Identification of 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin and pinitol alpha-D-galactosides in jojoba seed meal (Simmondsia chinensis)

The isolation and identification of two pinitol alpha-D-galactosides from jojoba meal are described. The products were isolated by a combination of preparative HPLC on silica gel and TLC on amino silica gel and were identified by MS, NMR spectroscopy, and chemical derivatization as 5-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 5-alpha-D-galactopyranosyl-D-pinitol and 2-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 2-alpha-D-galactopyranosyl-D-pinitol. The same preparative HPLC method on silica gel allowed a new simmondsin derivative to be isolated and identified as 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin mainly by NMR spectroscopy and high-resolution mass spectrometry.     

Gas chromatographic analysis of simmondsins and simmondsin ferulates in jojoba meal

A capillary gas chromatographic method was developed for the simultaneous determination of simmondsins and simmondsin ferulates in jojoba meal, in detoxified jojoba meal, in jojoba meal extracts, and in animal food mixtures.     

Reaction pathways of the diketonitrile degradate of isoxaflutole with hypochlorite in water

Isoxaflutole (IXF; Balance) belongs to a new class of isoxazole herbicides. Isoxaflutole has a very short half-life in soil and rapidly degrades to a stable and phytotoxic degradate, diketonitrile (DKN). DKN was previously discovered to rapidly react with hypochlorite (OCl-) in tap water, yielding the benzoic acid (BA) degradate as a major product, but the complete reaction pathway and mechanism have not been elucidated. Thus, the objectives of this work were to (1) determine the stoichiometry of the reaction between DKN and OCl-; (2) identify products in addition to BA; and (3) propose a complete pathway and reaction mechanism for oxidation of DKN by OCl-. Stoichiometry of the reaction showed a molar ratio of OCl-/DKN of 2. In addition, two previously uncharacterized chlorinated intermediates were identified under conditions in which OCl- was the limiting reactant. The proposed chemical structure of a chlorinated benzoyl intermediate was inferred from a series of HPLC/MS and HPLC/MS/MS experiments and the use of mass spectral simulation software. A chlorinated ketone intermediate was also identified using ion trap GC/MS. Two additional end products were also identified: cyclopropanecarboxylic acid (CPCA) and dichloroacetonitrile (DCAN). On the basis of the reaction stoichiometry, the structure of the chlorinated intermediates, and the identification of the products, two reaction pathways are proposed. Both pathways involve a two-step nucleophilic attack and oxidation of the diketone structure of DKN, leading to formation of BA, DCAN, and CPCA.     

Chemical studies on antioxidant mechanism of curcuminoid: analysis of radical reaction products from curcumin

In the course of studies on the antioxidant mechanism of curcumin, its radical reaction was investigated. Curcumin was reacted with radical species, which were generated from the pyrolysis of 2, 2'-azobis(isobutyronitrile) under an oxygen atmosphere, and the reaction products from curcumin were followed by HPLC. The reaction at 70 degrees C gave several products, three of which were structurally identified to be vanillin, ferulic acid, and a dimer of curcumin after their isolation. The dimer was a newly identified compound bearing a dihydrofuran moiety, and its chemical structure was elucidated using spectroscopic analyses, especially 2D NMR techniques. A mechanism for the dimer production is proposed and its relation to curcumin's antioxidant activity discussed. The time course and gel permeation chromatography studies of the reaction were also investigated, and the results indicate that the dimer is a radical-terminated product in the initial stage.     

