Furan formation from lipids in starch-based model systems, as influenced by interactions with antioxidants and proteins

The formation of furan upon sterilization of a lipid-containing starch gel was investigated in the presence of various antioxidants, namely, α-tocopherol, β-carotene, and ascorbic acid, with and without proteins. Results indicated that α-tocopherol did not significantly influence furan formation from oxidized lipids. β-Carotene, suggested previously to be a furan precursor itself, did influence the generation of furan in a concentration-dependent manner, although to a limited extent. Surprisingly, the presence of lipids seemed to limit the furan generation from β-carotene. Interestingly, the addition of ascorbic acid to the emulsions containing soybean or sunflower oils considerably enhanced the formation of furan from these oils. This was also the case when fresh oils were applied, shown previously to be nearly unable to generate furan. This observation can be explained by an intensified ascorbic acid degradation stimulated by the presence of lipids.     

Analysis of glycated and ascorbylated proteins by gas chromatography-mass spectrometry

Proteins or poly-L-lysine which were incubated in the presence of ascorbic acid, dehydroascorbic acid (ascorbylation), or various sugars (glycation) were analyzed by gas chromatography-mass spectrometry (GC-MS). To also detect more labile reaction products, the Maillard modified proteins or poly-L-lysine were enzymatically hydrolyzed and reacted with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide to form the N(O)-tert-butyldimethylsilyl (tBDMS) derivatives prior to GC analysis. Under these conditions, the known Maillard products N (epsilon)-(carboxymethyl)lysine (1), oxalic acid mono-N (epsilon)-lysinylamide (2), and N (epsilon)-(carboxyethyl)lysine (3) could be simultaneously detected and quantified in glycated and ascorbylated proteins. Additionally, N (epsilon)-(1-carboxy-3-hydroxypropyl)-L-lysine (4) was identified for the first time as a Maillard product of proteins. Under the conditions applied here, 4 was found only in ascorbylated proteins or poly-L-lysine, but not in glycated proteins. Maillard-modified poly-L-lysine was further subjected to high-performance liquid chromatography (HPLC) analysis after enzymatic hydrolysis and formation of the phenyl isothiocyanate derivatized amino acids. Using this method, N (epsilon)-formyl-L-lysine (5), which cannot be distinguished from 2 by GC-MS analysis, was identified for the first time as a glycation product. Compound 5 is mainly formed from ribose, lactose, and fructose. The indicated Maillard products were quantified in beta-lactoglobulin (GC-MS) or poly-L-lysine (HPLC) which were glycated or ascorbylated using different precursors.     

3-Hydroxy-4,5-dimethyl-2(5H)-furanone (Sotolon) causing an off-flavor: elucidation of its formation pathways during storage of citrus soft drinks.

Gas chromatography/olfactometry (GCO) and gas chromatography-mass spectrometry (HRGC-MS) revealed 3-hydroxy-4, 5-dimethyl-2(5H)-furanone (sotolon) to be responsible for the "burnt" and "spicy" off-flavor observed in citrus soft drinks during storage. Among the ingredients of citrus soft drinks, ethanol and ascorbic acid were identified as the essential precursors of sotolon. Two formation pathways were postulated by studies using (2)H (D)- and (13)C-labeled ethanol and ascorbic acid; i.e., sotolon is formed from two molecules of ethanol and carbons 2 and 3 of ascorbic acid (pathway 1), or it is generated from one molecule of ethanol and carbons 3-6 of ascorbic acid (pathway 2).     

Factors affecting thermally induced furan formation

Furan, a potential carcinogen, can be induced by heat from sugars, ascorbic acid, and fatty acids. The objective of this research was to investigate the effect of pH, phosphate, temperature, and heating time on furan formation. Heat-induced furan formation from free sugars, ascorbic acid, and linoleic acid was profoundly affected by pH and the presence of phosphate. In general, the presence of phosphate increased furan formation in solutions of sugars and ascorbic acid. In a linoleic acid emulsion, phosphate increased the formation of furan at pH 6 but not at pH 3. When an ascorbic acid solution was heated, higher amounts of furan were produced at pH 3 than at pH 6 regardless of phosphate's presence. However, in linoleic acid emulsion, more furan was produced at pH 6 than at pH 3. The highest amount of furan was formed from the linoleic acid emulsion at pH 6. In fresh apple cider, a product with free sugars as the major components (besides water) and little fatty acids, ascorbic acid, or phosphate, small or very low amounts of furan was formed by heating at 90-120 degrees C for up to 10 min. The results indicated that free sugars may not lead to significant amounts of furan formation under conditions for pasteurization and sterilization. Importantly, this is the first report demonstrating that phosphate (in addition to pH) plays a significant role in thermally induced furan formation.     

