography (GLC); and (3) preformed enzymes profiles. Species were distinguished by: hydrolysis of hippurate, starch and acsculin; fermentation of lactose, sucrose, rhamnose, xylose, arabinose, cellobiose, salicin, inulin and xylan; tolerance of Victoria blue 4R; and fluorescence. In GLC analysis, production of propionic, iso-butyric, iso-valeric and phenylacetic acids gave useful discrimination between species. Discriminatory enzyme profiles were obtained with tests for β-xylosidase, β-glucuronidase, β-glucosidase, α-fucosidase, β- N -acetylglucosaminidase and α-mannosidase. Correlation between the three approaches was good and an identification scheme was designed for non-pigmented Prevotella spp. Of the 91 clinical isolates from adult periodontal diseases, 63 gave results identical with those of reference strains and 21 showed only minor variation. The commonest species were P. buccae (42 isolates), P. veroralis ( I5 isolates) and P. oralis (eight isolates). Keywords: Prevotella ; oral flora; periodontal disease; identification; gas-liquid chromatography; enzyme tests.

A Modified Procedure for the Identification of Anaerobic Bacteria by High Performance Liquid Chromatography — Quantitative Analysis of Short-Chain Fatty Acids

We have developed a new rapid method for analysing volatile and non-volatile short-chain fatty acids using high-performance liquid chromatography. Within 50 min, 22 fatty acids in a standard mixture could be detected in a single chromatographic run. The fatty acids released by anaerobic bacteria in the culture media were ether-extracted and analysed with an Aminex HPX-87H column. Using a microprocessor-controlled chromatography unit, a quantitative analysis of the fatty acids produced in bacterial cultures was possible. Resolution, rapidity and sensitivity were improved as compared to previous methods by using an eluent of 5% acetonitrile in 0.01 N H2SO4 and changing the column temperature to 35 degrees C.

Low molecular fatty acids in the marine sediments of eutrophic coastal regions.

Analytical procedure using a High Performance Liquid Chromatography (HPLC) for the simultaneous determination of volatile and non-volatile fatty acids in marine sediments was examin-ed. The fatty acids were extracted from marine sediments by modified Miyoshi's method. Using Shimadzu gel SCR-101H column on HPLC system, low molecular fatty acids were identified clearly. The above procedure for the fatty acìds analyses was applied to the sediments of Uranouchi Inlet, Kochi Prefecture. Acetate, formate, propionate, and lactate were detected. Acetate was found in the highest concentration (7.1-29.6μmol/g dry sediment), followed by formate (2.5-11.0μmol/g dry sediment). Only trace amount of propinate was found regardless of season and depth of sediments, while lactate was detected only in the surface sediments during warmer seasons. No fatty acids were detected in the interstitial water. In general, the concentration of fatty acids in the sediments decreased with increasing depth. The concentration of fatty acids was closely correlated with that of carbohydrates in the sediments.

Preliminary study of the anaerobic bacteria isolated from subgingival plaque from sheep

A preliminary study was made to determine the genera of cultivable anaerobic bacteria which could be isolated from subgingival plaque of sheep. Samples were taken from 10 sheep on farms with a known record of broken mouth periodontitis. For assessment of the sampling technique, samples were also taken from freshly exposed tooth roots in killed sheep. The bacteria isolated on several selective and non-selective media were identified to genus level by a combination of Gram reaction, colony morphology and gas chromatographic analysis of volatile and non-volatile fatty acid metabolic end products. At least 10 different genera were isolated and these findings are discussed in relation to the bacteriology of human periodontitis and recent studies of sheep broken mouth periodontitis.

Gas chromatographic-mass spectral studies after methylation of metabolites produced by some anaerobic bacteria in spent media.

The gas chromatographic analysis of metabolites such as volatile fatty acids (VFAs) and non-volatile fatty acids (NVFAs) for identification of anaerobic bacteria is now widely performed. Cultures of anaerobes tested for NVFAs as methyl esters were found to contain several unidentified compounds not previously detected and/or reported with methylation procedures. Gas chromatographic-mass spectrometric studies demonstrated that these compounds correspond to the methyl esters of both saturated and unsaturated short-chain fatty acids, and also of 2-hydroxy and 2-oxo acids. The distribution of these acids among different species of anaerobes was determined and their amounts were measured. The effects of supplementing the culture medium with either glucose or amino acids on the production of these acids are described. The use of very polar stationary phases is suggested for a better separation of all NVFAs.

Gas-Liquid Chromatographic Determination of Propionic Acid Production Differentiates between the Food-Poisoning Strains and Toxigenic-Type Strains of Clostridium perfringens

Fatty acids of 32 strains belonging to five types of Clostridium perfringens were extracted from the supernatant liquid of cultures in various different media prepared with or without the addition of sugar and examined by application of the gas-liquid chromatographic technique. The major products in nearly all media were acetic, propionic and butyric acid among the volatile fatty acids and only lactic acid among the nonvolatile fatty acids. Quantitative comparison of the resulting chromatograms with regard to the presence and relative amounts of propionic acid as one of the large major peaks revealed clear differences between the toxigenic types and the food-poisoning strains of this organism. In cooked meat medium containing 2% fructose, all the toxigenic-type strains produced propionic acid, but the food-poisoning strains tested entirely lacked production of this acid. This differentiation characteristic may be useful in routine laboratory bacteriology.

'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora.

Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal beta-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora.

Dialysis culture for the production of Pseudomonas saccharophila alpha-amylase.