Isolation and characterization of an oxygen radical absorbance activity peptide from defatted peanut meal hydrolysate and its antioxidant properties

Defatted peanut meal hydrolysate (DPMH) was purified using ultrafiltration, gel filtration chromatography, and high-performance liquid chromatography. A tripeptide with strong oxygen radical absorbance capacity (ORAC) was isolated and identified as Tyr-Gly-Ser by ESI-MS/MS. It was then synthesized to measure its antioxidant properties in different systems. The ORAC value of Tyr-Gly-Ser was 3-fold higher than that of glutathione (GSH), and it displayed a stronger protective effect on linoleic acid peroxidation and H(2)O(2)-induced oxidative injury in rat pheochromocytoma line PC12 cells than GSH (p < 0.05). However, Tyr-Gly-Ser showed negligible DPPH radical scavenging activity, reducing power, and no metal chelating ability. The results suggested that Tyr-Gly-Ser displayed antioxidant activity via the hydrogen atom transfer mechanism, and the Tyr at the N-terminal was the hydrogen donor. The ORAC assay was recommended as a reliable and effective method to measure the antioxidant activity in the course of antioxidant peptide isolation.     

LC-MS investigation of oxidation products of phenolic antioxidants

Two oxidation systems were examined for the oxidation of three groups of phenolic antioxidants; five cinnamic acids, two benzoic acids, and two phenols characteristic of olive fruits. Periodate oxidation, which is reported to produce products similar to polyphenol oxidase, was contrasted with the reactivity of the Fenton system, an inorganic source of hydroxyl radicals. Reaction products were identified as various quinones, dimers, and aldehydes, but the nature of the products differed between the two oxidation systems. Structure-activity effects were also observed for the different phenols. All cinnamic acids in this study reacted with the Fenton reagent to produce benzaldehydes as the main products, with the exception of 5-caffeoylquinic acid. In contrast, periodate oxidation gave no reaction with some of the cinnamic acids. Quinone formation was observed for the two compounds, caffeic acid and 5-caffeoylquinic acid, possessing o-hydroxy groups. Caffeic acid was unusual in that dimer formation was the main initial product of reaction. Benzoic acids were readily oxidized by both systems, but no identifiable products were isolated. Oleuropein was oxidized by both oxidants used in this study, resulting in quinones in each system, whereas little or no oxidation of tyrosol was observed. This highlights the importance of conjugation between the alkene double bond and the hydroxy group. The results question the validity of many existing methods of testing antioxidant activity.     

Chemical studies on antioxidant mechanism of curcumin: analysis of oxidative coupling products from curcumin and linoleate

As a part of a research project on the antioxidant mechanism of natural phenolics in food components, curcumin, a turmeric antioxidant, was investigated in the presence of ethyl linoleate as one of the polyunsaturated lipids. During the antioxidation process, curcumin reacted with four types of linoleate peroxyl radicals. Six reaction products were observed in the reaction and subsequently isolated. Their structures were determined by physical techniques, revealing that they have novel tricyclic structures, including a peroxyl linkage. On the basis of the formation pathway for their chemical structures, an antioxidant mechanism of curcumin in polyunsaturated lipids was proposed, which consisted of an oxidative coupling reaction at the 3'-position of the curcumin with the lipid and a subsequent intramolecular Diels--Alder reaction.     

Maillard reaction of D-glucose: identification of a colored product with hydroxypyrrole and hydroxypyrrolinone rings connected by a methine group

Investigation of the colored products formed by the reaction of d-glucose with butylammonium acetate has been extended. The previously unknown 1-N-butyl-4-hydroxy-5-methyl-2-(N-butyl-3-hydroxy-5-(2-hydroxyethyl)pyrrolyl-2-methylidene)-2H-pyrrolin-3-one (2a) was isolated from the reaction mixture and identified after acylation by spectroscopic data. Butylaminammonium acetate was used as a model compound representing the lysine side chains of proteins.     

