Browning reactions of hydroxycinnamic acids and heterocyclic Maillard reaction intermediates - Formation of phenol-containing colorants.

Non-enzymatic conversion of phenolic compounds plays an important role during thermal processing of plant-based food such as coffee, cocoa, and peanuts. However, the more prominent Maillard reaction is mainly studied at a mechanistic level for carbohydrates and amino compounds to clarify reactions that contribute to ('classic') melanoidin formation, but the role of phenolic compounds in such reactions is rarely discussed yet. To understand their contribution to non-enzymatic browning, reactions between ubiquitous phenolic acids, such as caffeic acid and ferulic acid, and prominent heterocyclic Maillard intermediates, namely furfural, hydroxymethylfurfural, and pyrrole-2-carbaldehyde were investigated. Following incubation under roasting conditions (220 °C, 0-30 min), heterogenous products were characterized by high-resolution mass spectrometry, and, after isolation, by nuclear magnetic resonance spectroscopy. By this, color precursors were identified, and it was shown that in addition to aromatic electrophilic substitution, nucleophilic and condensation reactions are key mechanisms contributing to the formation of phenol-containing melanoidins.

Contribution of Hydroxycinnamic Acids to Color Formation in Nonenzymatic Browning Reactions with Key Maillard Reaction Intermediates.

The Maillard reaction is a vital part of food processing, involving a vast number of complex reaction pathways, resulting in high-molecular-weight colorants. So far, studies have been focused on the conversion of carbohydrates and amino compounds, but the literature elaborating the contribution of phenolic compounds to the formation of the colored end-products is still rare. The aim of this study was to characterize early reactions, underlying the formation of phenol-containing melanoidins. For this purpose, binary model systems of the prominent phenolic compounds caffeic acid and ferulic acid combined with α-dicarbonyl compounds typically formed in the Maillard reaction such as glyoxal, methylglyoxal, and diacetyl were analyzed after heat treatment. High-resolution mass spectrometry revealed that decarboxylation, aromatic electrophilic substitution, and nucleophilic addition are important reaction steps that lead to colored heterogeneous oligomers. Polymerization was favored for phenolic compounds with a high electron density in the aromatic system and for α-dicarbonyl compounds carrying aldehyde functions.

Colorants and Antioxidants Deriving from Methylglyoxal and Heterocyclic Maillard Reaction Intermediates.

The Maillard reaction is well known for producing antioxidant compounds alongside colored substances. Low-molecular-weight antioxidant intermediates such as maltol (MAL) or norfuraneol (NF) are well described, but it is still unclear which of these Maillard intermediates are the precursors of antioxidant and colored melanoidins-the so-called late stage Maillard reaction products. This study aimed to provide novel insights into the correlation between browning potential and antioxidant properties of reaction products formed during the heat treatment of prominent Maillard reaction intermediates. It was achieved by the incubation of binary reaction systems composed of methylglyoxal (MGO) or NF in combination with furfural (FF), MAL, and pyrrole-2-carbaldehyde (PA) at pH 5 and 130 °C for up to 120 min. Overall, it could be shown that the formation of colored products in the binary NF reaction systems was more efficient compared to those of MGO. This was reflected in an increased browning intensity of up to 400% and a lower conversion rate of NF compared to MGO. The colorants formed by NF and FF or PA (~0.34 kDa and 10-100 kDa) were also found to exhibit higher molecular weights compared to the analogue products formed in the MGO incubations (<0.34 kDa and 10-100 kDa). The incorporation of NF into these heterogenous products with FF and PA resulted in the preservation of the initial antioxidant properties of NF (p < 0.05), whereas no antioxidant products were formed after the incubation of MGO.

Characterization of Colorants Formed by Non-Enzymatic Browning Reactions of Hydroxycinnamic Acid Derivatives.

The browning of plant-based food is commonly understood to result from the enzymatic polymerization of phenolic compounds to pigments, called melanin. However, during the thermal treatment of food, enzymes are deactivated, and non-enzymatic reactions predominate. The extent of the contribution of phenolic compounds to these non-enzymatic reactions has been speculated ("melanin-like vs. melanoidin-like"), but the literature is limited. Therefore, the aim of the present study was to investigate the heat-induced reactions of caffeic acid (CA), para-coumaric acid (CS), ferulic acid (FA), hydrocaffeic acid (HC), and 5-O-caffeoylquinic acid (CGA) under dry conditions. The model systems were characterized by color formation, reactant conversion, and antioxidant properties. Reaction products were analyzed by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Decarboxylation could be classified as the driving force for the observed color formation and was significantly impacted by the substitution of the aromatic system. Reaction products were found to contribute to an increase in the antioxidant properties of the model systems. The oligomers described in this study could be incorporated into food melanoidins, contributing to the color and antioxidant properties observed in roasted food rich in phenolic compounds, such as coffee or cocoa.

