Early Maillard Reaction Research Articles

Light-Colored Maillard Peptides: Formation from Reduced Fluorescent Precursors of Browning and Enhancement of Saltiness Perception.

The browning formation and taste enhancement of peptides derived from soybean, peanut, and corn were studied in the light-colored Maillard reaction compared with the deep-colored reaction. The fluorescent compounds, as the browning precursors, were accumulated during the early Maillard reaction of peptides and subsequently degraded into dark substances, which resulted in a higher browning degree of deep-colored Maillard peptides (MPs), especially for the MPs derived from corn peptide. However, the addition of l-cysteine in light-colored Maillard reaction reduced the formation of deoxyosones and short-chain reactive α-dicarbonyls, thereby weakening the generation of fluorescent compounds and inhibited the browning of MPs. Synchronously, the peptides were thermally degraded into small peptides and amino acids, which were consumed less during light-colored thermal reaction due to its shorter reaction time at high temperature compared with deep-colored ones, thus contributing to a stronger saltiness perception of light-colored MPs than deep-colored MPs. Besides, the Maillard reaction products derived from soybean and peanut peptides possessed an obvious "kokumi" taste, making them suitable for enhancing the soup flavors.

Bioavailability evaluation of calcium, magnesium and zinc in Brazilian cheese through a combined model of in vitro digestion and Caco-2 cells

Cheese has important nutritional properties as a result of their protein, lipid and essential minerals content. This study analyzed the bioavailability of Ca, Mg and Zn in Brazilian cheeses, using the in vitro INFOGEST digestion method coupled with assays in Caco-2 cells. Furosine content (marker of the early Maillard reaction) was also evaluated. Two self-produced fresh cheeses, cow and goat Minas frescal; and two commercial matured goat cheeses, Blue and Pyramid, were analyzed. Mineral bioaccessibility after in vitro digestion was in the range of 44.4–74.7 %, 54.8–66.1 % and 18.2–38.7 % for Ca, Mg and Zn, respectively. Bioaccessibilty was significantly affected by the mineral and fat content level. The digested fresh cheeses (Minas frescal) showed the highest values of soluble Ca and Mg; and the Pyramid goat cheese presented the greatest Ca and Zn transport efficacy across Caco-2 cell monolayers. Furosine could be a useful indicator to evaluate cheese ripeness and its indirect effect on the mineral availability.

Acid-Induced Gelation of Enzymatically and Nonenzymatically Cross-Linked Caseins-Texture Properties, and Microstructural Insights.

Casein gels consist of a fractal organized network of aggregated casein particles. The gel texture thereby depends on the structure, the spatial distribution, and the interaction forces of the network's elementary building blocks. The aim of this study was to explore the technofunctional consequences of a possible specificity of Maillard reaction-induced cross-linking reactions on casein with respect to texture and microstructure of acid gels. Therefore, sodium caseinate glycated with lactose in the dry state (60 °C, aw 0.5) was compared with casein samples cross-linked with methylglyoxal, with glutaraldehyde, or via microbial transglutaminase, respectively, at similar levels of protein cross-linking as confirmed by size-exclusion chromatography under denaturing conditions. Casein gels prepared by acidification with glucono-δ-lactone were characterized concerning pH kinetics during gelation, mechanical texture properties under large deformation, and water-holding capacity, while viscometric properties of casein suspensions were obtained prior to gelation. The gel microstructure was captured by confocal laser scanning microscopy and evaluated by means of image texture analysis. All protein cross-linking reactions studied led to an enhanced gel strength which was accompanied by an increased interconnectivity of the gel network and a decrease in apparent pore sizes. Gels with more densely packed strands, as was the case for enzymatically modified casein, exhibited pronounced mechanical stability. The spontaneous destabilization of the gel network upon prolonged glycation reactions, which was not obviously displayed by microstructural features but connected to an increased viscosity and pronounced pseudoplastic flow of the unacidified suspension, suggests a limitation of particle rearrangements and the weakening of interparticle protein-protein interactions by additional structure attributes formed during the early Maillard reaction (glycoconjugation).