Mechanistic insights into furan formation in Maillard model systems

Furan has recently received considerable attention as a possibly carcinogenic compound occurring in thermally processed foods. Although several food constituents have been identified as furan precursors, multiple formation pathways remain unclear. Therefore, the mechanisms of furan formation in Maillard model systems were studied by means of the carbon module labeling (CAMOLA) technique. Under both roasting and pressure-cooking conditions, furan was formed from glucose via the intact skeleton, and its formation pathways from glucose alone were not amino acid-dependent. However, some amino acids, especially alanine and serine, did influence the furan production by providing an additional formation pathway. Furthermore, most amino acids enhanced the furan production from glucose. Roasting conditions produced 25-100 times higher amounts of furan as compared to pressure-cooking conditions. Surprisingly, in the alanine/glucose model systems, the relative importance of furan production from glucose alone and from the combination of a glucose-derived and an alanine-derived fragment changed completely over a limited time course of 60 min.     

Formation of furan from carbohydrates and ascorbic acid following exposure to ionizing radiation and thermal processing

This study was conducted to investigate the formation of furan from sugars, ascorbic acid, and organic acids as affected by ionizing radiation and thermal treatments. Results showed that both thermal treatments and irradiation induced formation of furan from ascorbic acid, fructose, sucrose, or glucose. Little furan was produced from malic acid or citric acid. The pH and concentration of sugars and ascorbic acid solutions had profound influences on furan formation due to either irradiation or thermal treatment. The rate of irradiation-induced furan formation increased with decreasing pH from 8 to 3. Approximately 1600 times less furan was formed at pH 8 as apposed to pH 3. At the same pHs, the amounts of furan formed from irradiation of ascorbic acid, fructose, and sucrose were always higher than from glucose. As pH decreased from 7 to 3, an increase in thermally induced furan was observed for sucrose and ascorbic acid solutions, but for glucose solution, less furan was formed at pH 3 than at pH 7. The levels of sugars commonly found in fruits and fruit juices, upon irradiation, would be high enough to potentially produce low parts per billion (ppb) levels of furan. The concentration of ascorbic acid at which a maximum of furan was produced upon irradiation was about 0.5 mg/mL, a level commonly found in some foods. Five furan derivatives were tentatively identified in thermally treated ascorbic acid solution, while one furan derivative was tentatively found in both irradiated and thermally treated samples.     

Origin and mechanistic pathways of formation of the parent furan--a food toxicant

Studies performed on model systems using pyrolysis-GC-MS analysis and (13)C-labeled sugars and amino acids in addition to ascorbic acid have indicated that certain amino acids such as serine and cysteine can degrade and produce acetaldehyde and glycolaldehyde that can undergo aldol condensation to produce furan after cyclization and dehydration steps. Other amino acids such as aspartic acid, threonine, and alpha-alanine can degrade and produce only acetaldehyde and thus need sugars as a source of glycolaldehyde to generate furan. On the other hand, monosaccharides are also known to undergo degradation to produce both acetaldehyde and glycolaldehyde; however, (13)C-labeling studies have revealed that hexoses in general will mainly degrade into the following aldotetrose derivatives to produce the parent furan-aldotetrose itself, incorporating the C3-C4-C5-C6 carbon chain of glucose (70%); 2-deoxy-3-ketoaldotetrose; incorporating the C1-C2-C3-C4 carbon chain of glucose (15%); and 2-deoxyaldotetrose, incorporating the C2-C3-C4-C5 carbon chain of glucose (15%). Furthermore, it was also proposed that under nonoxidative conditions of pyrolysis, ascorbic acid can generate the 2-deoxyaldotetrose moiety, a direct precursor of the parent furan. In addition, 4-hydroxy-2-butenal-a known decomposition product of lipid peroxidation-was proposed as a precursor of furan originating from polyunsaturated fatty acids. Among the model systems studied, ascorbic acid had the highest potential to produce furan, followed by glycolaldehyde/alanine > erythrose > ribose/serine > sucrose/serine > fructose/serine > glucose/cysteine.     

