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Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and Total Lysine through Stable Isotope Dilution Assay and Tandem Mass Spectrometry
Abstract
The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-l-lysine (CML), Nε-(Carboxyethyl)-l-lysine (CEL). A new analytical strategy was proposed which allowed to simultaneously quantify lysine, CML, CEL and the Nε-(2-Furoylmethyl)-l-lysine (furosine), the indirect marker of AP. The procedure is based on stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry. It showed high sensitivity and good reproducibility and repeatability in different foods. The limit of detection and the RSD% were lower than 5ppb and below 8%, respectively. Results obtained with the new procedure not only improved the knowledge about the reliability of thermal treatment markers, but also defined new insights in the relationship between Maillard reaction products and their precursors.
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... [111] Boiling and frying beef caused a ∼7.0and ∼15.5fold higher levels of CML, respectively, in compare to the raw beef. [112] [100] Sterilized milk 343 mg/kg protein RP-HPLC [104] Fried minced beef 61.1 mg/kg protein UPLC-MS/MS [100] Evaporated milk Up to 1015 mg/kg protein RP-HPLC and UPLC-MS/MS [100,104] Chicken breast, boiled 17.2 mg/kg protein UPLC-MS/MS [105] Condensed milk 205 mg/kg protein LC-MS/MS [101] Chicken breast, roasted 17.4 mg/kg protein UPLC-MS/MS [105] Infant formula (liquid) Up to 62.9 mg/kg protein LC-MS/MS and GC-MS [101,104] Chicken breast, fried 23.5 mg/kg protein UPLC-MS/MS [103] infant formula (powder) Up to 148 mg/kg protein LC-MS/MS and GC-MS [101,104,105] Coffee 84.1 mg/kg protein UPLC-MS/MS [103] Butter 37.1 mg/kg protein UPLC-MS/MS [79] Roasted peanut 5-77 mg/kg protein GC-MS [106] Coffee cream Up to 618 mg/kg protein RP-HPLC [101] Peanut puffs 61-63 mg/kg protein GC-MS [106] Whey cheese 1691 mg/kg protein RP-HPLC [101] Peanut butter 63-203 mg/kg protein GC-MS [106] Cheese 23.2 mg/kg protein UPLC-MS/MS [79] Unroasted almond 1.5 mg/kg a LC-MS/MS [107] Bakery products Bakery products Bread crust 58-94 mg/kg protein LC-MS/MS [108] Biscuits 50-117 mg/kg protein LC-MS/MS [108] Bread crumb 14-34 mg/kg protein LC-MS/MS [108] Cookies 5-35 mg/kg protein GC-MS [91] Corn flakes 6-8 mg/kg protein GC-MS [104] ...
... [111] Boiling and frying beef caused a ∼7.0and ∼15.5fold higher levels of CML, respectively, in compare to the raw beef. [112] [100] Sterilized milk 343 mg/kg protein RP-HPLC [104] Fried minced beef 61.1 mg/kg protein UPLC-MS/MS [100] Evaporated milk Up to 1015 mg/kg protein RP-HPLC and UPLC-MS/MS [100,104] Chicken breast, boiled 17.2 mg/kg protein UPLC-MS/MS [105] Condensed milk 205 mg/kg protein LC-MS/MS [101] Chicken breast, roasted 17.4 mg/kg protein UPLC-MS/MS [105] Infant formula (liquid) Up to 62.9 mg/kg protein LC-MS/MS and GC-MS [101,104] Chicken breast, fried 23.5 mg/kg protein UPLC-MS/MS [103] infant formula (powder) Up to 148 mg/kg protein LC-MS/MS and GC-MS [101,104,105] Coffee 84.1 mg/kg protein UPLC-MS/MS [103] Butter 37.1 mg/kg protein UPLC-MS/MS [79] Roasted peanut 5-77 mg/kg protein GC-MS [106] Coffee cream Up to 618 mg/kg protein RP-HPLC [101] Peanut puffs 61-63 mg/kg protein GC-MS [106] Whey cheese 1691 mg/kg protein RP-HPLC [101] Peanut butter 63-203 mg/kg protein GC-MS [106] Cheese 23.2 mg/kg protein UPLC-MS/MS [79] Unroasted almond 1.5 mg/kg a LC-MS/MS [107] Bakery products Bakery products Bread crust 58-94 mg/kg protein LC-MS/MS [108] Biscuits 50-117 mg/kg protein LC-MS/MS [108] Bread crumb 14-34 mg/kg protein LC-MS/MS [108] Cookies 5-35 mg/kg protein GC-MS [91] Corn flakes 6-8 mg/kg protein GC-MS [104] ...
... [111] Boiling and frying beef caused a ∼7.0and ∼15.5fold higher levels of CML, respectively, in compare to the raw beef. [112] [100] Sterilized milk 343 mg/kg protein RP-HPLC [104] Fried minced beef 61.1 mg/kg protein UPLC-MS/MS [100] Evaporated milk Up to 1015 mg/kg protein RP-HPLC and UPLC-MS/MS [100,104] Chicken breast, boiled 17.2 mg/kg protein UPLC-MS/MS [105] Condensed milk 205 mg/kg protein LC-MS/MS [101] Chicken breast, roasted 17.4 mg/kg protein UPLC-MS/MS [105] Infant formula (liquid) Up to 62.9 mg/kg protein LC-MS/MS and GC-MS [101,104] Chicken breast, fried 23.5 mg/kg protein UPLC-MS/MS [103] infant formula (powder) Up to 148 mg/kg protein LC-MS/MS and GC-MS [101,104,105] Coffee 84.1 mg/kg protein UPLC-MS/MS [103] Butter 37.1 mg/kg protein UPLC-MS/MS [79] Roasted peanut 5-77 mg/kg protein GC-MS [106] Coffee cream Up to 618 mg/kg protein RP-HPLC [101] Peanut puffs 61-63 mg/kg protein GC-MS [106] Whey cheese 1691 mg/kg protein RP-HPLC [101] Peanut butter 63-203 mg/kg protein GC-MS [106] Cheese 23.2 mg/kg protein UPLC-MS/MS [79] Unroasted almond 1.5 mg/kg a LC-MS/MS [107] Bakery products Bakery products Bread crust 58-94 mg/kg protein LC-MS/MS [108] Biscuits 50-117 mg/kg protein LC-MS/MS [108] Bread crumb 14-34 mg/kg protein LC-MS/MS [108] Cookies 5-35 mg/kg protein GC-MS [91] Corn flakes 6-8 mg/kg protein GC-MS [104] ...
N (6)-Carboxy-methyl lysine (CML), a biomarker of advanced glycation end products (AGE), is one of the hazardous substances from the food safety perspective. Recent researches have shown a direct relationship between consuming heat-treated foods with high carbohydrate, protein and fat content and increased levels of CML in food and body tissues which consequently has adverse effects on human health. In this review, the effect of several factors on CML formation was separately discussed in human body and food products. In the first part, the results of in vivo and in vitro researches including formation routes of biological CML; and impact of intrinsic and extrinsic factors on its formation were summarized. In the second part, the CML formation in the food products was reviewed emphasizing the role of processing/cooking method, food component and additives. Finally, the biological fate of dietary CML was discussed in which its accumulation and/or detoxification is associated with AGE receptors systems.
... The hydrophilic nature of AGEs makes it difficult to retain them on reversed-phase columns and therefore ion pairing agents are necessary to increase the interaction between the analytes and the column (Zhang, Huang, Xiao & Mitchell, 2011). Ion pairing agents, primarily nonafluoropentanoic acid (NFPA), are widely used in chromatographic analysis of AGEs in different research groups (Moeckel, Duerasch, Weiz, Ruck & Henle, 2016;Niquet-Léridon & Tessier, 2011;Troise, Fiore, Wiltafsky & Fogliano, 2015) because these are useful for increased retention of polar compounds on reversed-phase columns and increased resolution of isobaric and isomeric compounds. Nevertheless, these ion pairing agents have some disadvantages, being harmful to the column and instrument as well as decreasing the ionization efficiency. ...
... In general, the LOD values obtained in the present work are in accordance with the values of a previous method developed by our research group (Poojary et al., 2020), which was an LC-MS method using ion pair-RP-HPLC separation and Orbitrap MS detection. LOD values for furosine and CEL obtained in this study are also comparable to other ion pairing RP-HPLC-based methods (Troise et al., 2015). In one study published in 2016, where the authors used an amino column with a HILIC mode plus ion interaction, LOD values were reported for CML, CEL, MG-H1, GO-H1, GOLD, MOLD and pentosidine as 0.2-9 ng/mL in 1% aqueous formic acid (Nomi et al., 2016). ...
... Dairy products (UHT milk, liquid infant formula and powdered infant formula) and bakery products (corn flakes, cookies, salted sticks) had the highest concentrations of furosine. The levels of furosine in dairy foods are in accordance with previously published studies (Aktag, Hamzalıoglu & Gökmen, 2019;Troise et al., 2015), where it was shown that infant formula has comparatively higher concentrations than other UHTtreated dairy products. Similar findings in our study could be attributed to the higher concentrations of reducing sugars in infant formulas. ...
An analytical method was developed and validated for simultaneous identification and quantification of advanced glycation end products (AGEs), amino acid cross-links, lysine and arginine in foodstuffs based on acid hydrolysis, hydrophilic interaction chromatography and high-resolution mass spectrometry. The method proved to be sensitive, reproducible and accurate for furosine, N-Ɛ-(carboxymethyl)lysine, N-Ɛ-(carboxyethyl)lysine, methylglyoxal and glyoxal-derived hydroimidazolones (MG-H and GO-H isomers, respectively), glyoxal lysine dimer, lysinoalanine, lanthionine, lysine and arginine. LOD and LOQ values in water were found to be 0.9–15.5 ng/mL and 2.8–47 ng/mL, respectively, and increased to 1.4–60 ng/mL and 4.4–182 ng/mL in liquid infant formula. Recovery values ranged from 76–118% in four different food matrices. Microwave-assisted hydrolysis for 11 min had similar efficiency as conventional hydrolysis, which requires overnight incubation. Acid stability of each compound was determined during microwave and conventional hydrolysis, and showed that the MG-H1 isomer is partially converted to the MG-H3 isomer during acid hydrolysis.
... Analysis of furosine, CML, and CEL content was conducted on an Ultimate 3000 high-pressure liquid chromatograph (HPLC) (Thermo Scientific, USA) coupled to a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (San Jose, CA, USA) using the method of Troise, Fiore, Wiltafsky, and Fogliano (2015), with modifications. One millilitre of sample solution was mixed with 4 mL of 7.5 M hydrochloric acid (HCl) in a 10 mL heating tube and incubated at 110 • C for 20 h. ...
... To the best of our knowledge, there has not been other studies reporting CML and CEL contents in SPI-based systems. However, in powdered soybeanbased feed products, CML and CEL contents were found to be 9.94 ± 0.74 and 0.98 ± 0.04 mg/100 g of protein, and increased to around 76 and 2.41 mg/100 g of protein, after being incubated at 110 • C for 60 and 45 min, respectively (Troise et al., 2015). This means that the concentrations of CML in SPI and SPI-dextran systems in our study was of the same order of magnitude as the ones reported by Troise and co-workers; while the CML and CEL contents in SPI-glucose systems were much higher than those reported by Troise et al. (2015). ...