Growth and alpha-amylase synthesis of Pseudomonas saccharophila was shown to be inhibited by the accumulation of a mixture of nonvolatile fatty acids during nondialysis cultivation. Using dialysis culture a 9-fold increase in the level of biomass and an 8.5-fold increase in alpha-amylase yield was achieved.

'Normalization' of germfree mice with anaerobically cultured caecal flora of 'normal' mice.

Germfree (GF) mice were inoculated with a cultured flora from 10(-1), 10(-3), 10(-5), and 10(-7) dilutions of caecal contents from a 'normal' mouse. GF mice associated with a flora of a 'normal' mouse served as controls. The following intestinal parameters were determined: Colonization resistance (CR), Relative caecal weight (RCW), villus:crypt ratio (jejunum and ileum), IgA-producing cells (jejunum and ileum), beta-aspartyl glycine (faeces), volatile and non-volatile fatty acids (caecum) and bile acids (faeces). Only the 10(-1) culture was able to induce similar changes in the GF mice to a 'normal' flora. The GF + 10(-5) and GF + 10(-7) groups deviated markedly from the controls while the GF + 10(-3) group showed in general intermediate values between GF + SPF and GF + 10(-1) on the one hand and GF + 10(-5) and GF + 10(-7) on the other hand. beta-aspartyl glycine was present only in the GF + 10(-7) group. Scanning electron microscopy (SEM) of ileal contents revealed segmented filamentous organisms in the ileum of controls and the GF + 10(-1) group. The faecal flora consisted mainly of fusiform organisms. In the faeces of the 10(-5) and 10(-7) groups increasing amounts of non-bacterial matter were found, while in the faeces of the other groups virtually only bacteria were seen.

CHEMICAL COMPOSITION OF FLUE-CURED TOBACCO CULTIVARS WITH DIFFERENT SENSITIVITIES TO WEATHER FLECK

Experiments were conducted in 1976, 1977 and 1978 to examine the chemical composition of four flue-cured tobacco cultivars (Nicotiana tabacum L.) with a range of weather fleck tolerances. Weather fleck is associated with the air pollution injury to plants. The amount of surface wax was positively related to the weather fleck tolerance. Individual phenolic constituents, nonvolatile organic acids and fatty acids were not related to weather fleck tolerance in the tobacco cultivars used in the study.Key words: Weather fleck, flue-cured tobacco, surface waxes, phenolic constituents, organic and fatty acids

INFLUENCE OF APPLIED NITROGEN ON THE NONVOLATILE ORGANIC, FATTY, AND AMINO ACIDS OF FLUE-CURED TOBACCO

A field experiment was conducted in 1974 and 1975 on Fox loamy sand in Ontario to study the effects of different rates (0, 22.4, 44.8, and 67.2 kg/ha) of N fertilization on the nonvolatile organic acids, fatty acids, and protein amino acids of flue-cured tobacco (Nicotiana tabacum L. ’Delhi 34’). Nitrogen fertilization increased the concentration of the nonvolatile organic acids and amino acids, except oxalic acid and methionine. Increasing the rate of N fertilization decreased individual fatty acids except myristic and linolenic acids. The nonvolatile organic acids decreased with ascending stalk position but the reverse was true for the amino acids. Fatty acids did not change significantly with stalk position.

無芽胞嫌気性菌の実験的マウス単独感染皮下膿瘍と培養培地および患者検体中の低級脂肪酸のガスクロマトグラフィー (GLC) による比較について

Five percent mucin suspensions each containing Bacteroides fragilis, Fusobacterium varium, Peptococcus asaccharolyticus, and Peptostreptococcus anaerobius isolated from anaerobic infections were injected once subcutaneously into the dd strain of conventional mice weighing 14 to 16 g. Bacteroides asaccharolyticus Rm-1 and Veillonella alcalescens ATCC 17745 were injected in the same way. Injections consisted of 0.2 ml. Viable cell number of bacteria per ml of mucin suspensions were approximately 2 x108 colony forming units (CFU) of Bacteroides fragilis, 4×107 CFU of F. varium, 3×107 CFU of P. asaccharolyticus, 1×107 CFU of P. anaerobius, 2×108 CFU of B. asaccharolyticus, and 4×107 CFU of V. alcalescens. Injections of pure culture of each organism except B. asaccharolyticus produced subcutaneous abscess of back. Injection of pure culture of B. asaccharolyticus produced moist gangrene. The lesions were visible 8 to 10 days after injection of each organism. The injected organisms were recovered from each of the lesion and no contaminating aerobe or other anaerobes were found within these lesions. Pus obtained from subcutaneous abscess in mice were analyzed by the gasliquid chromatograph (GLC) equipped with flame ionization detectors for the presence of short chain fatty acids. Chromatographic patterns of volatile fatty acids had strongly resemblance to culture results, however non-volatile fatty acids had not. GLC analysis of clinical specimens of pus recovered mixed anaerobes revealed that combined chromatographic patterns of volatile fatty acids detected in pus of subcutaneous abscesses inmice were obtained.It is considered that GLC analysis of iso-butyric acid, butyric acid, iso-valeric acid, valeric acid, isocaproic acid and caproic acid, which were not detectable in clinical specimens of pus from aerobic infections, provides reliable means for the presumptive diagnosis of infections caused by anaerobes and for identification of the pathogen.

Rapid gas-chromatographic method for identification of metabolic products of anaerobic bacteria.

The volatile fatty acids, alcohols, nonvolatile fatty acids, and ketones produced by anaerobes can be separated and identified by using a single type of gas-chromatographic column. A rapid and simple procedure is described.