Diagnostic MS/MS fragmentation patterns for the discrimination between Schiff bases and their Amadori or Heyns rearrangement products

Schiff bases, the Amadori and Heyns rearrangement products are the most important isomeric intermediates involved in the early Maillard reaction; distinguishing between them by analytical mass spectroscopic techniques remains a challenge. Here we demonstrate that MS/MS fragmentation patterns can be used for the discrimination between glucose derived Schiff bases, Amadori, and Heyns compounds with glycine. An ESI-qTOF-MS system operated in the positive mode under both acidic and neutral conditions was employed to generate unique MS/MS fragmentation patterns of the molecules. Analysis of the MS data has indicated that acidic medium is suitable for generating characteristic and diagnostic ions. At high collision energy (20 eV), the spectrum of Schiff base was largely uninformative, whereas both Amadori and Heyns compounds undergo characteristic fragmentations with high diagnostic value. At low collision energy values (10eV), we observed formation of prominent diagnostic ions from the Schiff base precursor, as well as extensive dehydration reactions of all three molecules. Under acidic conditions, the diagnostic fragmentation pattern of the Amadori compound featured consecutive dehydration reactions. At higher values (20 eV) it underwent the α-fission at the carbonyl group and produced a prominent diagnostic ion [AA + H + CH2]+ at m/z 88. The Schiff base was found to preferentially undergo the retro-aldol degradation and produce diagnostic ions at m/z 118 [AA + H + diose]+ and m/z 140 [AA + Na + diose]+, together with their sugar complements at m/z 85 [tetrose + H–2H2O]+ and m/z 143 [tetrose + Na]+. In the case of Heyns compound, several diagnostic ions were also detected, including the ions at m/z 154 [M + H–2H2O–C2H4O2]+, m/z 170 [AA + Na + triose]+ and m/z 142 [AA + H + Furan]+.

Preliminary Assessment of Non-Enzymatic Browning and Antioxidant Activity in Sea Cucumber Derived Gelatin-Sugar Models

Four models were developed to assess the development of non-enzymatic browning and antioxidant activity in mixtures of sea cucumber derived gelatin (control), gelatin-glucose, gelatin-fructose and gelatin-glucosamine. All gelatin-sugar models were prepared in mixtures consisted of 5% w/v gelatin and 1.25% w/v sugar and compared against a control (5% w/v gelatin). The mixtures were incubated at 37 °C and 70 °C respectively for up to 24 h. The progress of non-enzymatic browning was monitored via changes in absorption at 280 nm, 320 nm and 420 nm. The gelatin-glucosamine model showed greater development in early Maillard reaction products (280 nm) and pre-melanoidins (320 nm) when incubated at 70 °C after 16 h of incubation. Meanwhile lower incubation temperature (37 °C) promoted browning development (420 nm) in all the models. The gelatin-glucosamine mixture incubated at 70 °C showed high potency in antioxidant activity (p<0.05), this is in accordance to the greater reactivity of glucosamine in the formation of Maillard reaction products at the early and intermediate stages. The results suggested the gelatin-glucosamine model can be further optimized to achieve high potency in antioxidant activity and a desired browning intensity to cater specific applications such as in food and biomaterials.

Association of Enzymatically and Nonenzymatically Functionalized Caseins Analyzed by Size-Exclusion Chromatography and Light-Scattering Techniques.

The influence of covalent protein modifications resulting from the Maillard reaction (glycation) of casein and lactose on the noncovalent association behavior of the protein was studied. Nonenzymatic cross-linking with methylglyoxal (MGO) and glutaraldehyde (GTA) as well as enzymatic cross-linking with microbial transglutaminase (mTG) was investigated in comparison. Molar mass, particle size, and conformational characteristics of nonmicellar casein associates as well as the extent of intraparticle protein cross-linking were examined utilizing size-exclusion chromatography (SEC) combined with UV detection and static and dynamic light scattering. Cross-linking resulted in the stabilization of a certain fraction of casein associates, with particle sizes of approximately 30 nm in radius of gyration (Rg), and promoted an incorporation of further casein molecules into those particles, yielding molar masses (Mw) of 1.0-1.2 × 106 g/mol. When caseins were additionally conjugated with lactose during the early Maillard reaction, a further growth of the associates up to approximately 50 nm in Rg with a Mw of 2.1 × 106 g/mol was observed. Furthermore, glycation reactions induced a transition from slightly elongated, random-coil structures toward more anisotropic conformations. Associates consisting of caseins cross-linked with GTA appeared to preserve the original particle conformation.

Milk-Derived Amadori Products in Feces of Formula-Fed Infants

Food processing of infant formula alters chemical structures, including the formation of Maillard reaction products between proteins and sugars. We detected early Maillard reaction products, so-called Amadori products, in stool samples of formula-fed infants. In total, four Amadori products (N-deoxylactulosyllysine, N-deoxyfructosyllysine, N-deoxylactulosylleucylisoleucine, N-deoxyfructosylleucylisoleucine) were identified by a combination of complementary nontargeted and targeted metabolomics approaches. Chemical structures were confirmed by preparation and isolation of reference compounds, LC-MS/MS, and NMR. The leucylisoleucine Amadori compounds, which most likely originate from β-lactoglobulin, were excreted throughout the first year of life in feces of formula-fed infants but were absent in feces of breastfed infants. Despite high inter- and intraindividual differences of Amadori products in the infants' stool, solid food introduction resulted in a continuous decrease, proving infant formula as the major source of the excreted Amadori products.

Angiotensin I-Converting Enzyme Inhibitory Effect of Chinese Soypaste Along Fermentation and Ripening: Contribution of Early Soybean Protein Borne Peptides and Late Maillard Reaction Products

Angiotensin I-converting enzyme inhibitory effect of Chinese soypaste was investigated during 4-month fermentation and ripening. This effect increased significantly at early stage and reached a plateau until the end of ripening, with an IC50 value of 0.436 mg/mL for the final product. Peptide and reducing sugar contents increased drastically during early fermentation and declined afterward. Maillard reaction took place continuously as indicated by consistent accumulation of precursor, intermediate, and final Maillard reaction products monitored by fluorescence, ultraviolet-absorbance and absorbance at 420 nm, respectively. During early fermentation, peptides were principal compounds responsible for the angiotensin I-converting enzyme inhibitory effect. During late ripening, angiotensin I-converting enzyme inhibition by Maillard reaction products could compensate for the loss of the effect caused by the utilization of peptides, and enable the total effect to remain at a good and steady level, suggesting an indispensable contribution of Maillard reaction products to angiotensin I-converting enzyme inhibitory effect of soypaste, particularly in products with prolonged maturation.

Characterization of concentrated agave saps and storage effects on browning, antioxidant capacity and amino acid content

Sap from agave plants (“aguamiel”) is traditionally consumed in Mexico as a fresh beverage, fermented or concentrated. Concentrated agave sap (CAS) is used as a sweetener but, due to heating, a brown color develops and intensifies during storage. Browning varies among CAS batches and this work was focused on its correlation with the chemical composition changes observed during 20 weeks of storage. The browning index (BI), measured as the optical density at 490nm (OD490nm) per gram of sample, increased 54.4% in the batch that initially had 57 OD490nm/g but in the other two batches that had a lower BI, the increase was less than 26.1%. Antioxidant capacity only increased in the batch with the highest BI going from 18 to 23Trolox equivalent μmol/g dry weight. Saponin content was different in the three batches (224.2–434.7 protodioscin equivalents/gram dry weight) but did not change after 20 weeks of storage. Browning index and antioxidant capacity were negatively correlated with free amino acid concentration, particularly serine, phenylalanine and lysine decreased 29.4, 50 and 30%, respectively. Browning was positively correlated to furosine, an early Maillard reaction derivative of lysine previously reported as a free radical scavenger.

Characteristics of Early Maillard Reaction Products by Electrospray Ionization Mass Spectrometry

Characteristics of Early Maillard Reaction Products by Electrospray Ionization Mass Spectrometry

Stability and bioactivity of a Bowman–Birk inhibitor in orange juice during processing and storage

The present research aimed to evaluate the feasibility of a soybean Bowman-Birk inhibitor (BBI) as a natural functional food ingredient. The influence of food processing, storage and digestion on the structure and bioactivity of BBI added to a food matrix was evaluated. The cytotoxic effect of the digested samples was also tested with the aim to provide information regarding the safe use of BBI as a functional food ingredient. Samples of freshly squeezed orange juice (OJ) and two orange juice model systems (OJ-ms) supplemented and non-supplemented with BBI were prepared and processed mimicking pasteurization and sterilization industrial conditions. Moreover, pasteurized samples were stored at 4 °C for two months. The samples were digested in vitro under oral-gastrointestinal physiological conditions. Results seem to indicate that the activity of BBI supplemented to the food matrix sufficiently survives thermal processing, storage and digestion processes. Changes in BBI bioactivity may be ascribed among others to the Maillard reaction. Formation of early Maillard reaction products also called Amadori products has been detected in the food sample. No cytotoxic effect was observed for the samples under study. In conclusion, our findings support that BBI has significant potential as a natural functional food ingredient in pasteurized orange juice stored at 4 °C.