... However, in powdered soybeanbased feed products, CML and CEL contents were found to be 9.94 ± 0.74 and 0.98 ± 0.04 mg/100 g of protein, and increased to around 76 and 2.41 mg/100 g of protein, after being incubated at 110 • C for 60 and 45 min, respectively (Troise et al., 2015). This means that the concentrations of CML in SPI and SPI-dextran systems in our study was of the same order of magnitude as the ones reported by Troise and co-workers; while the CML and CEL contents in SPI-glucose systems were much higher than those reported by Troise et al. (2015). Glucose can directly oxidize to generate GO and MGO and further react with lysine to form CML and CEL, whereas dextran has first to be degraded into glucose units, then to be oxidized to induce GO and MGO (Fig. S4 A-F). ...
Dry and subsequent wet heating were used to glycate soy proteins with dextran or glucose, followed by fractionation based on size and solubility. Dry heating led to protein glycation (formation of furosine, Nε-(carboxymethyl)-l-lysine, Nε-(carboxyethyl)-l-lysine, and protein-bound carbonyls) and aggregation (increased particle size); while subsequent wet heating induced partial unfolding and de-aggregation. The measurable free amino group content of soy proteins changed from 0.77 to 0.14, then to 0.62 mmol/g upon dry and subsequent wet heating; this non-monotonic evolution is probably due to protein structural changes, and shows that this content should be interpreted with caution as a glycation marker. After both heating steps, the smaller-sized water-soluble fractions showed higher surface activity than the larger insoluble ones, and dextran conjugates exhibited a higher surface activity than their glucose counterparts. We thereby achieved a comprehensive understanding of the properties of various fractions in plant protein fractions, which is essential when targeting applications.
... Analytical performance robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, carry over and matrix effects were evaluated by following the procedures previously reported focusing on CML, d 4 -CML and lysine. 20 All the acquisition parameters are summarised in Table S1. CML was measured by using internal standard technique. ...
... To determine the concentration of CML and its metabolites in urines, analytical strategy was based on hydrophilic interaction chromatography by taking advantage of analyte positive charge in acidic conditions: zwitterionic stationary phase effectively separated polar charged analytes and improved response and robustness in positive ion mode confirming analytical performances for lysine and CML. 18,20 Indeed, quality control samples (pooled urine samples spiked with internal standards) showed a relative Please do not adjust margins Please do not adjust margins standard deviation of less than 12% between sample batches and calibration curve batches. (Table S2). ...
During food processing most of the thermally-driven chemical reactions start off on the side chain amino group of lysine generating structurally modified compounds with specific metabolic routes. Upon human digestion, dietary Nε-carboxymethyllysine (CML) may enter the colon and undergo gut microbial metabolism. However, little is known about the in vivo metabolic fate of dietary CML and its relationship with the habitual diet. We explored by hydrophilic interaction liquid chromatography tandem mass spectrometry the metabolites of CML in urine samples collected from 46 healthy subjects and studied the associations with diet. Mean concentration of N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), N-carboxymethylaminopentanol (CM-APO), and the N-carboxymethyl-Δ1-piperideinium ion were 0.49 nmol mg-1 creatinine, 1.45 nmol mg-1 creatinine, 4.43 nmol mg-1 creatinine and 4.79 nmol mg-1 creatinine, respectively. The urinary concentration of CML, its metabolites and lysine were positively correlated. Dietary intake of meat products negatively correlated with urinary excretion of CML and CM-APA; conversely dietary plant-to-animal proteins ratio positively correlated with urinary CML and its metabolites. The identification and quantification of CML metabolites in urine and the associations with diet corroborate the hypothesis that CML, an advanced glycation end-product, can undergo further biochemical transformations in vivo. The gut microbiome may have a major role in human metabolism of dietary CML.
... This may be attributed to the complex structure and wide ranges of AGEs in food caused by the variety of amino acids and extensive source of carbonyl compounds. Notably, CML, CEL, and Pyr, the well-exploited AGEs, are frequently selected in laboratory studies as markers for AGEs (Michael Hellwig et al. 2016;Troise et al. 2015). They are all lysine-derived AGEs. ...
... GC-MS typically require dual derivatization of the carbonyl and amino groups of targets, which is cumbersome and time consuming (Dong et al. 2020a). Of the methods previously mentioned, HPLC-MS/MS is the most common approach for analyzing AGEs in foods in advantages of high sensitivity and accuracy, good selectivity, and simple operation (He et al. 2014;Scheijen et al. 2016;Troise et al. 2015). Therefore, the aim of the present work was to establish a fractional extraction, hydrolysis, and solid-phase extraction purification method coupled with UHPLC-MS/MS technique to determine CML, CEL, and Pyr in free or bound form in milk powder. ...
The objective of this study was to establish and validate an UHPLC-MS/MS method for simultaneous determination of advanced glycation endproducts (AGEs) in either free or bound form in milk powder. The target analytes in free form in milk powder were extracted by 1% trichloroacetic acid, while target analytes in bound form were hydrolyzed by hydrochloric acid to cleave the protein amide bond and consequently dissociated. After extraction and purification, Nε-(carboxymethyl)lysine (CML) and Nε-(1-carboxyethyl)lysine (CEL) were quantified by internal standard method and pyrraline (Pyr) was by external standard method. Results revealed that three target analytes displayed excellent linearity in their corresponding concentration ranges. Limits of quantifications (LOQs) were in the range of 20–95 μg/kg. The average recoveries of three target analytes spiked at three concentration levels were in the ranges of 81.8–107.2% with relative standard deviations (RSDs) of 3.5–8.1%. Finally, the described method was proved to be suitable for the quantification of these AGEs in milk powder products.
... Deux voies de synthèse majoritaires ont été identifiées quant à la formation de la CML. Dans les aliments, il s'agit de la voie de la glycation et de celle de la glycoxydation (Ahmed, Thorpe, & Baynes, 1986;Fan, 2005;Fu et al., 1996;Thorpe & Baynes, 2002;Troise, Fiore, Wiltafsky, & Fogliano, 2015). ...
... Les échantillons ont été préparés de la même façon que pour l'analyse de l'acrylamide. Les analyses de CML ont été réalisées par un autre prestataire (Laboratoire LABS-Laboratorio Fogliano/Vitaglione, Portici, Italie) selon la méthodologie décrite dans de récents travaux (Troise et al., 2015). ...
Les revêtements perfluorés sont utilisés comme anti-adhésifs dans des moules de cuisson pour pains de mie. Ils font l’objet de diverses interrogations relatives à leur vieillissement. Dans ce contexte, la problématique de ce travail a porté sur l’étude des conséquences du vieillissement de revêtements perfluoroalkoxy (PFA) sur leurs performances anti-adhésives. De plus, les conséquences de la perte des propriétés anti-adhésives des revêtements ont été évaluées en termes de risque chimique. La production de néoformés dans la croûte de pain au contact des moules a donc été étudiée. Pour répondre à la problématique, des moules ont été vieillis en conditions industrielles, puis à partir d’un protocole mis au point spécifiquement dans le cadre de cette étude, des mesures de forces ont pu être réalisées pendant le démoulage des pains. Par ailleurs, plusieurs formules de pains de mie ont été testées. Il s’agissait d’étudier d’une part l’effet de l’état physique des lipides sur le démoulage. De plus, l’effet d’autres constituants tels que protéines et fibres a été étudié (utilisation de farine complète).Le vieillissement en conditions industrielles n’affecterait que les propriétés physiques (rugosité) des revêtements PFA. Ces modifications de surface n’ont cependant pas entraîné de problème de démoulage en fin de cuisson. Par ailleurs, les marqueurs choisis de la réactivité (furane, acrylamide et CML) évoluent peu avec le vieillissement, et ce quelle que soit la formule. La CML montre toutefois une tendance à augmenter avec le vieillissement, ce qui pourrait malgré tout indiquer un effet du vieillissement des revêtements sur la réactivité du produit.Par ailleurs, pour caractériser plus spécifiquement l’adhésion de pâtes de farine au cours de la cuisson, une méthodologie basée sur des tests d’adhérence en simulateur expérimental sur rhéomètre a été mise au point. Ces tests d’adhérence ont été effectués avec des surfaces modèles en verre, choisies pour être représentatives des propriétés chimiques des revêtements PFA. Il apparaît que les propriétés adhésives des pâtes évoluent de la même façon que les propriétés viscoélastiques en fonction de la température. Par ailleurs, les propriétés interfaciales contribuent pour une part à l’évolution d’adhésion des pâtes avec la température. Les pâtes tendent à adhérer plus facilement sur des surfaces hydrophiles. D’ailleurs, sur ces surfaces, les ruptures peuvent devenir cohésives au-delà d’une certaine température, indiquant une diminution de leur hydrophobicité de surface.
... More than 20 AGEs have been identi ed in both intra-and extracellular tissue proteins [20]. As two typical AGEs, N -(carboxymethyl)lysine (CML, Fig. 1B) and N -(carboxyethyl)lysine (CEL, Fig. 1C) are mainly generated by the reactions between lysine, methylglyoxal (MGO) and glyoxal (GO) [21,22] and are often used as biomarkers to evaluate the contents of AGEs in foods [23,24]. ...
... The CEL level in the Shuanghuanglian powder injection was the lowest (0.38 ng/mL), while the CML level in the Ciwujia injection was the lowest (0.51 ng/mL). We deduced that high protein and fat levels raised the CML and CEL contents in the Shuxuetong injection, as its components were Hirudo and Lumbricus; likewise, high levels of CML and CEL were frequently generated during milk processing [24]. Moreover, the Ciwujia injection is rich in avones, anthraquinones and other secondary metabolites that could stabilize MGO and GO to limit the generation of CML and CEL during the Maillard reaction [36]. ...
Background: Traditional Chinese medicine injections (TCMIs) are widely applied to treat many chronic diseases. However, product quality problems occur occasionally due to unknown constituents in TCMIs. 5-hydroxymethylfurfural (5-HMF), NƐ-(carboxymethyl)lysine (CML) and NƐ-(carboxyethyl)lysine (CEL) are three compounds generated during food and Chinese medicinal herb processing and may be harmful to human health.
Methods: In this study, the contents of 5-HMF, CML and CEL were determined by high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). For 5-HMF, the separation was performed on a Hypersil ODS2 column (250 mm×4.6 mm, 5 µm), and the column temperature was set at 30℃. The mobile phase was composed of water-methanol (95:5) at a flow rate of 1.0 mL/min. For CML and CEL, separation was performed on a CORTECS HILIC UPLC column (2.1 mm×50 mm, 1.6 µm), and the column temperature was set at 40℃. The mobile phase was composed of acetonitrile-water (3:7) at a flow rate of 0.3 mL/min. Multiple-reaction monitoring mode was employed for analyte determination with positive ionization.
Results: The contents of 5-HMF in 16 TCMIs varied from 0.19 to 74.98 µg/mL, with a larger variation than the contents of CML and CEL. The Ciwujia injection had the highest content of 5-HMF, and the Qingkailing injection had the lowest 5-HMF content. The contents of CML and CEL among these TCMIs were 0.51-7.32 ng·mL⁻¹ and 0.38-5.49 ng·mL⁻¹, respectively. The contents of CML and CEL in the Shuxuetong injection were much higher than in the others.
Conclusions: The methods established in this study were simple, rapid and accurate and could provide a theoretical basis for the quality evaluation of TCMIs.
... As both AGEs and protein cross-links are known to reduce the nutritional value of food proteins and are linked with metabolic disorders in vivo, it is crucial to study the extent of their formation as a function of food matrix/processing or diseases associated with protein dysfunction. A few publication records are available on the analysis of free and protein-bound AGEs by chromatographic and mass spectrometric methods in the literature [25][26][27][28][29][30] . In addition, immunological and fluorescence assays are also employed for the determination of AGEs [ 31 , 32 ]. ...