Effect of ascorbic acid in dough: reaction of oxidized glutathione with reactive thiol groups of wheat glutelin

The reactions of oxidized glutathione generated from endogenous glutathione by the addition of ascorbic acid (AA) prior to dough mixing on free thiol groups of gluten proteins have been investigated. A small amount of (35)S-labeled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement. First, gluten was isolated from the dough, then the gliadins were extracted, and residual glutenin was partially hydrolyzed with thermolysin. After preseparation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography. Radioactive peptides were identified, isolated, sequenced, and assigned to amino acid sequences of gluten protein components. The isolated peptides contained exclusively the cysteine residues C(b) and C(x) of low molecular weight subunits of glutenin, which are supposed to be highly reactive in forming intermolecular disulfide bonds. From these results it can be assumed that the cysteine residues C(b) and C(x) of the low molecular weight subunits of glutenin are at least partly present in the thiol form in flour. During dough mixing they are converted to protein-protein disulfides or glutathione-protein mixed disulfides by thiol/disulfide interchange reactions. Oxidized glutathione necessary for this reaction is generated from glutathione by the action of AA. These results are in accordance with the major hypothesis about the mechanism of action of AA.     

Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction

The formation of mutagens after the heating of sugar-casein model systems at 120 degrees C was examined by the Ames test, using Salmonella typhimurium strain TA100. Several sugars (glucose, fructose, galactose, tagatose, lactose, and lactulose) were compared in their mutagenicities. Mutagenicity could be fully ascribed to Maillard reaction products and strongly varied with the kind of sugar. The differences in mutagenicity among the sugar-casein systems were caused by a difference in reaction rate and a difference in reaction mechanism. Sugars with a comparable reaction mechanism (glucose and galactose) showed a higher mutagenic activity corresponding with a higher Maillard reactivity. Disaccharides showed no mutagenic activity (lactose) or a lower mutagenic activity (lactulose) than their corresponding monosaccharides. Ketose sugars (fructose and tagatose) showed a remarkably higher mutagenicity compared with their aldose isomers (glucose and galactose), which was due to a difference in reaction mechanism.     

Antioxidant properties of kilned and roasted malts

Compounds possessing antioxidant activity play a crucial role in delaying or preventing lipid oxidation in foods and beverages during processing and storage. Such reactions lead to loss of product quality, especially as a consequence of off-flavor formation. The aim of this study was to determine the antioxidant activity of kilned (standard) and roasted (speciality) malts in relation to phenolic compounds, sugars, amino acids, and color [assessed as European Brewing Convention units (degrees EBC) and absorbance at 420 nm]. The concentrations of sugars and amino acids decreased with the intensity of the applied heat treatment, and this was attributed to the extent of the Maillard reaction, as well as sugar caramelization, in the highly roasted malts. Proline, followed by glutamine, was the most abundant free amino/imino acid in the malt samples, except those that were highly roasted, and maltose was the most abundant sugar in all malts. Levels of total phenolic compounds decreased with heat treatment. Catechin and ferulic acid were the most abundant phenolic compounds in the majority of the malts, and amounts were highest in the kilned samples. In highly roasted malts, degradation products of ferulic acid were identified. Antioxidant activity increased with the intensity of heating, in parallel with color formation, and was significantly higher for roasted malts compared to kilned malts. In kilned malts, phenolic compounds were the main identified contributors to antioxidant activity, with Maillard reaction products also playing a role. In roasted malts, Maillard reaction products were responsible for the majority of the antioxidant activity.     

Antioxidation mechanism studies of caffeic acid: identification of antioxidation products of methyl caffeate from lipid oxidation

As a part of a research project on the elucidation of the chain-breaking antioxidation mechanism of natural phenols in food components, caffeic acid, a polyphenolic acid widely distributed in edible plants, was investigated. The identification and time course analysis of the antioxidation reaction products from methyl caffeate were carried out in the ethyl linoleate oxidation system. The antioxidation reaction produced a quinone derivative of methyl caffeate as an antioxidation product during the initial stage, which was identified by (13)C NMR. The quinone, however, was not the final product, and a further reaction occurred to produce several new peroxides. The isolation and structure determination of the peroxides revealed that they had tricyclic structures, which consisted of ethyl linoleate, methyl caffeate, and molecular oxygen. On the basis of the formation pathway of these products, an antioxidation reaction mechanism of methyl caffeate, including the redox reaction of the caffeate and Diels-Alder reaction of the produced peroxides, was proposed.     

Thermal degradation of sulforaphane in aqueous solution.

