N-(1-deoxy-D-fructos-1-yl) fumonisin B(1), the initial reaction product of fumonisin B(1) and D-glucose

Incubation of fumonisin B(1) and D-glucose in aqueous solutions resulted in the formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) in addition to the previously reported N-(carboxymethyl) fumonisin B(1). N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) is the first stable product formed after the Amadori rearrangement of the Schiff base formed by the reaction of the primary amine of fumonisin B(1) and the aldehyde group of D-glucose. N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) was synthesized by reacting fumonisin B(1) with an excess of D-glucose in methanol and heating for 6 h at 64 degrees C. It was purified using C(18) and strong cation exchange solid-phase extraction cartridges and characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. Subsequently, N,N-dimethylformamide was found to be a better reaction solvent, requiring reaction for only 2-3 h at 64 degrees C and eliminating the formation of methyl esters. Alkaline hydrolysis of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) gave a mixture of hydrolyzed fumonisin B(1) and hydrolyzed N-(carboxymethyl) fumonisin B(1).     

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.     

Nonenzymatic browning of lactose and caseinate during dry heating at different relative humidities

Dry mixtures of lactose and caseinate were heated at 60 degrees C for up to 96 h at different relative humidities (RHs) ranging from 29 to 95%. The resulting nonenzymatic browning was studied by determining lactulosyl lysine formation in the caseinate (as measured by the conversion to furosine), amount of reacted lactose, loss of lysine, color formation, and fluorescent intensity. For each measurement, the maximum reaction occurred at intermediate RHs. While there is general agreement between the results obtained by different methods, discrepancies are understandable given the complex nature of nonenzymatic browning. It was shown that the degradation of the Amadori product, lactulosyl lysine, increased with RH. Moreover, the Maillard reaction, as opposed to caramelization of lactose, was the major pathway at all RHs. Visible browning occurred when the destruction of Amadori product became dominant, and interactions between sugar fragments and caseinate were not the rate-limiting steps in the nonenzymatic browning.     

Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts

The relations between the formation of acrylamide and color, pyrazines, or antioxidants in an asparagine/d-glucose browning model system under various conditions were investigated. The highest level of acrylamide was produced in the asparagine/glucose (1:3) system heated at 170 degrees C for 30 min (2629 microg/g asparagine). Color intensity increased with temperature and heating time. The formation of pyrazines increased steadily with an increase of temperature (140-170 degrees C) and heating time (15-60 min). Antioxidant formation varied among the samples heated under different conditions. A clear correlation between formation of acrylamide and browning color was obtained. The formation of acrylamide was linearly correlated with the formation of total pyrazines during the initial stages of the Maillard reaction. No obvious correlation between formation of acrylamide and antioxidants was observed. However, excess amounts of asparagine increased the formation of antioxidants, whereas excess amounts of glucose reduced its formation.     

Reaction of folic acid with reducing sugars and sugar degradation products

The reaction of folic acid with reducing sugars (nonenzymatic glycation) under conditions that can occur during food processing and preparation was studied by high-performance liquid chromatography with diode array detection. N-(p-Aminobenzoyl)-L-glutamic acid, a well-established oxidation product, was detected in the reaction mixtures. Furthermore, a new product was isolated and identified as N2-[1-(carboxyethyl)]folic acid (CEF). CEF was the main product that was formed by the nonenzymatic glycation of folic acid. For preparation, N2-[1-(carboxyethyl)]folic acid was obtained in high yields when folic acid and dihydroxyacetone (DHA), a sugar degradation product, were heated at 100 degrees C in phosphate buffer. Mixtures of folic acid and different sugars or DHA were heated under variation of reaction time and temperature, and CEF was quantified. Up to 50% of the vitamin was converted to CEF, with highest yields formed from maltose (49%) and lactose (43%).     