... The LOD and LOQ value of furosine, a well-known quantitative marker of early stage of Maillard reaction, was 3.18 and 9.51 ng/mL, respectively (in plasma). In a previous study, Troise and others [26] have reported similar LOD and LOQ values of 3 and 9 ng/mL, respectively, using an LC-triple quadrupole mass instrument. The LOD of most routinely investigated AGEs, CML and CEL, were 2.64 and 1.77 ng/mL (in water) or 8.46 and 0.30 ng/mL (in plasma), respectively. ...
Advanced glycation end products (AGEs) and protein cross-links have been extensively investigated in both food and biomedical fields over the past years. Although there are a few chromatographic and immunological methods for the analysis of selected AGEs, there is no method available for comprehensive simultaneous analysis of major AGEs found in processed foods and biological samples. In the present study, we have reported a validated UHPLC-MS/MS method for simultaneous identification and quantification of 15 different AGEs, furosine (an indicator of Amadori products), 2 protein-derived cross-links (lanthionine and lysinoalanine) and 2 amino acids (Lys and Arg). The analytes were separated on a reversed phase C-18 column and quantified accurately based on the isotope dilution method, where 9 stable isotope-labelled internal standards were used to quantify 20 different analytes using an Orbitrap mass analyzer. The method showed acceptable linearity, accuracy and precision. The LOD and LOQ values in plasma were in the range of 0.30-19.02 and 0.87-57.06 ng/mL, respectively. The recovery values at the three spiked levels were in the range of 71-110%, with some exceptions. The intraday and interday precision were in the range of 1.5-13.2%, however, quantification of N-ɛ-(carboxymethyl)lysine accompanied slightly higher interday precision (30.7%). The applicability of the method was successfully assessed by analyzing AGEs and protein cross-links in six different complex matrices including Ultra-High Temperature (UHT) processed milk, roasted chicken breast meat, roasted chicken skin, roasted pork liver, bovine plasma and perfusion liquid.
... Protein bound fructoselysine in high fat diet and low fat diet Protein bound fructoselysine in the two diet was indirectly quanti ed through furosine concentration according to the method of Troise et al. [73] by using a Nexera U-HPLC system coupled with a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan). In brief, a 0.5 g aliquot of each chow was added to 4 mL of HCl (7.4 M) (Thermo Fisher Scienti c). ...
Background: The human gut microbiome strongly influences host metabolism via fermentation of dietary components to metabolites that allow communication with peripheral tissues. Short chain fatty acids are among the most known microbial metabolites that signal to the host. Intestinimonas butyriciproducens is a prevalent commensal bacterium that has a unique capability of converting dietary fructoselysine to butyrate and acetate and has a completed fructoselysine catabolic pathway. Dietary fructoselysine is an abundant Amadori product formed in foods during processing and is part of food products rich in dietary advanced glycation end products which can be potentially toxic. Therefore, understanding the role of this bacterium and fructoselysine metabolism in metabolic health is highly relevant.
Results: We accessed associations of I. butyriciproducens with metabolic risk biomarkers via both strain and functional levels using a human cohort characterized by fecal metagenomic analysis. We observed that the level of the bacterial strain as well as fructoselysine fermentation genes were reversely associated with BMI, triglycerides, HbA1c and fasting insulin levels. We also investigated degradation capacity of fructoselysine within the Intestinimonas genus using a culture dependent approach and observed that I. butyriciproducens as a key player in the butyrogenic fructoselysine metabolism in the gut. To explore the function of I. butyriciproducens on host metabolism, we employed the diet-induced obesity mouse model to mimic the human metabolic syndrome. Oral supplementation of I. butyriciproducens counteracted body weight gain, hyperglycemia as well as adiposity. Moreover, within the inguinal white adipose tissue, bacterial administration reduced inflammation and promotes pathways involved in browning and insulin signaling. The observed effects are attributable to the formation of the short-chain fatty acids butyrate and acetate from dietary fructoselysine, as their plasma levels were significantly augmented by the bacterial strain, thereby contributing to systemic effects of the bacterial treatment.
Conclusions: I. butyriciproducens ameliorates host metabolism in the context of obesity and may thus be a good candidate for new microbiota-therapeutic approaches to prevent or treat metabolic diseases.
... The determination of CEL, CML, MG-H1 and furosine contents in the samples was performed according to the method of Troise et al. (2015) with modifications. In brief, a 1 mL aliquot of each model system was added to 4 mL of HCl (7.4 M) and then hydrolyzed at 110 • C, for 20 h. ...
Maillard reaction readily takes place in dairy products because of the association between thermal treatments, extended storage and the matrix composition. Along with the impairment of protein digestion, the formation of glycation and α-dicarbonyl compounds is a concern for quality attributes of whey proteins when used as ingredients. In this paper, we outline the capacity of brewer's spent grain melanoidins in reducing the accumulation of α-dicarbonyl compounds, thus controlling the formation of dietary advanced glycation end-products in accelerated shelf life at 35 °C. Results revealed that brewer's spent grain melanoidins targeted methylglyoxal and glyoxal reactivity leading to the reduction of N-ε-carboxymethyllysine and methylglyoxal-hydroimidazolone up to 27 and 60%, respectively. We here describe that the presence of melanoidins is instrumental in limiting the undesired effects of α-dicarbonyl compounds on whey proteins.
... This determination method, to 193 a certain level, had good reproducibility and high sensitivity. The detection limit was lower 194 than 5 ppb and RSD % was lower than 8 % (Troise et al., 2015). Using LC-(ESI)MS to 195 determine the AGEs contents generated by the cross linking of lysine and arginine in butter 196 biscuits and savory biscuits, the average concentrations of MODIC in butter biscuits reached 197 151 mg/kg protein (Biemel et al., 2001 can be detected and quantified by HPLC-DAD. ...
... Undeniably, the dry-heating furnishes longer stability and more ease of manipulation and preservation compared with wet-heating (Zheng et al., 2019). Currently, a myriad of studies was placed on monitoring the early glycation products derived from various glycated proteins, such as soybean products, wheat flourderived products, milk, and fish products (Troise et al., 2015;Troise et al., 2018;Bignardi et al., 2013;Artavia et al., 2018;Han et al., 2018). To the best of our knowledge, the information about characterising the protein glycation course by quantifying the formation of AGEs and the loss of amino acids in glycated proteins is relatively limited. ...
The formation of advanced glycation end products (AGEs), regarded as a potentially harmful substance, in the protein glycation course is still under investigation. This study investigated the glycation modification of silver carp myofibrillar protein (MF) with glucose via Maillard‐driven chemistry. The resultant conjugates and glycation extent were characterised by chemical indicators, such as Fourier transform infrared spectroscopy, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, colour development, and AGEs level. As the primary responsibility for glycation, the loss of Lysine (Lys) and Arginine (Arg) in MF with longer glycation time (>24 h) was more than 25%. While, the associated AGEs, including Nε‐carboxymethyl‐lysine (CML), Nε‐carboxyethyl‐lysine (CEL), and methylglyoxal‐derived hydroimidazolone 1 (MG‐H1), presented a gradually increasing tendency with glycation time. The kinetic studies indicated that the formation of CML and MG‐H1 presented a highly linear correlation with heating time, while the CEL development could be described as an exponential function of glycation time (r ≥ 0.95). Moreover, the correlation analysis among these chemical indicators indicated that colour development was positively correlated with AGEs (r ≥ 0.85), while negatively related to the loss of Lys and Arg (r ≤ −0.89). The findings may help to better control the glycation course of food protein based on the formation of AGEs.
... During storage, CEL levels increased by a factor of 3 for both DI-IF and IN-IF (Fig. 3), but were 3-4 times lower compared to CML concentrations. To the best of our knowledge, CEL has not been quantified in liquid IF before, but CML has generally been reported in higher values than CEL in many food products (Akillioglu & Lund, 2022;Troise, Fiore, Wiltafsky, & Fogliano, 2015;, which is in agreement with the CEL and CML levels detected in the present study. ...
The formation of Maillard reaction products, including Amadori compounds (determined as furosine), advanced glycation end products (AGEs), α-dicarbonyl and furfural compounds, as well as amino acid cross-links (lysinoalanine and lanthionine) was investigated in direct (DI) and indirect (IN) UHT-treated experimental liquid infant formula (IF) during storage at 40 °C. IN-IF had higher concentrations of all investigated compounds compared to DI-IF and low pasteurized IF. IN UHT treatment induced significantly higher concentrations of α-dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone and 3-deoxygalactosone) compared to DI, which facilitated increased formation of AGEs (N-Ɛ-(carboxymethyl)lysine, methylglyoxal- and glyoxal-derived hydroimidazolones) in unstored IFs. During storage for 6 months, concentrations of furosine and AGEs increased while α-dicarbonyl compounds decreased. Principal component analysis indicated that differences between IN-IF and DI-IF disappeared after 2 months of storage. IN-IF had higher concentrations of lysinoalanine and lanthionine and lower concentrations of available lysine and arginine than DI-IF indicating higher loss of protein quality in IN-IF.
... The recoveries of analytes were determined in triplicate at least three times for every examined condition. As reported previously, CML and CEL could be formed from reactive lipid oxidation products such as glyoxal and methylglyoxal, 34 and thus, the presence of lipid might cause CML and CEL formation during acid hydrolysis at 110°C. The defatting treatment of the sample was evaluated by measuring the chromatographic peak area of target analytes in treated samples with and without the defatting step. ...
In this work, a stable isotope dilution ultrahigh-performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated for simultaneous determination of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), and acrylamide (AA) in baked and fried foods. Ground food samples were extracted with acetone followed by two parallel assays. In assay A, a cleanup procedure based on dispersive solid-phase extraction was conducted for AA, free CML, and CEL analysis using the supernatant. In assay B, a multistep process including reduction, protein precipitation, acid hydrolysis, and solid-phase extraction was conducted for bound CML and CEL analysis using precipitation. The developed method was validated in terms of linearity, sensitivity (limit of detection, LOD; limit of quantitation, LOQ), accuracy, and precision. The results showed that the method had a wide linear range (0.25-500 ng/mL for CML and CEL, 0.5-500 ng/mL for AA), low LOD and LOQ (0.47-0.94 and 1.52-1.91 μg/kg, respectively), and good linearity (R2 > 0.999). The recovery test on baby biscuit and French fries samples showed the recovery rates of 90.2-108.3% for CML, 89.0-106.1% for CEL, and 94.5-112.3% for AA with satisfactory precision (relative standard deviation (RSD) < 10%). Finally, the developed method was successfully applied to 11 baked and fried food samples, and total CML, CEL, and AA contents varied in the ranges of 4.07-35.88 mg/kg, 1.99-14.49 mg/kg, and 5.56-506.64 μg/kg, respectively. Therefore, the isotope dilution UHPLC-QqQ-MS/MS method developed herein is promising for routine analysis of CML, CEL, and AA in baked and fried foods.
... CML, CEL, MG-H1 and furosine were determined according to Troise, Fiore, Wiltafsky, and Fogliano (2015) with some modifications and the furosine content was multiplied by a factor of 3.1 to calculate the amount of N-ε-fructosyllysine (Krause, Knoll, & Henle, 2003). Briefly, an aliquot of the WP/glucose, WP/GO and WP/MGO model systems (0.75 mL) was mixed with 3.25 mL of HCl (7.4 M) and then heated at 110 • C for 20 h. ...