Sulforaphane, a cancer chemopreventive agent identified from broccoli, was degraded in an aqueous solution at 50 and 100 degrees C. The reaction mixtures were extracted with methylene chloride and analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Dimethyl disulfide, S-methyl methylthiosulfinate, S-methyl methylthiosulfonate, methyl (methylthio)methyl disulfide, 1,2,4-trithiolane, 4-isothiocyanato-1-(methylthio)-1-butene, and 3-butenyl isothiocyanate were identified as volatile decomposition products. After methylene chloride extraction, the aqueous layer was dried and silica gel column chromatography was used to separate and purify the nonvolatile decomposition products. The major thermal degradation compound was determined by (1)H NMR, (13)C NMR, and FAB-MS as N, N'-di(4-methylsulfinyl)butyl thiourea. A possible mechanism for the formation of these products is proposed.     

Degradation of cyanidin 3-glucoside by caffeic acid o-quinone. Determination of the stoichiometry and characterization of the degradation products.

Caffeic acid o-quinone (CQ) was prepared by oxidation of caffeic acid with o-chloranil in organic media. The reaction between the purified CQ and cyanidin 3-glucoside (Cy 3-glc, o-diphenolic anthocyanin) was monitored by HPLC, and quantitative analyses were performed to establish the stoichiometry of the reaction. The results indicate that Cy 3-glc is degraded by a coupled oxidation mechanism with integration of CQ into the degradation products. The ratio of degraded Cy 3-glc to CQ incorporated into the condensation products was approximately 2.0. No brown products could be detected, only a slight orange color. Moreover, the addition of purified polyphenol oxidase to the slightly colored media resulted in the disappearance of the caffeic acid formed from the reaction of coupled oxidation (Cy 3-glc/CQ) and the formation of brown polymers. The degradation products were isolated by gel filtration on Sephadex G-25. The UV-vis spectra and chemical analysis (acidic hydrolysis) of the degradation products suggest that they resulted from the condensation of caffeic acid and Cy 3-glc. HPLC analysis showed that the partial purified fraction contained a mixture of complex condensation products.     

A Critical Assessment of the Quantification of Wheat Grain Arabinoxylans Using a Phloroglucinol Colorimetric Assay

Arabinoxylans (AX) of wheat (Triticum aestivum L.) play a critical role in processing, end‐use quality, and human health and nutrition. Consequently, an efficient, accurate method of AX quantification is desirable. The objective of this work was to evaluate a standard phloroglucinol colorimetric method for quantification of wheat AX. The method is based on the formation and spectrophotometric quantification of a phloroglucide product that results from the reaction of furfural produced during the condensation of pentose sugars with phloroglucinol. Method parameters, including reaction reagents and reaction times, were varied to identify areas for improved accuracy and consistency. Phloroglucide formation at three xylose concentrations was examined over time. The optimal reaction reagents and reaction times were determined based upon improved consistency in xylose quantification. The optimized method was used on xylose and arabinose standards and on whole meal wheat samples for total and water‐extractable AX content. Glucose was shown to be unnecessary in the reaction and was eliminated. A second‐order polynomial equation provided a slightly better fit to the nearly linear standard xylose curve. A reduced concentration of phloroglucinol of 10% was found to give equivalent results to the standard 20%. Optimum reaction time was 25 min, and it required the inclusion of all reagents. The phloroglucide product decreased in absorbance over time such that, within the range of xylose concentration examined, about 40–50% of the colored product was lost over 100 min; however, the rate of loss was linear over time. Four operators performed the optimized method on whole wheat meal samples for total and water‐extractable AX. Inter‐ and intraoperator variation was identified as an area requiring further study and improvement. However, all operators tended to rank the samples in a consistent manner. Compared with a gas chromatography–flame ionization detection method, the phloroglucinol method underestimated total AX by about 2.3% and water‐extractable AX by about 0.08%.     