Gas chromatography-olfactometry (GC-O) and proton transfer reaction-mass spectrometry (PTR-MS) analysis of the flavor profile of grana padano,parmigiano reggiano,and grana trentino cheeses

Gas chromatography-olfactometry (GC-O) and proton transfer reaction-mass spectrometry (PTR-MS) techniques were used to deduce the profile of odor-active and volatile compounds of three grana cheeses: Grana Padano (GP), Parmigiano Reggiano (PR), and Grana Trentino (GT). Samples for GC-O analysis were prepared by dynamic headspace extraction, while a direct analysis of the headspace formed over cheese was performed by PTR-MS. The major contributors to the odor profile were ethyl butanoate, 2-heptanone, and ethyl hexanoate, with fruity notes. A high concentration of mass 45, tentatively identified as acetaldehyde, was found by PTR-MS analysis. Low odor threshold compounds, e.g., methional and 1-octen-3-one, which contributed to the odor profile but were not detected by FID, were detected by PTR-MS. Principal component analysis on both GC-O and PTR-MS data separated the three cheese samples well and showed specific compounds related to each sample.     

Epicatechin carbonyl-trapping reactions in aqueous maillard systems: Identification and structural elucidation

Recently, our group reported via labeling experiments that epicatechin in Maillard reaction aqueous glucose-glycine model systems formed adduct reaction products with C2, C3, and C4 sugar fragments. In the current study, we investigated the identity of the sugar fragment precursors responsible for adduct generation by directly comparing the liquid chromatography-mass spectrometry properties of these reported epicatechin (EC)-sugar fragments adducts with those generated from reactions consisting of only EC and well-known Maillard-generated glucose fragments (i.e., glyoxal, glycolaldehyde, methylglyoxal, glyceraldehyde, etc.). The structural properties of an EC-methylglyoxal adduct reaction product were also analyzed by NMR. The most likely precursors for the C2, C3, and C4 sugar moiety of the EC-sugar fragment adducts were identified as glyoxal, hydroxyacetone, and erythrose, respectively. 1H NMR analysis of the EC-methylglyoxal indicated that the analyte underwent rapid conformational/constitutional exchange. Using cold temperature (-25 degrees C) two-dimensional NMR analyses (heteronuclear multiple bond coherence, heteronuclear multiple quantum coherence, and 1H-(1)H correlation spectroscopy), the structure of one of the isomers was reported to consist of a covalent linkage between the C1 position of the methylglyoxal and either the C6 or the C8 position of the EC A ring, presumably generated by hydroxyalkylation and aromatic substitution reactions.     

鳙鱼皮水解物美拉德反应产物抗氧化活性研究

为进一步利用鱼类加工副产物,采用微波技术预处理鳙鱼皮,碱性蛋白酶酶解制备鳙鱼皮水解物(FSH),将其与葡萄糖、木糖和核糖进行美拉德反应,评价美拉德反应产物的抗氧化活性和挥发性风味物质。结果表明,美拉德反应显著提高了鳙鱼皮水解物的抗氧化活性,核糖美拉德反应产物的抗氧化活性最高,在135℃、35 min条件下,核糖美拉德反应产物的总氧化活性为2 156 μmol·L^-1 FeSO₄,DPPH自由基清除能力达到96.53%,超氧阴离子自由基清除能力达到88.08%。采用固相微萃取-气相色谱-质谱联用(SPME-GC-MS)分析美拉德反应产物的挥发性风味物质发现,呈肉香味的吡嗪、呋喃类杂环化合物相对含量增加,从而改善了鳙鱼皮水解物的风味。本研究结果为制备食品天然抗氧化剂提供了理论依据,为鱼类加工副产物高值化开发与利用提供了参考。     

Sensory activity,chemical structure,and synthesis of Maillard generated bitter-tasting 1-oxo-2,3-dihydro-1H-indolizinium-6-olates

Thermal treatment of aqueous solutions of xylose, rhamnose, and l-alanine led to a rapid development of a bitter taste of the reaction mixture. To characterize the key compounds causing this bitter taste, the recently developed taste dilution analysis (TDA), which is based on the determination of the taste threshold of reaction products in serial dilutions of HPLC fractions, was performed to locate the most intense taste compounds in the complex mixture of Maillard reaction products. By application of this TDA, 26 fractions were obtained, among which seven fractions were evaluated with a high taste impact. LC/MS and NMR spectroscopy as well as synthetic experiments revealed the 1-oxo-2,3-dihydro-1H-indolizinium-6-olates 1-5 as the key compounds contributing the most to the intense bitter taste of the Maillard mixture. Calculation of the taste impact of these compounds based on a dose/activity relationship indicated that these five compounds already accounted for 56.8% of the overall bitterness of the Maillard mixture, thus demonstrating this class of 1-oxo-2,3-dihydro-1H-indolizinium-6-olates as the key bitter compounds. First synthetic studies on the relationship between the chemical structure and the human psychobiological activity of 1-oxo-2,3-dihydro-1H-indolizinium-6-olates revealed that substitution of the furan rings of 1 by 5-methylfuryl moieties (compounds 3-5) or by 5-(hydroxymethyl)furyl groups (compound 6) led to a significant increase of the bitter threshold. In contrast, the substitution of the oxygen atoms in the furan rings of 1 by sulfur atoms induced a significant decrease of the detection threshold of the 1-oxo-2,3-dihydro-1H-indolizinium-6-olate; for example, the thiophene derivative 7 showed the extraordinarily low bitter detection threshold of 6.3 x 10(-5) mmol/kg (water).     

Simultaneous quantitative analysis of maillard reaction precursors and products by high-performance anion exchange chromatography

A new analytical setup allowing the simultaneous analysis of precursors and products of the Maillard reaction is described. It is based on high-performance anion exchange chromatography with electrochemical (ECD) and diode array detectors (DAD) coupled in series. Chromatography and detection were optimized to permit simultaneous monitoring of compounds relevant to the Maillard reaction, such as the sugar, the amino acid, and the corresponding Amadori compound as well as the cyclic intermediates 5-(hydroxymethyl)-2-furaldehyde, maltol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. Separation was achieved on a CarboPac PA-1 column using a gradient of sodium acetate in aqueous sodium hydroxide. The Amadori compound, glucose, and glycine were monitored by an ECD operating in the integrated amperometry mode. The number of analyzed compounds was further increased by coupling the ECD with a DAD for the analysis of ultraviolet-active constituents. This method was successfully applied to model Maillard reaction mixtures based on glucose and glycine.     

Formation of Maillard reaction products during heat treatment of carrots

As indicators of the early stage of the Maillard reaction in carrots, N-(furoylmethyl) amino acids (FMAAs) formed during acid hydrolysis of the corresponding Amadori products were analyzed using RP-HPLC with UV detection. N(ε)-FM-Lys (furosine), FM-Gly, FM-Ala, FM-Val, FM-Ile, FM-Leu, and FM-GABA were identified using synthesized standard material by means of mass spectrometry. Furthermore, N(ε)-carboxymethyllysine (CML) and pyrraline were analyzed as indicators for advanced stages of glycation. For commercial samples with high water content, the formation of Amadori compounds predominates, whereas the advanced stage of Maillard reaction plays only a minor part. Carrot juices, baby food, and tinned carrots showed quite low rates of amino acid modification up to 5%. For dehydrated carrots, significantly higher values for Amadori products were measured, corresponding to a lysine derivatization of up to 58% and nearly 100% derivatization of GABA. Drying experiments revealed great differences in reactivity between the amino acids studied. Whereas furosine reached constant values quite quickly, some FMAAs showed a continuous increase with heating time, indicating that selected FMAAs can be used as a hallmark for the early Maillard reaction to control processing conditions.     

Sugar fragmentation in the maillard reaction cascade: formation of short-chain carboxylic acids by a new oxidative alpha-dicarbonyl cleavage pathway

The formation of short-chain carboxylic acids was studied in Maillard model systems (90 degrees C, pH 6-10) with emphasis on the role of oxygen and water. The total amount of acetic acid formed did not depend on the reaction atmosphere. In the presence of labeled dioxygen or water (18O2, H2 17O), labeled oxygen was partially incorporated into acetic acid. Thermal treatment of 1-deoxy-d-erythro-2,3-hexodiulose (1) and 3-deoxy-d-erythro-hexos-2-ulose in the presence of 17O-enriched water under alkaline conditions led to acetic and formic acid, respectively, as indicated by 17O NMR spectroscopy. The suggested mechanism involves an oxidative alpha-dicarbonyl cleavage leading to an intermediary mixed acid anhydride that releases the acids, e.g., acetic and erythronic acid, from 1. Similarly, glyceric and lactic acids were formed from 1-deoxy-3,4-hexodiuloses, corroborated by complementary analytical techniques. This paper provides for the first time evidence for the direct formation of acids from C6-alpha-dicarbonyls by an oxidative mechanism and incorporation of a 17OH group into the carboxylic moiety. The experimental data obtained support the coexistence of at least two newly described reaction mechanisms leading to carboxylic acids, i.e., (i) a hydrolytic beta-dicarbonyl cleavage as a major pathway and (ii) an alternative minor pathway via oxidative alpha-dicarbonyl cleavage induced by oxidizing species.     

Influence of thermally processed carbohydrate/amino acid mixtures on the fermentation by Saccharomyces cerevisiae

The production of alcoholic beverages such as Tequila, Mezcal, whiskey, or beer includes the fermentation of a mash containing Maillard reaction products. Because excessive heating of the mash can lead to complications during the following fermentation step, the impact of Maillard products on the metabolism of Saccharomyces cerevisiae was investigated. For this purpose, fermentation was carried out in a model system in the presence and absence of Maillard reaction products and formation of ethanol served as a marker for the progression of fermentation. We found that increasing amounts of Maillard products reduced the formation of ethanol up to 80%. This effect was dependent on the pH value during the Maillard reaction, reaction time, as well as the carbohydrate and amino acid component used for the generation of Maillard reaction products. Another important factor is the pH value during fermentation: The inhibitory effect of Maillard products was not detectable at a pH of 4 and increased with higher pH-values. These findings might be of relevance for the production of above-mentioned beverages.     

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.     

Antioxidant action of glutathione and the ascorbic acid/glutathione pair in a model white wine

Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine.     

Influence of epicatechin reactions on the mechanisms of Maillard product formation in low moisture model systems

The influence of the polyphenolic compound epicatechin on Maillard chemistry was investigated under simulated roast conditions (10% moisture at 220 degrees C for 10 min). Quantitative gas chromatography (GC) analysis indicated that the addition of epicatechin to glucose or fructose/glycine model systems significantly reduced the generation of hydroxyacetone, 2-methylpyrazine, 2,3,5-trimethylpyrazine, furfural, 2-acetylfuran, 5-methylfurfural, 2(5H)-furanone, 2-acetylpyrrole, and furfuryl alcohol. These analytes were reported to be primarily generated from intact C2, C3, C4, C5, and C6 sugar fragments based on gas chromatography/mass spectrometry quantitative isotopomeric analysis of a 1:1 13C6:12C6 hexose sugar/glycine model system. Liquid chromatography/mass spectrometry qualitative isotopomeric analysis of a 1:1 13C6:12C6 hexose sugar/glycine/epicatechin model systems confirmed epicatechin reacted with Maillard reactants in the model systems; two main reaction products were reported, epicatechin-C5 and -C6 sugar fragment adducts. In addition, LC/MS analysis of a model system consisting of only 3-deoxy-2-hexosulose and epicatechin identified 3-deoxy-2-hexosulose as a precursor of the epicatechin-C5 and -C6 sugar fragment adducts reaction products. These results imply that epicatechin quenched 3-deoxy-2-hexosulose (a key source C6 to C1 sugar fragments) and consequently inhibited Maillard product formation.     

Rye Flour Extraction Rate Affects Maillard Reaction Development,Antioxidant Activity,and Acrylamide Formation in Bread Crisps

This report shows the effect of rye flour extraction rate on Maillard reaction, antioxidant activity, and acrylamide formation during toasting of rye bread crisps. Four rye flours with extraction rates of 70, 85, 95, and 100% were tested. Maillard reaction development was studied by measuring browning development, hydroxymethylfurfural (HMF), and glucosilisomaltol (GIM) formation, as well as antioxidant activity. Results showed that HMF and GIM concentrations in toasted bread crisps were higher as the flour extraction rate increases. Antioxidant activity increased during toasting as a consequence of antioxidant Maillard reaction product formation. Acrylamide concentration was clearly affected by free asparagine content of flour, while no effect of dietary fiber and natural antioxidant content of flours had an effect on acrylamide formation. Overall data suggest that the rate of Maillard reaction is higher in whole flours because of their higher free amino acid and protein content.     

N(delta)-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine, a novel methylglyoxal-arginine modification in beer

N(delta)-(5-Hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine, or Argpyrimidine, was identified and quantified in beer by high-performance liquid chromatography (HPLC) and coupled gas chromatography-mass spectrometry (HRGC-MS). This novel fluorescent arginine Maillard modification represents the first amino acid modification reported in beer retaining the full backbone of the original amino acid. Two mechanisms of formation could be verified: the major pathway via methylglyoxal and the minor pathway via 5-deoxypentoses. Argpyrimidine concentrations, determined in 35 lager-type beer varieties, reached up to 27 nmol/L and could be positively correlated to beer color and wort content. Within this context, 5-deoxy-D-ribose was identified as a novel intermediate of the Maillard reaction of maltose by HRGC-MS and independent synthesis.