The control of Maillard reaction in foods is important to preserve protein nutritional quality. In this study, we investigated the effects of melanoidins obtained from different roasted cocoa beans toward the formation of dietary advanced glycation end-products (d-AGEs) in aqueous solution of whey protein (WP) and glucose, glyoxal and methylglyoxal at 35 °C and pH 7.0. Cocoa melanoidins (4 mg/mL) were more effective to inhibit glyoxal-derived d-AGEs than methylglyoxal-derived d-AGEs, with 74.4% and 48% reduction of N-ε-carboxymethyllysine and methylglyoxal-hydroimidazolone formation in WP/glyoxal and WP/methylglyoxal system, respectively. Furthermore, protein-bound lysine Amadori compound, measured through furosine, decreased down to 57.2% in presence of cocoa melanoidins in WP/glucose model system suggesting an effective control of the Maillard reaction in an early stage. These findings highlighted that cocoa melanoidins are functional ingredients able to mitigate protein glycation in dairy products during storage.
... Greifenhagen et al. found that Amadori modifications are potent CML precursors and can be efficiently transformed to CML within minutes during cooking [36]. However, CEL was mainly generated by the reaction of MGO with Lys residues [37]. The amount of protein-bound CML formed by the glycation of β-conglycinin with d-glucose at 180 °C for 7 min is 3.36 times higher than that formed at 100 °C for 30 min and 3.20 times higher than that formed at 121 °C for 15 min. ...
Glycation often occurs during the thermal processing of food, and may result in changes to the structure of proteins and decreases in nutritional value. A model system consisting of β-conglycinin and sugars (d-glucose, lactose) was used to investigate the effects of glycation on the structure of β-conglycinin and the formation of advanced glycation end products during the thermal processing of food. The results show that both d-glucose and lactose can modify β-conglycinin during thermal processing, including during boiling, sterilizing, and baking. As the degree of thermal processing increased, the molecular weight of glycated β-conglycinin increased significantly, and the secondary structure gradually changed from α-helix and β-sheet structures to random coil. The contents of free lysine (Lys) and arginine (Arg) residues in glycated β-conglycinin decreased along with the formation of protein-bound Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL). In addition, the major types of modification indicated by high-resolution mass spectrometry were d-glucose adduct, CML and CEL on Lys residues, and methylglyoxal-derived hydroimidazolone on Arg residues. The higher processing temperatures resulted in greater numbers of modification sites and greater contents of protein-bound AGEs.
... In order to obtain sufficient amount of substrate, FL was synthesized as previously reported (Troise, Wiltafsky, & Fogliano, 2015) starting from Fmoc-α-protected L-lysine that reacted specifically on its epsilon amino moiety with D-glucose in refluxed methanol and subsequently de-protected under basic conditions to afford the desired product. The identity, isotopic distribution and the elemental analysis (C 12 H 24 N 2 O 7 ) of the obtained FL was checked by direct infusion (3 µL/min) onto high resolution mass spectrometer (Exactive, Thermo Fisher, Bremen, Germany). ...
Our study aim is to investigate the role of Intestinimonas, Nε-fructosyllysine (FL)-degrading bacterium, in infants and adults. We used lysine and subsequently FL in anaerobic serial dilutions of stools of infants and adults to enrich lysine and FL-degrading species. The fecal microbiota of adults were able to ferment lysine and FL to butyrate. Different groups of Intestinimonas spp. were detected from all lysine enrichments whereas the FL enrichments consisted of broader taxonomic groups with a reduced abundance of Intestinimonas-related species. Remarkably, the capability to degrade FL was only observed in formula-fed but not in breast-fed infants, which may relate to high contents of FL in formulas after thermal treatment. This possibility was supported by analyzing metagenomic datasets of 3-month and 4-month infants. Our data indicate the key role of Intestinimonas-like bacteria in FL degradation in formula-fed infants and adults as a profound example of adaptation of intestinal bacteria to dietary components.
... In order to monitor CML, LYS and other MRPs, the UHT milk samples were treated according to the protocol of Troise, Fiore, Wiltafsky, and Fogliano (2015). Acidic hydrolysis was performed by adding 100 µL of milk sample and 4 mL of 6 M HCl to a screw capped flask with a PTFE septum. ...
Both the Maillard reaction (MR) and thermal treatment influence the nutritional value of milk. In this paper, the capability of polyphenolic berry extract (PBE) to inhibit MR in an ultra-high temperature (UHT) treated milk was investigated. Total polyphenol content and antioxidant capacity of blueberry (BE) and raspberry extracts (RE) were also tested. A gas chromatography-mass spectrometry (GC–MS) method was developed to monitor the MR product N ε-(carboxymethyl)-L-lysine (CML) and L-lysine (LYS). PBE was added to milk at 0.05 and 0.1% w/v prior to UHT processing. Data revealed that formation of CML was significantly reduced (23.4 ± 5.1%) by addition of 0.1% w/v BE. The final concentrations of LYS measured following the addition of PBE prior to thermal treatment were statistically similar to the control milk which was not subjected to thermal processing. Additionally, the metabolic profile of milk samples was investigated by GC–MS and visualised using ‘FancyTiles’.
... Nevertheless, extrusion tends to increase furosine content in soy. At least two research groups have evaluated furosine generation in soybean-based feed products obtained under severe thermal treatment conditions and reported values of 66.55 ± 0.37 and 108.01 ± 8.97 mg/100 g protein [61][62][63]. Our data (Table 3) is in agreement with the results by Chiang [63,64] in dry dog food stored for 12 weeks at 37.8 • C. Increased concentrations of furosine are undesirable in pet food as they indicate impaired lysine utilization by the animal and compromised weight gain [65]. ...
Costa Rican animal feed production is continually growing, with approximately 1,238,243 metric tons produced in 2018. Production-wise, pet cat and dog food are in fifth place (about 41,635 metric tons per year) amongst animal feeds, and it supplies up to 90% of the national market. Pet food production has increased as a response to the increase in the population of dogs and cats in Costa Rica, where 50.5% of households own at least one dog and indicates more responsible ownership in terms of feeding pets. Part of the process of making dry pet food involves a thermal process called extrusion, which is capable of eliminating the microbial load. However, extrusion can compromise nutritional quality to some extent by denaturing proteins, oxidizing lipids, and reducing digestibility. The objective of this study was to evaluate the quality and safety of dry pet food and to assess the effect of the extrusion process on digestibility and the quality of proteins, amino acids, and fatty acids. Pet food samples were collected before and after extrusion and were used to evaluate Good Manufacturing Practices (GMP), based on Central American Technical Regulation (RTCA 65.05.63:11). In general terms, weaknesses in infrastructure, documentary evidence, and post-process practices were observed in two Costa Rican feed manufactories. Feed safety was surveyed through the analysis of Salmonella spp., Escherichia coli, Listeria spp., Staphylococcus aureus, aerobic mesophilic microorganisms, fungi, and yeasts counts. The extrusion process effectively reduced pathogenic microorganisms, and showed no effect on the digestibility of dog food (p = 0.347), however, it could reduce the availability of some nutrients (e.g., amino acids, fatty acids). Furthermore, a retrospective diagnosis was made for puppy food (n = 68), dog food (n = 158), and cat food (n = 25), to evaluate the history of nutritional quality and safety. Finally, it can Animals 2019, 9, 980 2 of 25 be confirmed that the correct implementation of GMP allows feed manufacturers to deliver a product of optimum texture, smell, nutritional composition, and safety.
... N-ε-carboxyethyllysine (CEL), which is formed by the reaction between methylglyoxal and lysine, is a homolog of CML. Its content may be determined after acid hydrolysis or enzymatic hydrolysis by HPLC and LC-MS/MS methods [7,81,84,101,119,120]. ...
Glycation refers to the addition of a sugar moiety to a protein molecule and occurs during the Maillard reaction. Maillard reaction is initiated by the condensation of amino groups of proteins, peptides, and amino acids with carbonyl groups of reducing sugars. After subsequent elimination, degradation, and oxidation reactions in the later stages, so-called advanced glycation end products (AGEs) are formed. AGEs have been linked to many chronic and degenerative disorders and aging. The results of several animal and human studies confirm that dietary AGE levels have effects on AGE accumulation in body and further complications in degenerative diseases. In this chapter, following brief information about protein glycation, the consequences of glycation, and the contribution of dietary AGEs will be discussed. Formation routes of main glycation products in foods will be explained and their analysis methods will be summarized. Major mitigation strategies developed so far will be evaluated.
... [5,6]. Higher furosine concentrations have been reported in infant formulas which ranged from 471.9 mg/100 g to 639.5 mg/100 g [7][8][9]. Particularly, considering furosine is a by-product in heat treatment of food, it is widely used as a marker of protein loss and food nutritional quality [10]. ...
As one of the Maillard reaction products, furosine has been widely reported in a variety of heat-processed foods, while the toxicity of furosine on the reproductive system and related mechanisms are unclear. Here, we constructed an intragastric gavage male mice model (42-day administration, 0.1/0.25/0.5 g furosine/Kg body weight per day) to investigate its effects on mice testicle index, hormones in serum, and mice sperm quality. Besides, the lipid metabonomics analysis was performed to screen out the special metabolites and relatively altered pathways in mice testicle tissue. Mice primary sertoli cells were separated from male mice testicle to validate the role of special metabolites in regulating pathways. We found that furosine affected testicle index, hormones expression level and sperm quality, as well as caused pathological damages in testicle tissue. Phosphatidylethanolamine (PE) (18:0/16:1) was upregulated by furosine both in mice testicle tissue and in primary sertoli cells, meanwhile, PE(18:0/16:1) was proved to activate Cep55/NF-κB/PI3K/Akt/FOX01/TNF-α pathway, and as a functional protein in dairy products, lactoferrin could inhibit expression of this pathway when combined with furosine. In conclusion, for the first time we validated that furosine posed toxic effects on mice sperms and testicle tissue through upregulating PE(18:0/16:1) and activating Cep55/NF-κB/PI3K/Akt/FOX01/TNF-α pathway.
... [14]. Similarly, Troise et al. reported the CML concentration of 80-140 mg /kg protein regarding commercial powder infant formulas [30]. As it was explained before, infant formulas show higher sensitivity towards the Maillard reaction, and this is mainly due to their composition which mimics the composition of human milk as well as the application of rigorous heat treatments. ...
Although the literature on the Maillard reaction in infant formulas is extensive, most studies have focused on model systems, and in only a few cases on real food systems. Therefore, the objective of the present study was to determine the status of the Maillard reaction, both the early and advanced phases, in a variety of commercial infant formulas available on the Swedish market. Ten powder and liquid milk-based infant formulas from three manufacturers were selected to determine available lysine and CML contents, the two established indicators of the reaction. The products were also characterized with respect to protein content, carbohydrates composition, water content and water activity. In order to be able to compare the impact of different processing steps applied on powder and liquid formulas, the solid formulas contained similar ingredients as their corresponding liquid ones. Our findings showed that powder and liquid formulas contained similar available lysine concentrations regardless of the manufacturer, showing 27.14–36.57% decrease in the available lysine, compared to the reference skim milk powder in this study. The CML concentrations were in a broad range of 68.77–507.99 mg / kg protein. In the case of one manufacturer, liquid infant formulas had significantly higher CML content, compared to the powder products (p < 0.05). The results from this study are a step taken towards better understanding of the extent of the Maillard reaction in real complex systems of infant formulas.
... 24 The compounds were eluted at 300 μL/min through the following Analytical performances such as robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, carryover, and matrix effects were evaluated by following the procedures previously reported. 25 Typical retention times were 4.5 and 5.3 min for lysine and CML, respectively. Quantitation of CML and lysine was performed by using the internal standard technique, and results are reported as mmol/L. ...