Bound fumonisin B1: analysis of fumonisin-B1 glyco and amino acid conjugates by liquid chromatography-electrospray ionization-tandem mass spectrometry

To study the formation of fumonisin artifacts and the binding of fumonisins to matrix components (e.g., saccharides and proteins) in thermal-treated food, model experiments were performed. Fumonisin B(1) and hydrolyzed fumonisin B(1) were incubated with alpha-d-glucose and sucrose (mono- and disaccharide models), with methyl alpha-d-glucopyranoside (starch model), and with the amino acid derivatives N-alpha-acetyl-l-lysine methyl ester and BOC-l-cysteine methyl ester (protein models). The reaction products formed were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of d-glucose with fumonisin B(1) or hydrolyzed fumonisin B(1) resulted in the formation of Amadori rearrangement products. Whereas conjugates were found following the reaction of sucrose, methyl alpha-d-glucopyranoside, and the amino acid derivatives with fumonisin B(1), the heating with hydrolyzed fumonisin B(1) yielded no artifacts. For structural determination, the stable reaction product formed by heating of methyl alpha-d-glucopyranoside (as starch model) with fumonisin B(1) was purified and identified by nuclear magnetic resonance spectroscopy as the diester of the fumonisin tricarballylic acid side chains with methyl alpha-d-glucopyranoside. These model experiments demonstrate that fumonisins are able to bind to polysaccharides and proteins via their two tricarballylic acid side chains.     

Radical-assisted melanoidin formation during thermal processing of foods as well as under physiological conditions

Color-generating reactions of protein-bound lysine with carbohydrates were studied under thermal as well as under physiological conditions to gain insights into the role of protein/carbohydrate reactions in the formation of food melanoidins as well as nonenzymatic browning products in vivo. EPR spectroscopy of orange-brown melanoidins, which were isolated from heated aqueous solutions of bovine serum albumin and glycolaldehyde, revealed the protein-bound 1,4-bis(5-amino-5-carboxy-1-pentyl)pyrazinium radical cation (CROSSPY) as a previously unknown type of cross-linking amino acid leading to protein dimerization. To verify their formation in foods, wheat bread crust and roasted cocoa as well as coffee beans, showing elevated nonenzymatic browning, were investigated by EPR spectroscopy. An intense radical was detected, which, by comparison with the radical formed upon reaction bovine serum albumin with glycolaldehyde, was identified as the protein-bound CROSSPY. The radical-assisted protein oligomerization as well as the browning of bovine serum albumin in the presence of glycolaldehyde occurred also rapidly under physiological conditions, thereby suggesting CROSSPY formation to be probably involved also in nonenzymatic glycation reactions in vivo.     

alpha-Dicarbonyl compounds formed by nonenzymatic browning during the dry heating of caseinate and lactose

A method using high-performance liquid chromatography with UV and electrospray ionization mass spectrometry detection was developed for monitoring the alpha-dicarbonyl compound profiles generated from nonenzymatic browning using o-phenylenediamine (OPD) as a trapping agent. The alpha-dicarbonyl compounds were generated by the "dry" reaction of sodium caseinate and lactose heated at various relative humidities (RHs). The proportions of alpha-dicarbonyls formed were different for samples heated at low, intermediate, and high RHs. This study shows that relatively large amounts of 3-deoxypentosulose and galactosyl 2-pentosulose are produced under high RHs, while galactosyl hexosulose and 1,4-dideoxyhexosulose are elevated under low RH conditions. Both caramelization and Maillard reaction pathways contributed to the generation of alpha-dicarbonyls.     

Use of 2-furoylmethyl derivatives of GABA and arginine as indicators of the initial steps of maillard reaction in orange juice

The formation of 2-furoylmethyl derivatives of GABA (2-FM-GABA) and arginine (2-FM-Arg) as early indicators of nonenzymatic browning in different types of orange juice was studied. In dehydrated orange juice, the presence of 2-FM-GABA and 2-FM-Arg was detected from the first day of storage at 30 degrees C. In this type of juice, the content of these two compounds increased with temperature (30, 50 degrees C) and time (1-7 days) of storage. A noticeable increase in 5-hydroxymethylfurfural was only observed after 4 days of storage at 50 degrees C. No formation of 2-FM-GABA and 2-FM-Arg was detected in liquid orange juice heated under conditions similar to those used in the industry. These furoylmethyl derivatives were also found in commercial orange juice made from concentrates. A slight increase in their concentration was observed in the two samples stored during 8 months at room temperature. According to the results obtained, 2-FM-GABA and 2-FM-Arg contents could be suitable indicators to assess the main modifications due to Maillard reaction produced during the manufacture and/or storage of orange juice concentrates.     