Testing isotopic labeling with [13C6]glucose as a method of advanced glycation sites identification

The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid. However, the diversity of the structures of AGEs makes detection of these compounds more difficult. The aim of this study was to test a new method of AGE identification based on isotope 13C labeling. The model protein (hen egg lysozyme) was modified with an equimolar mixture of [12C6]glucose and [13C6]glucose and then subjected to reduction of the disulfide bridges followed by tryptic hydrolysis. The digest obtained was analyzed by LC–MS. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed identification of 38 early Maillard reaction products and five different structures of the end glycation products. This isotopic labeling technique combined with LC–MS is a sensitive method for identification of advanced glycation end products even if their chemical structure is unknown.

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.

Quantification of Amadori products in cheese

To obtain basic information about the extent of the early Maillard reaction in commercially available cheese varieties of varying degrees of maturity, Amadori products (APs) formed between reducing sugars and the e-amino group of lysine (e-APs) or the α-amino of selected amino acids (α-APs) were analyzed. APs were determined as the corresponding N-(2-furoylmethyl) amino acids (FMAAs) after acid hydrolysis using RP–HPLC with UV-detection as well as mass spectroscopy. Identification of the FMAAs resulting from the α-APs of lysine, valine, leucine, and isoleucine was achieved for the first time for cheese samples. The content of furosine, which is a hallmark for e-APs, ranged from to 3–75 mg/100 g protein corresponding to 24–591 μmol e-AP/100 g protein. e-AP levels declined during ripening in all investigated cheeses, depending on the cheese type and stage of maturation. The mean content of four α-APs ranged from 138 to 681 μmol/100 g protein. Lowest concentrations were found in long-term ripened cheese samples, indicating a putative degradation of α-APs and e-APs during advanced stages of maturation. The ratio of α-APs to e-APs might be a useful indicator of cheese ripening.

Acrylamide in Foods: Chemistry and Analysis. A Review

Acrylamide is a potential cause of a wide spectrum of toxic effects and is classified as probably “carcinogenic in humans”. The discovery of acrylamide in human foods has given rise to extensive studies exploring its formation mechanisms and levels of exposure and has spurred search into suitable analytical procedures for its determination in foodstuffs. However, the exact chemical mechanisms governing acrylamide formation are not yet known and cheap, convenient, and rapid screening methods are still to be developed. Acrylamide in food is produced by heat-induced reactions between the amino group of asparagine and the carbonyl group of reducing sugars along with thermal treatment of early Maillard reaction products (N-glycosides). Similarly, the decarboxylated Schiff base and decarboxylated Amadori compounds of asparagine as well as the Strecker aldehyde have been proposed as direct precursors and intermediates of acrylamide. Corresponding chromatographic methods are used to determine various structural groups present in Maillard reaction model systems. Gas chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry analysis are both acknowledged as the main, useful, and authoritative methods for acrylamide determination. This review is an attempt to summarize the state-of-the-art knowledge of acrylamide chemistry, formation mechanisms, and analytical methods. Special attention is given to comparison of different chromatographic techniques, particularly the novel, simple, and low-cost methods of its determination.

Development of the Maillard reaction in foods cooked by different techniques. Intake of Maillard-derived compounds

Specific and non-specific Maillard reaction (MR) indices such as CIELab colour, browning measurement, furosine, hydroxymethylfurfural (HMF) and furfural, as well as the nutrient content, were analysed for commonly consumed dishes, to test the effects of different culinary treatment on dishes composed of the same ingredients. In addition, the consumption of early MR products (MRP), Amadori compounds, HMF and furfural from a normal serving of these dishes was calculated. As expected, recipes including frying, apart from their particular composition, led to significantly higher values of furosine and HMF, ranging from 4.40 to 175 and from 0.30 to 22.7 mg/kg, respectively; consequently they provided the highest levels of Amadori compounds and HMF intake (0.42–26.8 and 0.02–2.38 mg/serving, respectively). Even so, MRP intake/serving was not very high in comparison with levels reported in the bibliography on some individual foods typically studied in terms of MR development, suggesting that the culinary treatments used do not make a great contribution to the daily MRP consumption.