Structural characterization of polar melanoidins deriving from Maillard reaction intermediates - A model approach.

Chemical conversions of reducing sugars and amino compounds induce the formation of heterogenous, high-molecular-weight colorants ('melanoidins') with widely unknown chemical structures. Model experiments of reactive intermediates have proven to be suitable for unravelling the formation mechanisms of colored reaction products. Here, the active methylene norfuraneol was selected and incubated individually as well as in combination with glyoxal, glycolaldehyde, and acetaldehyde at elevated temperatures. Photometric and chromatographic methods as well as mass spectrometry were used to analyze the colored reaction products and reveal the reactivity of different carbonyls regarding the formation of heterogenous oligomers. Aqueous solutions of norfuraneol and glyoxal exceeded the color formation of all other model reaction systems and it could be shown that the initial reactants as well as their degradation products were incorporated into the colorants. The colored oligomers described herein were composed of carbohydrate-based intermediates of the Maillard reaction and defined as melanoidin precursors or pre-melanoidins.

Arabinoxylan-Based Microcapsules Being Loaded with Bee Products as Bioactive Food Components Are Able to Modulate the Cell Migration and Inflammatory Response-In Vitro Study.

The aim of the research was to use bioactive heteropolysaccharides isolated from rye bran to obtain innovative systems for the controlled release of bioactive compounds. The core of the obtained encapsulates was honey and royal jelly. It was shown for the first time that preparations effectively ameliorated inflammatory response in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, decreasing the secretion of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and nitric oxide (NO). The in vitro digestion process revealed that bee products' encapsulates were stronger oxidative stress reducers and had sustained ability to reduction in inflammation state mediators. The lack of inhibitory effect on migration rate of human microvascular endothelial cells (HMEC-1) endothelial cells and mouse embryonic fibroblasts (NIH-3T3), both cell models involved in wound healing process, additionally identified these preparations as agents potentially used in the management of inflammatory response. In the process of a simulated digestion in vitro, the innovative microcapsules showed 85% higher biostability and two to ten times better bioavailability, compared to natural bee products.

Novel transformation products from glucosinolate-derived thioglucose and isothiocyanates formed during cooking.

Glucosinolates are plant secondary metabolites occurring in Brassicaceae plants. Upon tissue disruption, these compounds can be enzymatically hydrolyzed into isothiocyanates, which are very reactive and can react with nucleophiles during thermal processes such as boiling. Here, a novel type of glucosinolate degradation product was identified resulting from the reaction of thioglucose with the isothiocyanates sulforaphane or allyl isothiocyanate during aqueous heating. The two heterocyclic compounds 4-hydroxy-3-(4-(methylsulfinyl)butyl)thiazolidine-2-thione and 3-allyl-4-hydroxythiazolidine-2-thione were isolated and their structure elucidated by NMR spectroscopy and high-resolution mass spectrometry. Based on a set of chemical experiments, a reaction mechanism was proposed. Finally, the formation of the two 3-alk(en)yl-4-hydroxythiazolidine-2-thiones was quantified in boiled cabbage samples using a standard addition method in which 92 pmol/g and 19 pmol/g fresh weight of the sulforaphane and allyl isothiocyanate derivatives were found, respectively.

Systematic Studies on the Antioxidant Capacity and Volatile Compound Profile of Yellow Mealworm Larvae (T. molitor L.) under Different Drying Regimes.

The yellow mealworm (Tenebrio molitor L., Coleoptera: Tenebrionidae) is an edible insect and due to its ubiquitous occurrence and the frequency of consumption, a promising candidate for the cultivation and production on an industrial scale. Moreover, it is the first insect to be approved by EFSA 2021 following the Novel Food Regulation. Industrial production of mealworms necessitates optimized processing techniques, where drying as the first postharvest procedure is of utmost importance for the quality of the final product. The focus of the present study was to analyse the chemical composition, antioxidant capacity, volatile compound profile and colouring of mealworm larvae dried in various regimes (freeze-drying, microwave drying, infrared drying, rack-oven drying and high-frequency drying). Proximate composition and fatty acid profile were similar for all dried larvae. Freeze dried larvae were predominantly marked by lipid oxidation with significantly higher peroxide values, secondary/tertiary oxidation products in the headspace GC-MS profiles and lower antioxidant capacity. High-temperature treatment in the rack oven-and to some extent also infrared or microwave drying-led to mealworm larvae darkening and the appearance of volatile Maillard secondary products such as 2-methylpropanoic acid, 2-/3-methylbutanoic acid and alkylpyrazines. High-frequency drying as a new emerging technology in insect processing was the most cost-effective method with energy costs of solely 0.09 Є/kg T. molitor L. leading to final larval material characterized by both lipid oxidation and nonenzymatic Maillard-browning.