Glucose reaction with fumonisin B1 partially reduces its toxicity in swine

Acute and subacute intraperitoneal doses of fumonisin B(1) (FB(1)) were administered to test the efficacy of the FB(1)-glucose reaction products in detoxifying FB(1) in swine. In the acute study at 11 mumol of FB(1)/kg of body weight, five of six pigs administered FB(1) and four of six pigs administered FB(1)-glucose died from acute pulmonary edema. Analysis of weight gain, serum aspartate aminotransferase and gamma-glutamyltransferase, total cholesterol, and pathological evaluation did not provide evidence of protection against FB(1) toxicity by the FB(1)-glucose reaction products. In the subacute study at 5.5 mumol of FB(1)/kg of body weight, one pig administered FB(1) died from liver damage. Analysis of serum aspartate aminotransferase, gamma-glutamyltransferase, and total bilirubin showed protection against FB(1) toxicity by the FB(1)-glucose reaction products. The levels of sphinganine and sphinganine/sphingosine ratios in serum and liver as well as pathologic findings provided definitive evidence of protection against the FB(1) toxic effects by this detoxification procedure (p < 0.05).     

Comparison of the ion exclusion chromatographic method with the Monier-Williams method for determination of total sulfite in foods

Experimental data comparing the alkali extraction/ion exclusion chromatographic method with the Monier-Williams method for determination of total sulfite are presented in (a) enzymatic and nonenzymatic browning systems, (b) vegetables containing naturally occurring sulfite, and (c) a carbohydrate-type food additive, erythorbic acid. Excellent agreement, with a linear correlation coefficient of 0.99, was observed in fresh potato samples homogenized with sulfite and allowed to react for different time intervals (enzymatic browning system). A good overall correlation was observed in dehydrated, sulfited apple samples heated for different times (nonenzymatic browning system); however, as heating time increased, higher results were obtained by the Monier-Williams method than by the alkali extraction/ion exclusion chromatographic method. The results of determining sulfite in the alkali trapping solution following acid distillation or acid treatment without heat suggested that this deviation was due to a fraction of sulfite bound to the browning reaction products in such a way that it was released by acid distillation but not by alkali extraction or acid treatment without heat. Similar behavior was demonstrated in cabbage with naturally occurring sulfite, which was released by acid distillation but not by alkali extraction or acid treatment without heat. The ion exclusion chromatographic method could overcome interference by the volatile caramelization reaction products in the Monier-Williams determination of erythorbic acid.     

Effect of the pyrrole polymerization mechanism on the antioxidative activity of nonenzymatic browning reactions

The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic browning reactions is changing at the same time that the browning (by the pyrrole polymerization mechanism) is being produced. The antioxidative activities of eight model pyrroles (pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole, pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde) as well as the browning reaction of 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and the dimer (DIM) produced during HMP browning were determined. The results obtained suggest that the AAs observed in nonenzymatic browning reactions are the result of the AAs of the different oxidized lipid/amino acid reaction products formed. Thus, the different pyrrole derivatives produced in these reactions had different AAs, and the highest AAs were observed for alkyl-substituted pyrroles without free alpha-positions. Because some of these pyrrole derivatives are implicated in nonenzymatic browning production and this browning production implies the loss of hydroxyl groups and the transformation of some pyrroles with one type of substitution into others, changes in AA during browning production were observed, and the resulting DIM derivative was more antioxidant than HMP or higher polymers. These results explain the AA observed in fatty acid/protein mixtures after slight oxidation and suggest that, when the pyrrole polymerization mechanism is predominant, slightly browned samples may be more antioxidant than samples in which nonenzymatic browning has been highly developed.     