Assessment of heat treatment of dairy products by MALDI‐TOF‐MS

The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS. The lactosylation rate of alpha-lactalbumin increased with increasing reaction temperature and time. The results correlated well with established markers for heat treatment of milk (concentration of total soluble protein, soluble alpha-lactalbumin and beta-lactoglobulin at pH 4.6, and fluorescence of advanced Maillard products and soluble tryptophan index; r=0.969-0.997). The method was also applied to examine commercially available dairy products. In severely heated products, protein pre-purification by immobilized metal affinity chromatography improved spectra quality. Relative quantification of protein lactosylation by MALDI-TOF-MS proved to be a very fast and reliable method to monitor early Maillard reaction during milk processing.

Structure of D-Fructosamine Hydrochloride and D-Fructosamine Hydroacetate

D-Fructosamine derivatives are key intermediates of the early Maillard reaction and have been a subject of numerous studies in food and health sciences due to their implication in the nutritional and organoleptic quality of foods, as well as to complications in diabetes and renal disease. We report the crystal structure analyses of 1-deoxy-β -D-fructopyranos-1-ylamine hydrochloride (1) and -hydroacetate (2) salts. The carbohydrate rings adopt the normal 2C5 pyranose chair conformation in 1 and 2. Bond lengths and valence angles in 1 and 2 compare well with the average values from related pyranose structures. There are two conformationally nonequivalent molecules in the asymmetric unit in 1. All hydroxyl and ring oxygen atoms, ammonium groups, and chloride ions in 1 are involved in an extensive three-dimensional hydrogen bonding network. The hydrogen bonding network in 2 is formed by one type of infinite chain with attached antidromic cycles.

Influence of Cheese Type and Maturation Time on the Early Maillard Reaction in Cheese

Formation and degradation of Amadori products (APs) originating from the early Maillard reaction during maturation of three different commercial cheeses, namely Cheddar, Emmentaler and Gouda, was investigated. APs were analysed as the corresponding <I>N</I>-(furoylmethyl) amino acids formed after acid hydrolysis. The contents of furosine, which is a hallmark for Amadori products resulting from derivatisation of lysine at the ε-amino group (ε-APs), ranged from 4 to 20 mg/100 g protein, corresponding to 33 to 159 μmol of lysine Amadori product per 100 g protein in the cheese samples at the start of the ripening period. Furosine contents declined during ripening in all investigated cheeses, in which cheese type and the stage of ripening influenced the rate of furosine decline. In contrast to this, all detectable N-terminal APs (α-AP) decreased at similar rates. The mean total content of these substances ranged from 12 to 48 μmol/100 g protein. The ratio between ε-APs and α-APs can be used as an indicator for the cheese ripening.

Kinetics of Maillard reactions in model infant formula during UHT treatment using a static batch ohmic heater

The impact of an ultrahigh temperature (UHT) treatment on amodel infant formula (IF) was examined by assessing the advancement of Maillard reactions during a thermal treatment by ohmic heating. The heating and holding steps of the heat treatment were carried out in a static batch ohmic heater equipped with a nitrogen counter-pressure system, allowing the reaching of five temperature levels from 100 to 140 °C. A heat treatment was characterised by monitoring a holding time at a given temperature. Samples were taken during heating and holding steps and analysed for early Maillard reaction products (MRPs), such as furosine, advanced MRPs, such as advanced glycated end products (AGEs) and carboxymethyllysine (CML), and final MRPs such as melanoidins. The results show that Maillard reactions in model IF are highly dependent from time-temperature treatment. In compliance with other classical heating techniques, the selected chemical markers followed a pseudo-zero order of reaction under isothermal conditions. However, the contribution of the heating step can be significant as MRPs could be detected from the first seconds of the UHT treatment.