Modifications of lysine contribute to the amount of dietary advanced glycation end-products reaching the colon. However, little is known about the ability of intestinal bacteria to metabolize dietary N-ε-carboxymethyllysine (CML). Successive transfers of fecal microbiota in growth media containing CML were used to identify and isolate species able to metabolize CML under anaerobic conditions. From our study, only donors exposed to processed foods degraded CML and anaerobic bacteria enrichments from two of them used 77% and 100% of CML. Oscillibacter and Cloacibacillus evryensis increased in the two donors after the second transfer highlighting bacteria from these taxa could be candidates for anaerobic CML degradation. A tentative identification of CML metabolites produced by a pure culture of Cloacibacillus evryensis was performed by mass spectrometry: carboxymethylated biogenic amines and carboxylic acids were identified as CML degradation products. The study confirmed the ability of intestinal bacteria to metabolize CML under anoxic condition.
... As one of the typical MRPs [1], furosine (C 12 H 18 N 2 O 4 , 254.28 g/mol) was identified as ε-N-(2-furoylmethyl)-l-lysine in 1968 [2,3]; it is broadly found in a variety of foods including dairy products, cereals, honey, bakery products, etc., and its content is directly related to the degree of heating treatment and to the storage time [4]. Furosine was proved to be one of the most stable derivatives of the Amadori compounds and is also well known as a reliable marker and indicator of the nutritional value of heat-treated food, which always reflects the degree of protein loss and freshness [5,6]. ...
As one of the typical Maillard reaction products, furosine has been widely reported in a variety of heat-processed food. Though furosine was shown to be toxic on organs, its toxicity mechanism is still unclear. The present study aimed to investigate the toxicity mechanism of furosine in liver tissue. An intragastric gavage mice model (42-day administration, 0.1/0.25/0.5 g/kg of furosine per day) and a mice primary hepatocyte model were employed to investigate the toxicity mechanism of furosine on mice liver tissue. A metabonomics analysis of mice liver, serum, and red blood cells (RBC) was performed. The special metabolic mediator of furosine, lysophosphatidylcholine 18:0 (LPC (18:0)) was identified. Then, the effect of the upstream gene phospholipase A2 gamma (PLA2-3) on LPC (18:0), as well as the effect of furosine (100 mg/L) on the receptor-interacting serine/threonine-protein kinase (RIPK)1/RIPK3/mixed lineage kinase domain-like protein (MLKL) pathway and inflammatory factors, was determined in liver tissue and primary hepatocytes. PLA2-3 was found to regulate the level of LPC (18:0) and activate the expression of RIPK1, RIPK3, P-MLKL, and of the inflammatory factors including tumor necrosis factor α (TNF-α) and interleukin (IL-1β), both in liver tissue and in primary hepatocytes. Upon treatment with furosine, the upstream sensor PLA2-3 activated the RIPK1/RIPK3/MLKL necroptosis pathway and caused inflammation by regulating the expression of LPC (18:0), which further caused liver damage.
... 24,25 The presence of AGEs has also been demonstrated in IFs. 22,26 Elevated levels of modified protein species (e.g., di-Tyr, DOPA, 3-hydroxytyrosine, protein carbonyls, alcohols, and AGEs) have been detected in IFs (e.g., see refs 6, 19, and 27−29), as well as in other milk-protein preparations and in a wide range of foods (e.g., see refs 29 and 30). These species have also been detected in diseased and aged human tissues (reviewed in refs 5 and 31), but whether and how dietary sources contribute to these pools is not fully understood. ...
Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study markers of glycation, furosine (1.7-3.5 µg mg-1 protein) and Nɛ-(carboxymethyl)lysine (28-81 ng mg-1 protein), were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine pre-adipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side-chain oxidation and glycation products (1 and 10 µM), resulted in a loss (up to 40 %; p<0.05) of cell thiols and decreased metabolic activity compared with controls. In contrast, Caco-2 cells showed a stimulation (10-50 %; p<0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% (p<0.05) increase in protein carbonyls was detected on incubation with 200 µM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.
Glycation enhances plant protein techno-functionality; however, digestibility and the equilibrium between peptides absorbed and those reaching the colon can be altered. This study evaluated how undigested glycated lentil proteins, potentially reaching the colon affect the gut microbiota using batch fermentation and the Simulator of Human Intestinal Microbiome Ecosystem (SHIME®). Lentil protein-fructose mixtures were incubated at 60 °C for 0, 24, or 48 h (conjugates labelled LP+Fr0, LP+Fr1, LP+Fr2). Maillard reaction markers increased by over 10-fold and in vitro protein digestibility decreased by 23.5 % after 48-h incubation. Short- and branched-chain fatty acids produced by 48 h-fermentation of the insoluble hydrolysates of conjugates were comparable. LP+Fr2 hydrolysates caused 42 % relative increase in Bacteroidetes in the proximal colon of Donor 1 whereas 26 % increase was observed with LP+Fr0 hydrolysates. Bacteria population profile in the colon sections was differentially modulated depending on the donor. Our findings show that the extent of glycation does not affect short- and branched-chain fatty acid levels produced in the colon, while the effect on microbiota population is dependent on host and colon section.
The prevalence of diabetes mellitus is increasing in resource limited settings. Simultaneously, there has been an increase in the number of novel therapies for the management of diabetes mellitus. However, use of novel antidiabetic therapies is limited because of major market access challenges in resource limited settings. Niching products to those patients with the highest absolute risk for major adverse cardiovascular outcomes, and thus most likely to benefit from the therapy, are less likely to have negative budget impact for funders. To improve access, and reduce morbidity and mortality, requires alignment amongst key stakeholders including patient advocacy groups, health care professional councils, national departments of health, the pharmaceutical industry, treasury and finance departments.
Objective:
The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N-ε-carboxymethyl-L-lysine (CML) in vitro, testing N-ε-carboxy[D2]methyl-L-lysine (d2-CML), and N-ε-carboxy[4,4,5,5-D4]methyl-L-lysine (d4-CML) as internal standards.
Method:
During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated.
Result:
A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented.
Conclusion:
Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way.
Brown fermented milk (BFM) is favored by consumers in the dairy market for its unique burnt flavor and brown color. However, Maillard reaction products (MRPs) from high temperature baking are also noteworthy. In this study, tea polyphenols (TP) were initially developed as potential inhibitors of MRPs formation in BFM. The results showed that the flavor profile of BFM did not change after adding 0.08% (wt/wt) of TP, and its inhibition rates on 5-hydroxymethyl-2-furaldehyde (5-HMF), glyoxal (GO), methylglyoxal (MGO), Nε-carboxymethyl lysine (CML) and Nε-carboxyethyl lysine (CEL) were 60.8%, 27.12%, 23.44%, 57.7% and 31.28%, respectively. After 21 d of storage, the levels of 5-HMF, GO, MGO, CML and CEL in BFM with TP were 46.3%, 9.7%, 20.6%, 5.2%, and 24.7% lower than the control group, respectively. Moreover, there was a smaller change in their color and the browning index was lower than that of the control group. The significance of this study was to develop TP as additives to inhibit the production of MRPs in brown fermented yogurt without changing color and flavors, thereby making dairy products safer for consumers.
Advanced glycosylation end products (AGEs) are a series of complex compounds which generate in the advanced phase of Maillard reaction, which can pose a non-negligible risk to human health. This article systematically encompasses AGEs in milk and dairy products under different processing conditions, influencing factors, inhibition mechanism and levels among the different categories of dairy products. In particular, it describes the effects of various sterilization techniques on the Maillard reaction. Different processing techniques have a significant effect on AGEs content. In addition, it clearly articulates the determination methods of AGEs and even discusses its immunometabolism via gut microbiota. It is observed that the metabolism of AGEs can affect the composition of the gut microbiota, which further has an impact on intestinal function and the gut-brain axis. This research also provides a suggestion for AGEs mitigation strategies, which are beneficial to optimize the dairy production, especially innovative processing technology application.
Thermal treatment is a key step during infant formula (IF) processing which causes protein glycation and formation of dietary advanced glycation end-products (dAGEs). This study aimed to evaluate the glycation degree in IF in relation to the ingredients of the formula. dAGEs concentrations have been determined by UPLC-MS/MS in a range of commercial cow-based, goat-based, and soy-based IF. Results indicated that the protein source, protein composition, and amount and type of carbohydrates determines the level of protein glycation in IFs. The investigated soy-based formula had significant higher concentrations of arginine and arginine-derived dAGEs than cow-based and goat-based formulas. IF containing hydrolyzed proteins had higher dAGEs concentrations than those containing intact proteins. Lactose-containing formula was more prone to glycation than those containing sucrose and maltodextrin. Data showed glycation degree in IF cannot be estimated by a single compound, but the complete picture of the dAGEs should be considered.
The advanced glycation end products (AGEs) are formed in baked products through the Maillard reaction (MR), which are thought to be a contributing factor to chronic diseases such as heart diseases and diabetes. Lotus seedpod oligomeric procyanidins (LSOPC) are natural antioxidants that have been added to tough biscuit to create functional foods that may lower the risk of chronic diseases. The effect of LSOPC on AGEs formation and the sensory quality of tough biscuit were examined in this study. With the addition of LSOPC, the AGEs scavenging rate and antioxidant capacity of LSOPC-added tough biscuits were dramatically improved. The chromatic aberration (ΔE) value of tough biscuits containing LSOPC increased significantly. Higher addition of LSOPC, on the other hand, could effectively substantially reduced the moisture content, water activity, and pH of LSOPC toughen biscuits. These findings imply that using LSOPC as additive not only lowers the generation of AGEs, but also improves sensory quality of tough biscuit.
The occurring of glycation reaction and protein-protein interaction in the energy appetizers caused browning and hardness instability while storing these appetizers, leading to the loss of consumer acceptability. Amassing among anthocyanins and proteins could mitigate the appetizers' instability. We, therefore, investigated the anti-aggregation and ant-glycoxidation impacts of mulberry anthocyanins combined with ultrasonic treatment (UT) pre-texturization in an energy appetizer model throughout storage via multi-dimensional methods, containing UPLC-ESI-MS/MS, SDS-PAGE, FTIR, texture analyser, and a molecular docking analysis. Results noted that UT-anthocyanins significantly downgraded the browning progress, advanced glycation end-products, and/or N-(carboxymethyl)-l-lysine intensities of energy appetizers after 45 d of storage at 45 °C. UT-anthocyanins also relegated the protein insolubility, accumulation, oligomerization, and glycoxidation throughout the late storage. A molecular docking analysis evidenced that cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside networked with β-lactoglobulin subunits via H-bonding and π-π forces. This binding hindered some glycoxidation residues of β-lactoglobulin the lysyl residues. Finally, these findings recommended that the UT-anthocyanins could be employed as an encouraging antiglycative approach to alleviate AGEs-creation and other consequent undesirable fluctuations in protein-rich food patterns, thereby enhancing the energy appetizer's post-processing stability during storage.
A kinetic model was proposed by using a multiresponse kinetic modeling approach for Maillard and caramelization reactions in low moisture foods at neutral pH containing a moderate amount of amino acid and sucrose but a restricted amount reducing sugar. The change in the amount of sucrose, protein-bound lysine, free amino acids, and certain products of Maillard reaction was monitored during roasting of sunflower seed, pumpkin seed, flaxseed, peanut, and almond at 160 and 180 °C. A slightly different model was proposed for pumpkin seed due to its difference in compositional and physicochemical characteristics as expressed by principal component analysis. Accordingly, 3-deoxyglucosone formation via sugar degradation; 5-hydroxymethylfurfural formation from 3-deoxyglucosone and only in pumpkin seeds the conversion of N-ε-fructoselysine to glyoxal and Heyns product to 1-deoxyglucosone were found to be quantitatively important. N-ε-carboxymethyllysine and N-ε-carboxyethyllysine mainly originated through oxidation of N-ε-fructoselysine and the reaction of methylglyoxal with lysine residue, respectively.
Advanced glycation end products (AGEs) are a diverse group of compounds formed endogenously and exogenously due to non-enzymatic glycation of proteins and lipids. Although the effects of heating on AGE concentrations in foods are known, few studies have been published addressing the effects of new processing technologies on AGE formation. This work focuses on the current scientific knowledge about the impacts of novel technologies on AGE formation in food products. Most studies do not measure AGE content directly, evaluating only products of the Maillard reaction. Moreover, these studies do not compare distinct operational conditions associated with novel technologies. This lack of information impacts negatively the establishment of process–composition relationships for foods with safe AGE dietary intakes. Overall, the outcomes of this review suggest that the use of novel technologies is a promising alternative to produce food products with a lower AGE content.
The impact of lactose to whey protein ratio on processed-induced protein modifications was investigated in powdered infant formula. Model samples were prepared with different lactose to whey protein ratios and different total protein concentrations; protein modifications were evaluated before, during and after processing. Lab-scale equipment was used to mimic commercial manufacturing, and a powdered young-child formula was included to elucidate cumulative effects of commercial unit operations. Maillard-related and structural protein modifications were affected differently by unit operations, both in model samples and commercial formula. Maillard reaction products (furosine, α-dicarbonyls and advanced glycation end products) increased with increased lactose content of model samples, whereas absence of lactose facilitated formation of disulphide-linked aggregates, lysinoalanine and lanthionine. The lowest level of protein modifications was observed at a 30:70 lactose to whey protein ratio, suggesting partial dry blending of lactose as a feasible approach to improve protein quality in powdered infant formula.
Abstrac
The aim of this study was to investigate the occurrence of Maillard reaction products of different types of commercially available cheeses. Cheeses are the commonly consumed dairy products characterized by their many different flavors, textures and aromas. Pasteurization, fermentation and ripening are the key steps of cheese manufacturing, which might lead to the formation of some harmful products. In this study, furosine, furfurals, two “representative” advanced glycation end products (AGEs) were analyzed by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). The content of furosine in cheeses were the highest, followed by AGEs, while levels of furfurals were relatively low. The contents of these Maillard reaction products in hard and semi-hard cheeses were significantly higher than those in soft and semi-soft cheeses. Finally, the correlation between proximate composition and Maillard reaction products was further analyzed.
Various harmful Maillard reaction products such as lactulosyl-lysine (furosine), furfurals, and advanced glycation end products (AGEs) could be formed during the thermal processing of dairy products, which could lead to various chronic diseases. In this review, the furosine, furfurals, and AGEs formation, occurrence, analysis methods, and toxicological and health aspects in various dairy products were summarized to better monitor and control the levels of harmful Maillard reaction products in processed dairy products. It was observed that all types of dairy products, including raw milk, contain harmful Maillard reaction products, with the highest in whey cheese and condensed milk. High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the common method for the determination of furosine and furfurals and AGEs in dairy products, respectively. However, the simple, rapid, environment-friendly, and accurate methods of determination are still to be developed. Incorporating resveratrol, pectin oligosaccharides (POS) in milk are effective methods to inhibit AGEs formation. This review provides a guide not only for consumers regarding the selection and consumption of dairy products, but also for monitoring and controlling the quality of dairy products.
Infant formula contains thermal processing contaminants, such as dietary advanced glycation end‐products (dAGEs), glycidyl esters (GEs), 2‐monochloropropane‐1,3‐diol esters and 3‐monochloropropane‐1,2‐diol esters (MCPDEs). This systematic review aimed to gain insights into the occurrence of these contaminants in different types of infant formula, to understand potential effects of the formulation and processing of infant formulas on these contaminants, as well as into possible mitigation strategies. The occurrence of dAGEs in infant formula depends on the recipes and processing conditions. Hydrolyzed protein formulations promote dAGEs formation in infant formula since peptides are more prone to glycation than intact proteins, which is reflected in high dAGEs concentration in hypoallergenic infant formula. Different carbohydrates in recipes result into different glycation extents of infant formula: maltodextrin containing formulas contained less dAGEs than those with lactose. Concerning mitigation strategies, applying ultra‐high‐temperature (UHT) treatment during milk processing leads to less dAGEs formation than using in‐bottle sterilization. Although data are limited, evidence showed that encapsulation of raw ingredients or the use of antioxidants or enzymes in recipes is promising. The occurrence of MCPDEs and GEs in infant formula fully depends on the vegetable oils used in the recipe. High levels of these contaminants can be found when relatively high amounts of palm oils or fats are used. The mitigation of MCPDEs and GEs should therefore be performed on fats and oils before their application to infant formula recipes. Data and knowledge gaps identified in this review can be useful to guide future studies.
Drying is a widely used technique in food manufacturing to preserve food stability by minimizing physical and chemical changes during storage. In dried pasta production, drying is a crucial operation for the quality of the end-product. The traditional drying methods use low temperature (less than 60 °C) and long treatment times (for up to 60 h depending on the pasta shape); but on large-scale retail trade production, the high/very high temperature (75–100 °C or ≥ 100 °C) and short-time (for 10 to 3 h) drying processes are widely employed. A lot of studies prove that drastic heat treatment promotes the Maillard reaction causing changes in the final nutritional and organoleptic properties of pasta. Furosine, an amino acid derived from the acid hydrolysis of Amadori compounds formed during the early stage of the Maillard reaction, has been extensively used as a marker of the heat damage. This review aims to carry out an in-depth investigation of the scientific literature about the role of furosine as a marker of quality. Particularly, to valorise on the market durum wheat dried pasta that has not undergone to thermal stress and also to identify potential commercial fraud. Additionally, volatile compounds, formed during the advanced and final stage of Maillard reaction and responsible for several food properties, were considered due to the possibility to use them in combination with furosine for food security and food quality purposes.
Because the conventional methods for furosine analysis are time-consuming, a modified method is presented to improve analysis efficiency. Microwave-assisted HCl hydrolysis was performed at 140, 150, and 160°C for 10 to 200 min, with 6, 8, or 9 M HCl. The hydrolysate purification process was carried out using only paper and membrane filtration. An ultra-performance liquid chromatography (UPLC) system was used to achieve rapid analysis of furosine. The results showed that microwave-assisted HCl hydrolysis at 8 M and 160°C led to a stable furosine yield and took only 40 min. The UPLC analysis was completed in 8 min. The modified method was validated and obtained limit of detection at 3 µg/L, limit of quantitation of 10 µg/L, linearity range of 0.2 to 5.0 mg/L, 80.5 to 94.2% recoveries from spiked samples, and coefficients of variation of 2.2 to 6.8%. The modified method is rapid and reliable for the determination of furosine in milk.
In the acute toxicity model, 30 mice were randomly divided into 6 groups, control (without any treatment) and furosine-treated groups (4 h and 12 h group), by both orally administeration and tail vein injection. Furosine with the dosage of 0.24 g/kg body weight (b.w.) was administrated into the mice, and the liver and kidney tissue were dissected out. Organ index and biochemical indicators were obtained, and the concentration of furosine in liver and kidney tissue was quantified. Comparing with the control, furosine caused significant changes of biochemical indicators in liver and kidney tissue in 4 h and 12 h groups. This study for the first time provided evidences that furosine with the dosage of 0.24 g/kg posed acute adverse biological effects on the health of animals, and suggested that liver and kidney were the toxicity target organs.
The correlation between potato components and Maillard reaction derivative harmful products (MRDHPs) formation during heat-processing was assessed in nine commercial potato varieties in China. Principal component analysis (PCA) combined with canonical correlation analysis (CCA) approach was performed to explore their relationships. The variables contributing most to the PCA results were extracted for CCA, and the results indicated that several amino acids, including lysine, tryptophan, alanine, phenylalanine, aspartate, and glutamate, have significant impacts on acrylamide and β-carboline heterocyclic amine formation. Moreover, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, α-solanine, and α-chaconine were also important factors associated with acrylamide and β-carboline heterocyclic amine formation. Optimally using raw potato varieties based on the impacts of these factors can help control MRDHP formation during thermal processing. For the first time, such approach was applied, which may be a useful tool for discovering the correlation of food components and MRDHPs.
A rapid ultra-performance liquid chromatography (UPLC) method with simplified sample preparation procedures was developed for detection of furosine in milk. In tuning the sample preparation, nitrogen purging was not required for acid hydrolysis and the duration of acid hydrolysis was shortened. Purification by solid-phase extraction was also found to be unnecessary. UPLC parameters were optimized. The retention time of furosine was 1.866 min, with good separation from impurities in milk. The method was validated and shown to have good sensitivity, accuracy, precision, repeatability, and reproducibility. The limit of detection was 0.01 mg/L and the limit of quantification was 0.025 mg/L. The range of linearity was 0.5–5.0 mg/L (R² = 0.9998). Milk samples spiked with different furosine levels showed recoveries of 83.96%–93.51%, with an overall mean recovery of 89.68 ± 0.06%. The coefficients of variation for intraday, inter-day, and inter-laboratory determinations were 0.2%–1.6%, 0.5%, and 4.0%–8.1%, respectively. The proposed method was utilized to quantify furosine in pasteurized, extended shelf life, and ultra-high-temperature-treated milk samples, which demonstrated that this novel UPLC method is effective for rapid determination of furosine in milk samples.
α-Dicarbonyl compounds (α-DCs), which act as precursors for advanced glycation end products in foods and in vivo, are toxic compounds formed from carbohydrates via caramelization and the Maillard reactions during thermal processing of foods. This chapter introduces the species, formation pathways, and analytical methods of α-dicarbonyl compounds, and their occurrence in foods. Their mitigation strategies in foods are also discussed.
The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.
Foods are often heat processed and may contain advanced glycation end products (AGE). One of the most widely studied AGE is N ϵ-(carboxymethyl)lysine (CML); nevertheless, knowledge on dietary CML is fragmentary. This study aimed to review current scientific knowledge on analytical methods to determine CML contents in food, chemical pathways of CML formation in food, occurrence of CML in food, and health implications of dietary exposure to CML. Chemical analyses of CML in food products are carried out by immunochemical assays and instrumental methods, but the former method may interfere with the food matrix. CML is formed in food through various chemical pathways, depending on food ingredients and processing conditions. The compound is present in many cooked foods, with relatively high concentrations in carbohydrate-rich foods and dairy products. Dietary CML is very likely to impair human health, but full cause-effect evidence is not available yet. More studies on metabolic effects and impact of food-derived CML on human health should be performed. Food production should be optimized to minimize CML concentrations, while maintaining acceptable microbiological safety and organoleptic properties of the final food product. To this end, more insights into effects of food composition and processing conditions on CML formation are necessary.
The formation of the Amadori products
(APs) is the first key step of Maillard reaction. Only few papers have dealt with simultaneous quantitation of amino acids and corresponding APs (1-amino-1-deoxy-2-ketose). Chromatographic separation of APs is affected by several drawbacks mainly related to their poor retention in conventional reversed phase separation. In this paper, a method for the simultaneous quantification of amino acids and their respective APs was developed combining high-resolution mass spectrometry with ion-pairing liquid chromatography. The limit of detection was 0.1 ng/mL for tryptophan, valine and arginine, while the limit of quantification ranged from 2 to 5 ng/mL according to the specific sensitivity of each analyte. The relative standard deviation % was lower than 10 % and the coefficient of correlation was higher than 0.99 for each calibration curve. The method was applied to milk, milk-based products, raw and processed tomato. Among the analyzed products, the most abundant amino acid was glutamic acid (16,646.89 ± 1,385.40 µg/g) and the most abundant AP was fructosyl-arginine in tomato puree (774.82 ± 10.01 µg/g). The easiness of sample preparation coupled to the analytical performances of the proposed method introduced the possibility to use the pattern of free amino acids and corresponding APs in the evaluation of the quality of raw food as well as the extent of thermal treatments in different food products.
The Maillard reaction, which can occur during heat processing of pet foods or ingredients, is known to reduce the bioavailability of essential amino acids such as lysine due to the formation of early and advanced Maillard reaction products (MRP) that are unavailable for utilisation by the body. Determination of the difference between total and reactive lysine by chemical methods provides an indication of the amount of early MRP present in foods, feeds and ingredients. Previous research reported that the difference between total and reactive lysine in pet foods can be up to 61·8 %, and foods for growing dogs may be at risk of supplying less lysine than the animal may require. The endogenous analogues of advanced MRP, advanced glycation endproducts, have been associated with age-related diseases in humans, such as diabetes and impaired renal function. It is unknown to what extent advanced MRP are present in pet foods, and if dietary MRP can be associated with the development of diseases such as diabetes and impaired renal function in pet animals. Avoidance of ingredients with high levels of MRP and processing conditions known to favour the Maillard reaction may be useful strategies to prevent the formation of MRP in manufactured pet food. Future work should further focus on understanding the effects of ingredient choice and processing conditions on the formation of early and advanced MRP, and possible effects on animal health.
Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (β-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the β-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.
To determine Nε -carboxymethyllysine (CML) in foods a RP-HPLC method after derivatisation with o-phthaldialdehyde was developed. To prevent an overestimation of the CML values by the formation of CML from Amadori products during hydrolysis a borohydride reduction precedes the hydrolysis. A comparison of the determination with and without reduction shows that during hydrolysis 2–12 times more CML than originally present can be formed. With the analytical conditions described in this paper it is possible to obtain measurable amounts of this trace substance in spite of the much higher values for other amino acids. The CML contents in selected processed food items varied between 11 mg in a preparation from mixed cereals for infants to 408 mg/kg protein in a processed malt product. CML is suitable as indicator of heat damage in processed or stored foods, being more stable than the Amadori compounds determined, e.g. in form of furosine.
The Maillard reaction products (MRPs) most widely used as markers of the nutritional quality of foods are furosine, N(epsilon)-carboxymethyllysine (CML), hydroxymethylfurfural, pyrraline, pentosidine and pronyl-lysine. One of the MRPs identified first was furosine, which was quantified in foods 40 years ago as a chemical indicator of the Amadori compound N(epsilon)-fructoselysine. Since then, furosine has gained broad attention by food chemists and biomedical researchers, as its formation upon heat treatment is well characterised. Moreover, it represents the Amadori products from early Maillard reactions in which amino acids react with reducing carbohydrates, resulting in a loss of their availability. This is of importance for the essential amino acid lysine, which is also the limiting amino acid in many proteins. In order to evaluate the nutritional quality of a protein, the concomitant analysis of free - and nutritionally available - lysine and the amount of lysine reacted to form the respective MRP is essential, even for mildly processed foods. The other chemical markers of heat treatment such as CML, pyrraline, pentosidine or pronyl-lysine seem to be useful markers of the advanced stages of Maillard reactions. Compared to the conditions in which furosine is formed, these compounds are generated under more severe conditions of heat treatment. However, the concentrations analysed are significantly lower than those of furosine. Therefore, the nutritional evaluation of a food protein should include not only furosine, but also other chemical markers of heat treatment such as, for example, CML, pyrraline and pentosidine.
In food science the Maillard reaction is well known to cause degradation of amino acids and an overall decrease in the nutritional value of foods that have been subjected to heat in processing. There has been evidence more recently of the endogenous formation of some Maillard reaction products (MRPs) in biological systems and their association with pathophysiological conditions including diabetes, renal disease and cardiovascular disease. Several studies have suggested that dietary MRPs increase the in vivo pool of MRPs after intestinal absorption and contribute to the development of diabetes and related complications. This review focuses on the animal and human studies which have assessed the eventual implications of dietary MRPs on human health, highlighting the different diets tested, the experimental designs and the biomarkers selected to estimate the health effects. The results of these studies are compared to those of the recently published ICARE study. In this latter study an accurate determination of the MRP content of the diets was achieved, allowing the calculation of the contribution of individual food groups to daily MRP intakes in a regular western diet.
The manuscript reviews beneficial aspects of food processing with main focus on cooking/heat treatment, including other food-processing techniques (e.g. fermentation). Benefits of thermal processing include inactivation of food-borne pathogens, natural toxins or other detrimental constituents, prolongation of shelf-life, improved digestibility and bioavailability of nutrients, improved palatability, taste, texture and flavour and enhanced functional properties, including augmented antioxidants and other defense reactivity or increased antimicrobial effectiveness. Thermal processing can bring some unintentional undesired consequences, such as losses of certain nutrients, formation of toxic compounds (acrylamide, furan or acrolein), or of compounds with negative effects on flavour perception, texture or colour. Heat treatment of foods needs to be optimized in order to promote beneficial effects and to counteract, to the best possible, undesired effects. This may be achieved more effectively/sustainably by consistent fine-tuning of technological processes rather than within ordinary household cooking conditions. The most important identified points for further study are information on processed foods to be considered in epidemiological work, databases should be built to estimate the intake of compounds from processed foods, translation of in-vitro results to in-vivo relevance for human health should be worked on, thermal and non-thermal processes should be optimized by application of kinetic principles.
The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.
The chemistry of the key intermediate in the Maillard reaction, the Amadori rearrangements product, is reviewed covering the areas of synthesis, chromatographic analyses, chemical and spectroscopic methods of characterization, reactions, and kinetics. Synthetic strategies involving free and protected sugars are described in detail with specific synthetic procedures. GC- and HPLC-based separations of Amadori products are discussed in relation to the type of columns employed and methods of detection. Applications of infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy for structural elucidation of Amadori products are also reviewed. In addition, mass spectrometry of free, protected, and protein-bound Amadori products under different ionization conditions are presented. The mechanism of acid/base catalyzed thermal degradation reactions of Amadori compounds, as well as their kinetics of formation, are critically evaluated.
The limit of detection (LOD) for any analytical procedure, the point at which analysis is just feasible, may be determined by a statistical approach based on measuring replicate blank (negative) samples or by an empirical approach, consisting of measuring progressively more dilute concentrations of analyte. The limit of quantitation (LOQ), or concentration at which quantitative results can be reported with a high degree of confidence, may likewise be determined by either approach. We used both methods to determine LOD and LOQ for forensic gas chromatographic-mass spectrometric (GC-MS) analyses of abused drugs. The statistically determined LOD and LOQ values for these assays underestimated the LOD because of the large imprecision associated with blank measurements and the inability of blank samples to meet typical GC-MS acceptance criteria. The empirical method provided much more realistic LOD values, supported by reasonable experimental data, and are 0.5-0.03 the magnitude of the corresponding statistical LODs. The empirical LODs and LOQs are identical for these GC-MS assays. The observations made here about the LOD/LOQ for specific forensic GC-MS procedures are generally applicable to any type of analysis.
Thermal processing and Maillard reaction (MR) affect the nutritional and sensorial qualities of milk. In this paper an olive mill wastewater phenolic powder (OMW) was tested as a functional ingredient for inhibiting MR development in ultrahigh-temperature (UHT)-treated milk. OMW was added to milk at 0.1 and 0.05% w/v before UHT treatment, and the concentration of MR products was monitored to verify the effect of OMW phenols in controlling the MR. Results revealed that OMW is able to trap the reactive carbonyl species such as hydroxycarbonyls and dicarbonyls, which in turn led to the increase of Maillard-derived off-flavor development. The effect of OMW on the formation of Amadori products and N-ε-(carboxymethyl)-lysine (CML) showed that oxidative cleavage, C2-C6 cyclization, and the consequent reactive carbonyl species formation were also inhibited by OMW. Data indicated that OMW is a functional ingredient able to control the MR and to improve the nutritional and sensorial attributes of milk.
N
ε-(carboxymethyl) lysine (CML) and N
ε-(carboxyethyl) lysine (CEL) are two advanced glycation end products. Few studies have focused on the simultaneous determination of CML and CEL content in foods, especially in Eastern foods. In this study, a stable isotope dilution LC–MS/MS method was developed for the simultaneous determination of CML and CEL in foods. The CML and CEL contents in three cereal foods consumed in China were determined by the developed method. Sample preparation consisted of lyophilization, defatting, grinding, reduction, protein precipitation, acid hydrolysis, and solid-phase extraction. The limit of quantification for CML and CEL was 4 and 3 ng/g, respectively. CML and CEL content in fried dough stick was determined for the first time. CML and CEL contents in fried dough sticks were 28.06–66.69 and 10.67–30.22 μg/g of fried dough sticks, respectively. The highest CML and CEL contents in biscuits were 117.53 and 46.09 μg/g of biscuits, respectively. CML and CEL contents in bread crusts were higher than those in bread crumbs.
The potential adverse effects on health of diet-derived advanced glycation end-products (AGEs) is of current interest, due to their proposed involvement in the disease progression of diabetic and uraemic conditions. However, accurate information about levels of AGEs in foods is lacking. The objective of this investigation was to determine the level of one particular AGE, Nε-(carboxymethyl)lysine (CML), a marker of AGE formation, in a wide range of foods commonly consumed in a Western style diet. Individual foods (n = 257) were mixed, lyophilised, ground, reduced, fat-extracted, hydrolysed, and underwent solid-phase extraction. Extracts were analysed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Cereal (2.6 mg/100 g food) and fruit and vegetable (0.13 mg/100 g food) categories had the highest and lowest mean level of CML, respectively, when expressed in mg/100 g food. These data can be used for estimating potential consumer intakes, and provide information that can be used to educated consumers on how to reduce their CML intake.
Infant formulas are supplemented with iron and ascorbate for nutritional purposes, but activation of the advanced Maillard reaction by iron and production of hydroxyl radicals by the iron-ascorbate system may damage the proteins with possible nutritional loss. In this study, we evaluate the deleterious actions of iron and ascorbate, namely lysine damage and tryptophan oxidation on whey-lactose samples incubated at 60 °C. Iron-catalysed ascorbate oxidation enhanced lysine blockage mainly by ascorbylation, whereas lactosylation was little influenced. A three-fold increase in the accumulation rate of fluorescent advanced Maillard products (AMP) was observed in the presence of iron/ascorbate and AMP fluorescence was well correlated to carboxymethyllysine concentration in the samples. The tryptophan degradation rate was 2.5-fold higher in the presence of iron-ascorbate and the loss of this amino-acid was exponentially correlated with AMP fluorescence, similarly whatever iron-ascorbate was present or not. Application of milder heat treatments to infant formulas would allow to decrease the extent of these undesirable reactions.
beta-Lactoglobulin (beta-Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine beta-Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants.
We have redetermined the structure of beta-Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways.
Incorrect threading of the sequence in the published structures of beta-Lg affects four of the nine beta strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the beta-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5 degrees, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the beta strand with the free thiol, each of which shows genetic variation.
Nε-carboxymethyl-lysine (CML) is considered to be an important marker of the Maillard reaction. In the present investigation a liquid chromatography coupled to ion trap tandem mass spectrometry has been developed for the analysis of CML and lysine in drink mixes. To ensure the best specificity of the method, porous graphitic carbon packing material was used for the chromatographic separation and two transition reactions were recorded per analyte. With the stable-isotope dilution technique employed in this method the recovery ranged from 100% to 111.4% for CML. All other performance characteristics tested were also satisfactory. The method was applied to the analysis of chocolate-flavoured drink mixes after sodium borohydride reduction and acid hydrolysis. In this food category, CML content varied from 8.1 to 131.9μg/g powder or 95 to 3527μg/g protein. Among the food items analysed by different laboratories chocolate-flavoured drink mixes appear to have one of the highest CML contents.