Reactions of monosaccharides during heating of sugar-casein systems: building of a reaction network model

The Maillard reaction is important during the heating and processing of foods for its contribution to food quality. To control a reaction as complex as the Maillard reaction, it is necessary to study the reactions of interest quantitatively. In this paper the main reaction products in monosaccharide-casein systems, which were heated at 120 degrees C and pH 6.7, were identified and quantified, and the reaction pathways were established. The main reaction routes were (i) sugar isomerization, (ii) degradation of the sugar into carboxylic acids, and (iii) the Maillard reaction itself, in which not only the sugar itself but also its reaction products react with the epsilon-amino group of lysine residues of the protein. Significant differences in reaction mechanism between aldose and ketose sugars were observed. Ketoses seemed to be more reactive in the sugar degradation reactions than their aldose isomers, and whereas the Amadori product was detected as a Maillard reaction intermediate in the aldose-casein system, no such intermediate could be found in the ketose-casein system. The reaction pathways found were put together into a model, which will be evaluated by kinetic modeling in a subsequent paper.     

Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis)

A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.     

Production of Free Amino Acid and Short Peptide Fertilizer from Rapeseed Meal Fermentation Using Bacillus flexus NJNPD41 for Promoting Plant Growth

Free amino acids and short peptides (FAPS) that play an important role in plant nutrition can be extracted from rapeseed meal (RSM) by microbial enzymolysis. The enzymolytic activity of one bacterial strain isolated from RSM for the production of FAPS fertilizer (FAPSF) via solid-state fermentation, as well as its detoxification activity for isothiocyanates and oxazolidinethione, was investigated in this study. The strain NJNPD41 isolated from RSM piled-soil possessed effective proteolytic activity and was identified as Bacillus flexus on the basis of its morphological, physiological, and biochemical properties, as well as 16S rDNA analysis. Compared to the uninoculated control, inoculation with the strain NJNPD41 significantly increased the yield of total free amino acids and short peptides by 115% and decreased the content of isothiocyanates and oxazolidinethione by 53% and 60%, respectively, after solid-state fermentation. The pot and field experiments showed that FAPSF significantly promoted eggplant growth and enhanced the fruit yield and quality of eggplant compared to the control. In conclusion, this study provided a novel method for the high-value utilization of RSM to produce high-quality and low-toxicity FAPSF using the newly isolated strain B. flexus NJNPD41.     

Detection of Wheat Preharvest Sprouting Using a Pregelatinized Starch Substrate and Centrifugation

Wheat sprouting in the field before harvest is a serious negative quality attribute. Even low levels of preharvest sprouting affect the economic value of the grain. Unreleased test lines of wheat should be screened for resistance to preharvest sprouting. However, screening large numbers of test lines is relatively time‐consuming or expensive, depending on the existing method used. A new screening test for preharvest sprouting was developed and compared with the viscograph and α‐amylase activity (AAA) methods. The new method used the activity of sprout‐related elevation in α‐amylase to partially degrade added pregelatinized starch. The hydrolytic products were centrifuged and the weight of the centrifugate was expressed as a percentage of the original weight of the added pregelatinized starch plus the original meal or flour weight. The result reflected the AAA on pregelatinized starch (AAAPGS) as a measure of the degree of preharvest sprouting. The AAAPGS test had less standard error and was more sensitive at low levels of preharvest sprouting than the AAA method. Three grinders to produce wheat meal were compared for their effect on AAAPGS values. Flours produced slightly lower AAAPGS values than meals, but the coefficients of variation of each were comparable and both were less than that of the AAA method. The lowest levels of sensitivity to preharvest sprouting that could be detected by the AAA and AAAPGS methods were identified as areas of uncertainly, below which very low levels of preharvest sprouting could not be differentiated from sound, unsprouted background values. The new AAAPGS method was equally rapid and will be more economical than the AAA method or the viscograph when used for preharvest sprouting susceptibility of large numbers of samples.     

Collaborative study of a screening method for the detection of aflatoxins in mixed feeds, other agricultural products, and foods.

A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 mug aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10-25 mug total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.