Formation of melanoidins - Aldol reactions of heterocyclic and short-chain Maillard intermediates.

In the course of the Maillard reaction, reducing sugars and amino compounds are converted to colorants, whose chemical structures are still mostly unknown. Active methylene compounds like norfuraneol that can initiate aldol condensation reactions are considered as key intermediates in this reaction. The aim of the present study was to characterize color formation of norfuraneol with different carbonyl compounds and to identify the underlying mechanisms of the reaction. Norfuraneol was incubated with methylglyoxal or diacetyl at elevated temperatures and the resulting reaction mixtures were analyzed by means of high-resolution mass spectrometry. It was demonstrated that aldol reactions lead to the formation of heterogeneous carbohydrate-based oligomers, which are likely to contribute to the elevated browning observed in the reaction mixtures. Furthermore, redox reactions were identified as another important part of the reaction, resulting in an increasing number of double bonds in the detected reaction products.

High-Resolution Mass Spectrometry Analysis of Melanoidins and Their Precursors Formed in a Model Study of the Maillard Reaction of Methylglyoxal with l-Alanine or l-Lysine.

Despite more than 100 years of research, formation of food melanoidins from carbohydrates and amino acids in the course of the Maillard reaction is still not fully understood. Experiments with relevant precursors are commonly used to limit the pathways of the complex reaction and to elucidate the formation mechanisms of the colored end-products. Here as a simple model, methylglyoxal was incubated with l-alanine or l-lysine in aqueous solutions at 100 °C and pH 5. The reaction mixtures were analyzed for color formation, molecular weight distribution, and conversion of methylglyoxal. High-resolution mass spectrometry was used to characterize the variety of products formed. With the help of Kendrick and van Krevelen analyses, the complex data sets were investigated for common substructures and reaction patterns. This study revealed that methylglyoxal forms oligomers via aldol reaction under involvement of its prevalent reaction products such as formaldehyde, acetaldehyde, acetol, and aminoacetone with amino acids.

The Formation of Methyl Ketones during Lipid Oxidation at Elevated Temperatures.

Lipid oxidation and the resulting volatile organic compounds are the main reasons for a loss of food quality. In addition to typical compounds, such as alkanes, aldehydes and alcohols, methyl ketones like heptan-2-one, are repeatedly described as aroma-active substances in various foods. However, it is not yet clear from which precursors methyl ketones are formed and what influence amino compounds have on the formation mechanism. In this study, the formation of methyl ketones in selected food-relevant fats and oils, as well as in model systems with linoleic acid or pure secondary degradation products (alka-2,4-dienals, alken-2-als, hexanal, and 2-butyloct-2-enal), has been investigated. Elevated temperatures were chosen for simulating processing conditions such as baking, frying, or deep-frying. Up to seven methyl ketones in milk fat, vegetable oils, and selected model systems have been determined using static headspace gas chromatography-mass spectrometry (GC-MS). This study showed that methyl ketones are tertiary lipid oxidation products, as they are derived from secondary degradation products such as deca-2,4-dienal and oct-2-enal. The study further showed that the position of the double bond in the precursor compound determines the chain length of the methyl ketone and that amino compounds promote the formation of methyl ketones to a different degree. These compounds influence the profile of the products formed. As food naturally contains lipids as well as amino compounds, the proposed pathways are relevant for the formation of aroma-active methyl ketones in food.

Melanoidin formed from fructosylalanine contains more alanine than melanoidin formed from d-glucose with L-alanine.

In this study the elemental compositions of melanoidin formed at 160 °C from d-glucose (Glc) and l-alanine (Ala) as well as from fructosylalanine - the corresponding Amadori rearrangement product - were compared. Specific chemical bonds were probed by FTIR spectroscopy. This approach tackles the different chemical pathways for melanoidin formation via the Amadori rearrangement in contrast to the reaction from Glc/Ala. Melanoidins formed from fructosylalanine contain about twice as much nitrogen and therefore amino acid as compared to melanoidin from Glc/Ala and exhibit higher absorption in the UV/Vis. Consequently, melanoidins formed from Glc/Ala contain more sugar degradation products with lower absorption due to a smaller size of the conjugated double bond network.

Melanoidins Formed by Heterocyclic Maillard Reaction Intermediates via Aldol Reaction and Michael Addition.

Thermal treatment of food leads to the formation of melanoidins by reactions of carbohydrates with free amino acids or proteins in the late stage of Maillard reaction. The aim of this study was the identification of reaction mechanisms responsible for the formation of melanoidins involving active methylenes with the heterocyclic structure and the structural characterization of the resulting products. For this purpose, norfuraneol was incubated with the aldehydes pyrrole-2-aldehyde or furfural at 125 °C. With the help of high-resolution mass spectrometry, the structural composition of oligomers of up to fifteen C5-units could be described for the first time. Aldol and Michael reactions could be identified as crucial steps for the formation of new C-C-bonds. With increasing heating time, the elimination of water from the products was facilitated, and oxidation reactions of integrated reductone structures lead to the expansion of conjugated double-bond systems responsible for the color formation of the samples.

Basic Structure of Melanoidins Formed in the Maillard Reaction of 3-Deoxyglucosone and γ-Aminobutyric Acid.

Melanoidins are formed in foods during processing through the Maillard reaction between carbohydrates and amino compounds. The aim of this study was to draw conclusions about the formation mechanism and the structure of melanoidins formed at low water contents and low temperatures. In the Maillard reaction of d-glucose and γ-aminobutyric acid at low water contents 3-deoxyglucosone is the most important intermediate. Therefore, we used the reaction of 3-deoxyglucosone with γ-aminobutyric acid or ß-alanine as a simplified model system. The degradation of 3-deoxyglucosone and the color formation of the formed melanoidins were determined. In addition, the reaction mixture was analyzed with high-resolution mass spectrometry and a Kendrick analysis was applied. Oligomers consisting of up to four molecules of 3-deoxyglucosone and three amino acids and their respective dehydration products with furanoidic structure were detected. The melanoidin structure of C-C linked monomeric units postulated by Kroh et al. could be confirmed.

PCA-based identification and differentiation of FTIR data from model melanoidins with specific molecular compositions.

Melanoidins formed from different carbohydrates, such as d-glucose, d-fructose, and d-xylose, and their typical degradation products, such as hydroxymethylfurfural, furfural, glyoxal, and methylglyoxal, with l-alanine were analyzed with Fourier transform infrared spectroscopy (FTIR). Characteristic infrared bands were identified representing spectral differences between the investigated melanoidin species due to their different molecular compositions. With the help of principal components analysis (PCA) the IR data allowed for a fast discrimination between the different model melanoidins. From this study it is inferred that the intensity and relative absorption wavelength of CO single versus CO double bonds are characteristic features of the investigated melanoidins. Melanoidins formed from carbohydrates exhibit less carbonyl functions in comparison to melanoidins from the degradation products, the situation is opposite for the CO bond. The amount of CO is additionally correlated with a higher absorption at 420 nm indicating that strong colored melanoidins contain more carbonyl functions.

Structural characterization of melanoidin formed from d-glucose and l-alanine at different temperatures applying FTIR, NMR, EPR, and MALDI-ToF-MS.

The aim of this study was to identify specific chemical bonds and characteristic structures in melanoidins formed from d-glucose and l-alanine between 130 and 200 °C. The results might be used to control the type and amount of melanoidin produced during food processing. For this purpose, complementary techniques, such as FTIR, NMR, EPR, and MALDI-ToF, were employed. At 160 °C color, solubility and UV/Vis absorption change characteristically and consequently, structural transformations could be observed in FTIR and NMR spectra. For example, sharp signals of N-H, C-N, and C-H oscillations in the l-alanine spectrum are prone to inhomogeneous broadening in melanoidins prepared above 150 °C. These changes are caused due to formation of heterogeneous macromolecular structures and occur during condensation reactions that lead to an increasing loss of water from the melanoidins with increasing temperatures. Additionally, MALDI-ToF-MS indicates the polymerization of glyoxal/glyoxylic acid and EPR shows the formation of radical structures.

Formation of Reactive Intermediates, Color, and Antioxidant Activity in the Maillard Reaction of Maltose in Comparison to d-Glucose.