Formation of Maillard reaction products (MRPs) including 5-hydroxymethylfurfural (HMF) and acrylamide has been an intensive area of research in recent decades. The presence of reactants such as sodium chloride may influence the Maillard reaction (MR) pathways through the dehydration of various key intermediates. The aim of this work was to test the potential of ingredient encapsulation to mitigate the MR by investigating the case of sodium chloride encapsulation on the HMF formation in cookies. Thirteen cookies were prepared with recipes containing free or encapsulated NaCl. Increasing NaCl concentration from 0 to 0.65% increases HMF concentration up to 75%, whereas in the presence of encapsulated NaCl the reduction of HMF varied from 18 to 61% due to the inhibition of sucrose pyrolytic decomposition and the fructofuranosyl cation formation. Data demonstrated that the more heat-resistant the lipid-based coating was, the more pronounced the reduction of HMF formation. The results showed that encapsulation represents a useful approach to prevent the formation of potentially harmful compounds in thermally processed foods.
Many different types of organic reactions lead to the production of brown pigments at moderate temperatures. In spite of many reviews of the subject, there has been no comprehensive organization of the reactions. In this review some relationships are shown to exist among the carbonyl-amino, the nonamino, and the oxidative types of browning. Recent findings have provided the basis for an integration of the several isolated partial theories of browning (Maillard, sugar fission, ascorbic acid, furaldehyde) heretofore proposed. The significance of the occurrence of the Amadori rearrangement in the Maillard reaction is stressed, and a mechanism for browning in sugar-amine systems based upon the rearrangement is outlined. Attention is directed to the little-studied but important role of dehydrogenated reductones in both enzymatic and nonenzymatic browning reactions. Investigations of browning reactions in model systems during the past 3 years are reviewed, with the pertinent older studies, and the results of most of these are shown to fit into the proposed scheme of reactions. A classified directory to the major part of 201 references on browning in nitrogenous model systems (1940 to March 1953) is included.
A new method for qualitative and quantitative analysis of furosine in food products by capillary electrophoresis coupled to tandem mass spectrometry (CE-MS(2) ) has been optimized and validated. In analytical conditions, the pH value of the run buffer was similar to that of the hydrolyzed samples, which helps in avoiding interactions of the analyte with silanol groups of the inner wall of the fused silica capillary. In all previous CE methods proposed in literature, no SPE treatment has been used. The method has been applied to the analysis of food products, such as flour, having low amounts of furosine. Flour samples of different origin (wheat, chestnut, lupin, einkorn, chickpeas) have been investigated. Different food products such as pasta, milk, and tigelle bread (a typical Italian unleavened bread) were also analyzed. Some of the samples have also been analyzed by reversed-phase HPLC, and when compared to CE-MS, data obtained showed good agreement. CE-MS, in comparison to HPLC, showed advantages in terms of time of analysis and cost. The validation procedure provided good values for LOD and LOQ (respectively 0.07 and 0.25 mg L(-1) ), recovery (77 and 97%), and precision investigated in terms of intraday repeatability (RSD%: 4-6% for peak areas; 1-2% for migration times) and intermediate precision (below 16% for peak areas, and below 7% for migration times). Therefore, the technique reported can be proposed as a powerful analytical method and a valid alternative to common traditional analytical techniques.
The effects of recipe formulation in terms of leavening agents (ammonium and sodium bicarbonates), sugars (sucrose and glucose), initial moisture content, and baking conditions (temperature and time) on furosine formation in cookies were studied. The cookies were baked at different temperatures for different times to monitor the progress of the early stage of the Maillard reaction. Change in furosine levels as an indicator of the Amadori products showed a typical kinetic behavior with a rapid increase to an apparent maximum followed by exponential decrease during baking. Initial water activity of cookie dough had no remarkable effect on the apparent maximum, but lowering the water activity decreased the time required to attain it. In addition, levels of furosine in the final product are highly correlated to the initial water content of dough at the same baking conditions. The levels of furosine attained were significantly lower in cookies composed of sucrose than in cookies composed of glucose. Early stage of the MR is rapidly overcome during baking as the Amadori product degraded in the advanced stage. Among the leavening agents, ammonium bicarbonate was the most effective for the progress of the Maillard reaction in cookies during baking. (C) 2008 Elsevier Ltd. All rights reserved.
Slices of wheat bread were toasted for different times until a distinct intensity of brown colour was reached. Two assays were carried out: prolonged toasting times (5–60 min) and reduced toasting times (0.5–5 min). The browning indicators (furosine, available lysine, hydroxymethylfurfural (HMF), colour and absorbance at 284 and 420 nm) were determined. The precision of all indicators used was high (CV < 4%). No furosine or HMF was detected in the dough before baking. The furosine content increased until 7 min (299 mg per 100 g protein) and then decreased to 2.9 mg per 100 g protein at 60 minutes. For the first toasting times (0.5, 1 and 2 min) the furosine content decreased slightly. Available lysine reached losses of 50% after 25 min of heating. The toasting of bread increased HMF values from 12 to 2025 mg kg ⁻¹ for the assay at prolonged times of heating and from 1.3 to 4.2 mg kg ⁻¹ at reduced times (0.5–5 min). The HMF content decreased (1000 mg kg ⁻¹ ) when the sliced bread was toasted until it burnt. Colour (Δ E , 100 − L *) and absorbance at 284 and 420 nm always increased. High linear correlations ( r ² > 0.860) were obtained between browning indicators and time ( A 284 /time, A 420 /time, 100 − L */time and HMF/time).
© 2001 Society of Chemical Industry
The behavior of different lysine Amadori compounds during acid hydrolysis was investigated in order to determine the molar yield of furosine and the other hydrolysis products. Based on this, the conversion factors for calculating the content of Amadori compound and lysine modification before hydrolysis can be derived. For that purpose, the peptide-bound Amadori compounds Nk-(1-deoxy-D-fructosyl)-, Nk-(1-deoxy-D-tagatosyl)-, Nk-(1-deoxy-D-lactulosyl)- and Nk-(1-deoxy-D-maltulosyl)hippuryllysine as well as free Nk-(1-deoxy-D-fructosyl)lysine were synthesized. For isolation of peptide-bound Amadori compounds, an optimized enzyme-enhanced reversed phase-high pressure liquid chromatography procedure was developed. Pyridosine and Nk-(carboxymethyl)hippuryllysine were synthesized as reference materials. After acid hydrolysis with 6 M hydrochloric acid the, molar yields of furosine were determined to be 32% for fructosyllysine, 34% for lactulosyl- and maltulosyllysine and 42% for tagatosyllysine. Hydrolysis with 8 M hydrochloric acid resulted in a higher yield of furosine for Amadori compounds containing a fructosyl-moiety, 46% for fructosyllysine, 50% for lactulosyllysine and 51% for maltulosyllysine. Compared with this, the molar yield for furosine was not affected by concentration of hydrochloric acid in the case of tagatosyllysine. Based on these conversion factors a reliable calculation of the amount of Amadori compound or lysine modification and with it the evaluation of the progress of the "early" Maillard reaction in foods and biological samples is possible via the quantification of lysine and furosine after acid hydrolysis.
Maillard reaction (MR) is one of the main chemical event occurring during baking. To study the reaction in bakery products,
a dry model system is more useful than an aqueous one. In this work, the effects of formulation and processing conditions
in a crisp bread system were investigated to test the effects of different additives on both the overall reaction and the
formation of MR products such as 5-hydroxymethyl-2-furaldehyde (HMF) and acrylamide. Cylindrical dough made up of flour, water
and yeast was baked at 180°C for 35min and the slices were toasted at different times/temperatures combinations. Browning
and water content were monitored along with the kinetic of formation of chemical indicators such as HMF and acrylamide also
calculating rate constants and activation energy. These parameters were also monitored in systems added with glycine and asparaginase.
During toasting water content follows an exponential trend, being the rate of water loss faster in the initial stage of toasting
and at higher temperature. Browning was more intense when toasting at higher temperature and a linear correlations between
browning (ΔL*, ΔE*), HMF and acrylamide concentration were observed when toasting at 180°C. HMF and acrylamide content increased
with the toasting time and temperature. Their concentrations were strongly dependent on the water content of the final product,
and both the addition of glycine and asparaginase are effective in reducing acrylamide content. The addition of glycine enhanced
the browning of toasted bread, and slightly increased HMF content at any toasting temperature. The system characterized in
this work represents a suitable tool to study the development of the MR in dry systems.
Lipid and protein components of infant formulas can be subjected to modification during product manufacturing. In this study, protein and lipid modifications of several infant formulas, as well as pasteurized and UHT milk samples were evaluated. The levels of secondary lipid oxidation products (malondialdehyde and hexanal), proteins modified by early (furosine, measurement of protein glycation) and advanced (carboxymethyllysine) Maillard reactions, and oxidized and cross-linked proteins (protein carbonyls, dityrosine, lysinoalanine) were measured. Both lipid oxidation and protein modifications were affected by the processing conditions, but no correlation was observed. Therefore, it is of prime importance to monitor several markers of these modification pathways to better assess the heat treatments. Hydrolyzed proteins from hypoallergenic formulas were shown to be particularly sensitive to oxidative modifications. Further studies on well-defined systems (i.e., precise product composition and industrial treatments) are required to better understand the relationships between lipid oxidation and protein modification.
Infant formulas are milk-based products, which are adapted to the composition of human milk. To ensure microbiological safety and long shelf life, infant formulas usually undergo rigid heat treatment. As a consequence of the special composition and the heat regimen, infant formulas are more prone to thermally induced degradation reactions than regular milk products. Degradation reactions observed during milk processing comprise lactosylation yielding the Amadori product lactulosyllysine, the formation of advanced glycation end products (AGEs), and protein-free sugar degradation products, as well as protein or lipid oxidation. Several methods have been developed to estimate the heat impact applied during the manufacturing of infant formulas, including indirect methods such as fluorescence analysis as well as the analysis of defined reaction products. Most studies confirm a higher degree of damage in infant formulas compared to regular milk products. Differences between various types of infant formulas, such as liquid, powdered or hypoallergenic formulas depend on the analyzed markers and brands. A considerable portion of protein degradation products in infant formulas can be avoided when process parameters and the quality of the ingredients are carefully controlled. The nutritional consequences of thermal degradation products in infant formulas are largely unknown.
Dietary fibre consumption may help to control appetite and to reduce calorie intake. Underlying molecular mechanisms were not fully investigated. The aim of this study was to evaluate the effect of barley beta-glucans on short-term appetite and on satiety-related hormones in healthy subjects. Fourteen volunteers were selected and randomly assigned to have isocaloric breakfasts including a 3% beta-glucan-enriched bread (betaGB) or a control bread (CB). Post-breakfast individual self-records of appetite ratings and measure of calorie intake at an ad libitum lunch as well as measure of blood glucose, insulin, ghrelin and PYY concentrations, were performed. betaGB determined a significant higher reduction of hunger and increase of fullness and satiety than CB. Accordingly, a 19% reduction of energy intake at lunch subsequent to betaGB consumption compared to CB, was recorded. A 23% lower AUC(60-180) of plasma ghrelin and a 16% higher total AUC of PYY response after betaGB than CB consumption, independent from insulin response, was found. Glucose response was also blunted by betaGB vs CB. Barley beta-glucans were able to control appetite in the short term by modulating sensations and reducing energy intake. Data suggested for the first time that satiety effect of beta-glucans are mediated by ghrelin and PYY.