Nonenzymatic browning kinetics of a carbohydrate-based low-moisture food system at temperatures applicable to spray drying

Effects of water contents on nonenzymatic browning (NEB) rates of amorphous, carbohydrate-based food model systems containing L-lysine and D-xylose as reactants were studied at different temperatures (40, 50, 60, 70, 80, and 90 degrees C) applicable to spray drying conditions. Water sorption was determined gravimetrically, and data were modeled using the Brunauer-Emmett-Teller and Guggenheim-Anderson-deBoer equations. Glass transition, Tg was measured by DSC. NEB was followed spectrophotometrically. The rate of browning increased with water content and temperature, but a lower T-Tg was needed for browning at decreasing water content. Water content seemed to affect the activation energy of NEB, and higher water contents decreased the temperature dependence of the NEB. At higher temperatures, the NEB became less water content dependent and enhanced browning in spray-drying. The temperature dependence of nonenzymatic browning could also be modeled using the Williams-Landel-Ferry (WLF) equation, but the WLF constants were dependent on the water content.     

Identification and quantification of aminophospholipid-linked Maillard compounds in model systems and egg yolk products

While the Maillard reaction of free amino acids and proteins is a well-established process, no defined structures from the nonenzymatic browning of aminophospholipids in foodstuffs have been described so far. Phosphatidylethanolamine (PE)-linked glucosylamines (Schiff-PE), Amadori products (Amadori-PE), 5-hydroxymethylpyrrole-2-carbaldehydes (Pyrrole-PE), and carboxymethyl (CM-PE) as well as carboxyethyl (CE-PE) derivatives were detected and quantified by liquid chromatography- electrospray mass spectrometry (LC-(ESI)MS). Model incubations of soy-PE and D-glucose were employed to firmly establish the LC-(ESI)MS procedure. Analyses of spray-dried egg yolk powders and lecithin products derived therefrom show one-fourth of the native D-glucose content of egg yolk to be transformed to Amadori-PE, corresponding to a PE derivatization quota of 11-15.5 mol %. Schiff-PE and Pyrrole-PE were present only in low amounts, no CM-PE and CE-PE could be identified in any of the investigated samples. The high glycation rate of egg yolk PE will influence the emulsifying properties and perhaps even the oxidation resistance of the respective products.     

Comparative methyl linoleate and methyl linolenate oxidation in the presence of bovine serum albumin at several lipid/protein ratios

The oxidation of methyl linoleate (LMe) and methyl linolenate (LnMe) in the presence of bovine serum albumin (BSA) in the dark at 60 degrees C was studied to analyze the role of the type of fatty acid and the protein/lipid ratio on the relative progression of the processes involved when lipid oxidation occurs in the presence of proteins. The disappearance of the fatty acid, the formation of primary and secondary products of lipid peroxidation, the loss of amino acid residues, the production of oxidized lipid/amino acid reaction products, and the development of color and fluorescence were studied as a function of incubation time in protein/lipid samples at 10:1, 6:1, and 3:1 w/w ratios. The incubation of LMe and LnMe in the presence of BSA at 60 degrees C rapidly produced lipid peroxidation and protein damage. Although reaction rates were much faster for LnMe than for LMe, both fatty acids had similar behaviors, and LnMe seemed to be only slightly more reactive than LMe for BSA by producing a higher increase of protein pyrroles in the protein and the development of increased browning and fluorescence. The protein/lipid ratio also influenced the relative progress of the reactions implicated. Thus, a lower protein/lipid ratio increased sample oxidation and protein damage. This also produced an increased browning, in accordance with the mechanisms proposed for browning production by oxidized lipid/protein reactions. On the contrary, browning of extracted lipids increased at higher protein/lipid ratios. This opposite tendency allowed evaluation of the overall significance of the different browning processes implicated in the final colors observed, concluding that color changes observed in BSA/lipid samples were mostly a consequence of oxidized lipid/protein reactions.     

Fate of fumonisin B(1) in corn kernel steeping water containing SO(2)

Six 100 ppm fumonisin B(1) (FB(1)) solutions were prepared by dissolving pure standard in six different solvents containing SO(2). Two of the solvents contained 0.2 or 0.4% SO(2) in distilled water. The other four solvents were obtained by steeping corn kernels at 60 degrees C in a 0.2% SO(2) aqueous solution for 6, 12, 24, or 48 h. After the addition of FB(1), all solutions were maintained at 60 degrees C for 7 days. Fumonisin B(1) content in each solution was determined in triplicate by HPLC. Steeping corn kernels in 0.2% solution at 60 degrees C for 6 h seems to be the most effective treatment to decrease the amount of FB(1).     