In this study, the Maillard reaction of maltose and d-glucose in the presence of l-alanine was investigated in aqueous solution at 130 °C and pH 5. The reactivity of both carbohydrates was compared in regards of their degradation, browning, and antioxidant activity. In order to identify relevant differences in the reaction pathways, the concentrations of selected intermediates such as 1,2-dicarbonyl compounds, furans, furanones, and pyranones were determined. It was found, that the degradation of maltose predominantly yields 1,2-dicarbonyls that still carry a glucosyl moiety and thus subsequent reactions to HMF, furfural, and 2-acetylfuran are favored due to the elimination of d-glucose, which is an excellent leaving group in aqueous solution. Consequently, higher amounts of these heterocycles are formed from maltose. 3-deoxyglucosone and 3-deoxygalactosone represent the only relevant C6-1,2-dicarbonyls in maltose incubations and are produced in nearly equimolar amounts during the first 60 min of heating as byproducts of the HMF formation.

Browning Potential of C6-α-Dicarbonyl Compounds under Maillard Conditions.

In this work, the three major C6-α-dicarbonyl compounds glucosone (GLUC), 1-deoxyglucosone (1-DG), and 3-deoxyglucosone (3-DG) were synthesized and examined under Maillard conditions (aqueous solutions with the addition of l-alanine at 130 °C and pH 5/8). For the first time, the resulting color formation, antioxidant activity, and generation of short-chained α-dicarbonyls were investigated and compared to incubations of d-glucose and d-fructose. An additive effect on the formation of color, an antagonistic effect on the generation of α-dicarbonyl compounds, and a synergistic effect on the antioxidant activity could be observed for the 1-DG/GLUC combination. Despite their common degradation products, different extinctions could be measured, with 3-DG showing the strongest color formation, followed by GLUC and 1-DG. The analyzed α-dicarbonyl compounds have no direct impact on the formation of color but are precursors for most of the colored compounds. The main difference between the three substances is their ability to form different heterocyclic degradation products, such as pyranones (1-DG), furanones (1-DG), furans (GLUC and 3-DG), and the corresponding N-heterocycles in the presence of amino components. This seems to be the main reason for their varying browning potential and antioxidant activity.

Antioxidant Properties of Heterocyclic Intermediates of the Maillard Reaction and Structurally Related Compounds.

It is well established that a wide range of reductones is formed in the course of the Maillard reaction and that these substances contribute to the oxidative stability of food. The aim of this study was to analyze 12 important heterocyclic intermediates with and without reductone structure as well as structurally related substances under equal conditions to compare their antioxidant properties in detail. For this purpose, five methods were selected including photometrical methods such as the trolox equivalent antioxidant capacity assay and an electron paramagnetic resonance spectroscopic method. Reductones with furan-3-one structure and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one were reducing in all assays, whereas isomaltol and maltol did not react in assays based on the reduction of metal ions because of their complexing abilities. The introduction of protecting groups to the free hydroxyl functions of selected reductones could nearly eliminate their reducing abilities. In addition, the oxidation products of the different reductive heterocycles were compared after treatment with iodine. Mainly short-chained organic acids such as lactic, glycolic, and glyceric acid are formed as result of the degradation, which indicates 1,3-dicarbonyl cleavage reactions of corresponding tricarbonyl compounds as intermediates of the oxidation.

Antioxidant capacity of 1-deoxy-D-erythro-hexo-2,3-diulose and D-arabino-hexo-2-ulose.

The antioxidant capacity of two 1,2-dicarbonyl compounds, 1-deoxy-d-erythro-hexo-2,3-diulose (1-deoxyglucosone) and d-arabino-hexo-2-ulose (d-glucosone), was investigated. Both compounds are key intermediates of the Maillard reaction, and both possess a reductone-like structure. The reductive potential of the reductones was measured with the trolox equivalent antioxidant capacity (TEAC) assay and the Folin-Ciocalteu reagent (FCR) assay. Their antioxidant capacity set them apart from their precursors and other typical Maillard reaction products. Using electron paramagnetic resonance (EPR) spectroscopy, the special radical scavenging behavior of 1-deoxyglucosone and d-glucosone was measured. Both exhibited a slow, but constant, scavenging ability over the course of several hours, even days. It was postulated that this characteristic behavior is caused by the isomeric composition and the transformation to the particular antioxidant form. Reaction mixtures of 1-deoxyglucosone showed a correlation between the decrease of antioxidant properties and the decomposition of 1-deoxyglucosone.