The green oat story: possible mechanisms of green color formation in oat products during cooking.

Consumers occasionally report greenish colors generated in their oat products when cooking in tap water. Here we have investigated pH and ferrous (Fe(2+)) ion as possible mechanisms for this color change. Steel-cut oat groats can turn brown-green color when cooked in alkaline conditions (pHs 9 to 12). Extraction of this color with methanol, and high-pressure liquid chromatography indicated a direct association of this color with the phenolic acid or avenanthramide content of the oat. The presence of 50 mM NaHCO(3) in water will cause oat/water mixtures to turn alkaline when cooked as CO(2) is driven off, generating OH(-) ion. Although tap water rarely, if ever, contains so much bicarbonate, bicarbonate is used as a leavening agent in baking applications. Industrial interests using baking soda or alkaline conditions during oat processing should be aware of possible off color generation. We have also found that as little as 10 ppm Fe(2+) will turn oat products gray-green when cooked. The aleurone stained darker than the starchy endosperm. Other divalent cations, such as Ca(2+) or Mg(2+) had no effect on cooked oat color. As much as 50 ppm Fe(2+) may be found in freshly pumped well water, but Fe(2+) reacts quickly with oxygen and precipitates as Fe(OH)(3). Thus, some freshly pumped well water may turn oats green when cooked, but if the water is left under atmospheric conditions for several hours, no discoloration will appear in the cooked oats.

Monoterpene synthases from common sage (Salvia officinalis). cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase.

Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of the p-menthane (1,8-cineole), pinane (alpha- and beta-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1, 8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-alpha-pinene, (+)-camphene, and (+/-)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (-)-alpha-pinene, (+)- and (-)-beta-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of gamma-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.

Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina.

The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus.

Synthesis and cannabinoid receptor binding activity of conjugated triene anandamide, a novel eicosanoid.

A polyenoic fatty-acid isomerase (PFI) from a red marine alga was used to convert anandamide (5Z,8Z,11Z,14Z-eicosatetraenoyl-N-ethan olamide) to the 5Z,7E,9E,14Z-eicosatetraenoyl-N-ethanol amide isomer. This novel eicosanoid, termed conjugated triene anandamide (CTA), was assessed for its ability to bind to the cannabinoid receptor in rat brain membrane preparations. CTA is a high affinity cannabimimetic substance whose novel structure provides new insight into structure-activity relationships of cannabinoid receptor ligands. These experiments illustrate the utility of enzymes isolated from marine organisms in the development of pharmacological probes.

Structure and biosynthesis of novel conjugated polyene fatty acids from the marine green alga Anadyomene stellata.

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga, Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation--16:5, 18:4, 20:5, 20:6, and 22:7--was accomplished by organic extraction followed by a combination of vacuum and high-performance liquid chromatography. One of these that was a novel substance (22:7) was characterized as 4Z,7Z,9E,11E,13Z,16Z,19Z-do cosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoi c acid) resulted in substantially increased synthesis of unique tetraene compounds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.

Biosynthesis of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina.

The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)- eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)- eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme.

Laser-induced fluorescence detection strategies for sodium atoms and compounds in high-pressure combustors.

A variety of laser-induced fluorescence schemes were examined experimentally in atmospheric pressure flames to determine their use for sodium atom and salt detection in high-pressure, optically thick environments. Collisional energy transfer plays a large role in fluorescence detection. Optimum sensitivity, at the parts in 10(9) level for a single laser pulse, was obtained with the excitation of the 4p-3s transition at 330 nm and the detection of the 3d-3p fluorescence at 818 nm. Fluorescence loss processes such as ionization and amplified spontaneous emission were examined. A new laser-induced atomization/laser-induced fluorescence detection technique was demonstrated for NaOH and NaCl. A 248-nm excimer laser photodissociates the salt molecules present in the seeded flames prior to atom detection by laser-induced fluorescence.

Time resolved laser induced fluorescence of the NH radical in low pressure N(2)O flames.

Total removal rate constants from the NH(A(3)II(i)) electronic state have been obtained in low pressure (~14-Torr) N(2)O flames. The fluorescence decay constants and quantum yields Phi depend on the temperature and composition at each position interrogated in the flame. In similar conditions, Phi for NH(A(3)II(u)) is significantly greater than that for other radicals CH(A(2)Delta and B(2)Sigma(-)) and OH(A(2)Sigma(+)). A small decrease (~5%) in the electronic removal is observed with rotational excitation increasing from N' = 2 to 12 in the A state. Estimates of collisional quenching cross sections at flame temperatures are made for the important combustion species, H(2)O, C(3)H(8), C(2)H(4), and C(2)H(2).

Laser-induced fluorescence determination of temperatures in low pressure flames.

Spatially resolved temperatures in a variety of low pressure flames of hydrogen and hydrocarbons burning with oxygen and nitrous oxide are determined from OH, NH, CH, and CN laser-induced fluorescence rotational excitation spectra. Systematic errors arising from spectral bias, time delay, and temporal sampling gate of the fluorescence detector are considered. In addition, we evaluate the errors arising from the influences of the optical depth and the rotational level dependence of the fluorescence quantum yield for each radical. These systematic errors cannot be determined through goodness-of-fit criteria and they are much larger than the statistical precision of the measurement. The severity of these problems is different for each radical; careful attention to the experimental design details for each species is necessary to obtain accurate LIF temperature measurements.

Modulary effects of temperature on antibody response and specific resistance to challenge of channel catfish, Ictalurus punctatus, immunized against Edwardsiella ictaluri.

The effects of various temperature treatments on the level of the humoral antibody response in channel catfish immunized with formalin killed Edwardsiella ictaluri was determined in laboratory controlled experiments. Immunized fish that were held at 25 degrees C for 30 days and 12 degrees C for an additional 30 days had higher antibody titers, and were more protected upon challenge, than immunized fish held at 25 degrees C for 60 days. Also immunized catfish held at 25 degrees C for 5 or 10 days followed by 12 degrees C water had higher antibody titers than immunized fish held at 12 degrees C or 25 degrees C for 60 days. In a field experiment carried out during winter and spring (February-May) fingerling channel catfish were vaccinated with E. ictaluri using intraperitoneal injection or immersion with either sonicated or whole cell preparations. Following challenge, the fish vaccinated by immersion in the sonicated preparation had 11.8% mortality whereas the groups immersed in whole cell bacterin, injected with the whole cell bacterin in adjuvant, or injected with sonicate showed 24.6, 57.9 and 41.7% mortality, respectively. Although the fish vaccinated by immersion with the sonicated bacteria had lower antibody titers than those vaccinated by the other methods the immersion vaccinates were more protected against challenge with the pathogen.

Cathodoluminescence of biological molecules, macromolecules and cells.

Cathodoluminescence observations on biological compounds are compared with previously established data from ultra violet and visible light excitation studies. The comparison demonstrates that the same molecules are responsible for the luminescence properties of macromolecules independent of the type of exciting radiation. Natural cathodoluminescence was also observed from cells. Moreover, advantages gained by the absorption of strongly cathodoluminiscent dyes into cells are demonstrated.