Contribution of pyrrole formation and polymerization to the nonenzymatic browning produced by amino-carbonyl reactions

Recent studies have hypothesized that pyrrole formation and polymerization may be contribute to the nonenzymatic browning produced in both oxidized lipid/protein reactions and the Maillard reaction. To develop a methodology that would allow investigation of the contribution of this browning mechanism, the kinetics of formation of color, fluorescence, and pyrrolization in 4, 5(E)-epoxy-2(E)-heptenal/lysine and linolenic acid/lysine model systems were studied. In both cases similar kinetics for the three measurements were observed at the two temperatures assayed (37 and 60 degrees C), and there was a high correlation among color, fluorescence, and pyrrolization measurements obtained as a function of incubation time. Because the color and fluorescence production in the 4,5(E)-epoxy-2(E)-heptenal/lysine system is a consequence of pyrrole formation and polymerization, the high correlations observed with the unsaturated fatty acid also suggest a contribution of the pyrrole formation and polymerization to the development of color and fluorescence observed in the fatty acid/lysine system. Although the contribution of other mechanisms cannot be discarded, all of these results suggest that when the pyrrole formation and polymerization mechanism contributes to the nonenzymatic browning of foods, a high correlation among color, fluorescence, and pyrrolization measurements should be expected.     

Antioxidant properties of malt model systems

The aim of this study was to investigate the effect of kilning and roasting temperatures on antioxidant activity of malt model systems prepared from combinations of glucose, proline, and ferulic acid. Model systems (initial a(w) = 0.09, 6% moisture) were heated at 60 degrees C for up to 24 h, at 90 degrees C for up to 120 min, and at 220 degrees C for up to 15 min. The antioxidant activity of the glucose-proline-ferulic acid model system increased significantly on heating at 60 degrees C for 24 h or at 90 degrees C for 120 min. In contrast, the glucose-proline, ferulic acid-glucose, and ferulic acid-proline systems presented either nonsignificantly increased or unchanged antioxidant activity. The antioxidant activity of both the glucose-proline-ferulic acid and glucose-proline model systems increased significantly after heating at 220 degrees C for 10 min, followed by a significant decrease at 15 min. The data suggest that (1) at 60 degrees C, ferulic acid reacts with Maillard reaction products, resulting in a significant increase in antioxidant activity; (2) at 90 degrees C, the antioxidant activity of the glucose-proline-ferulic system comes from both ferulic acid and Maillard reaction products; and (3) at 220 degrees C, the major contributors to antioxidant activity in glucose-proline-ferulic acid and glucose-proline systems are glucose-proline reaction products.     

Reduction of fumonisin B1 in corn grits by single-screw extrusion

This study was designed to determine the efficacy of extrusion in reducing fumonisin B1 in corn flaking grits in the presence and absence of glucose. In addition, degradation products of fumonisin B1 during extrusion were identified and quantitated with a mass balance approach. Uncontaminated clean corn grits, grits spiked with 30 microg/g fumonisin B1, and grits fermented with Fusarium verticillioides M-2552 (40-50 microg/g fumonisin B1) were extruded in the presence and absence of glucose (10%, w/w) using a single-screw extruder. Extrusion decreased fumonisin B1 by 21-37%, whereas the same process with added glucose further decreased fumonisin B1 by 77-87%. LC-fluorescence and LC-MS showed that most fumonisin in the extruded samples without added glucose was the fumonisin B1 form, whereas the main degradation product in grits extruded with glucose was N-(deoxy- d-fructos-1-yl)fumonisin B1. The formation of hydrolyzed fumonisin B1 was not significant during extrusion. Results suggest that extrusion in the presence of glucose may reduce fumonisin B1 in corn grits significantly.     

Nonenzymatic browning reaction of essential amino acids: effect of pH on caramelization and Maillard reaction kinetics.

The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods.