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The Maillard reaction products (MRPs) 3-deoxyglucosone (3-DG) and 5-hydroxymethylfurfural (HMF), which are formed during the thermal processing and storage of food, come into contact with technologically used yeasts during the fermentation of beer and wine. In order for the yeast cells to work efficiently, handling of the stress-inducing carbonyl compounds is essential. In the present study, the utilization of 3-DG and HMF by 13 Saccharomyces yeast strains (7 brewer’s yeast strains, 1 wine yeast strain, 6 yeast strains isolated from natural habitats) was investigated. All yeast strains studied were able to metabolize 3-DG and HMF. 3-DG is mainly reduced to 3-deoxyfructose (3-DF) and HMF is completely converted to 2,5-bishydroxymethylfuran (BHMF) and 5-formyl-2-furancarboxylic acid (FFCA). The ratio of conversion of HMF to BHMF and FFCA was found to be yeast strain-specific and no differences in the HMF stress tolerance of the yeast strains and species were observed. After incubation with 3-DG, varying amounts of intra- and extracellular 3-DF were found, pointing to a faster transport of 3-DG into the cells in the case of brewer’s yeast strains. Furthermore, the brewer’s yeast strains showed a significantly higher 3-DG stress resistance than the investigated yeast strains isolated from natural habitats. Thus, it can be shown for the first time that Saccharomyces yeast strains differ in their interaction of 3-DG induced carbonyl stress. Graphical abstract

The aim of this study was to investigate the amino acid composition and the amount of individual glycation compounds in hot trub formed during boiling of wort prepared from different malts. Compared to the initial amino acid composition of the used malts, some Maillard reaction products (namely MG-H1, pyrraline) and hydrophobic amino acids (leucine, isoleucine, valine, phenylalanine) accumulated in the hot trub, whereas hydrophilic amino acids remained in the boiled wort. For MG-H1, a threefold increase was observed during wort boiling, whereas the other Maillard reaction products, namely CML, CEL, pyrraline and maltosine increased only slightly (1.1–2-fold). Furosine as a hallmark for peptide-bound Amadori compounds showed a small decrease. The results suggest that mainly glycated amino acids derived from small dicarbonyl compounds such as methylglyoxal and glyoxal are formed during wort boiling. Furthermore, the studies indicate that the modification of the protein structure as a result of the Maillard reaction has an influence on the hydration of the denatured proteins during the wort boiling process, thus affecting the coagulation process and, therefore, precipitation of the hot trub. The work carried out contributes to the understanding of the chemical reactions influencing the amino acid and Maillard reaction product transfer from malt to beer. Graphical abstract

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αs1 K34–αs2 Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins. Graphical abstract

For peanuts, roasted at 170 °C, the formation of selected glycerol-bound oxidized fatty acids (GOFAs), namely 9-oxononanoic acid (9-ONA), azelaic acid (AZA) and octanoic acid, was observed by GC-MS (EI). The content of octanoic acid as well as AZA increased with continuous roasting time (from 59 mg/kg peanut oil to 101 mg/kg peanut oil and from not detectable to 8 mg/kg peanut oil, respectively), whereas the content of 9-ONA initially decreased from 25 mg/kg peanut oil to 8 mg/kg peanut oil (20 min) and increased again up to 37 mg/kg peanut oil following roasting for 40 min. Due to its aldehyde function, 9-ONA could contribute to amino acid side chain modifications as a result of lipation, which could directly influence the functional properties of peanut proteins. Both 9-ONA and octanoic acid are potential markers of thermal processes. Furthermore, in model experiments using methyl linoleate and methyl oleate, up to 18 oxidized fatty acids could be identified as methyl esters, 9-ONA as well as octanoic acid as major components and a faster formation of GOFAs under roasting conditions (170 °C, 20 min). In addition, 9-ONA contributes to the formation of AZA and octanoic acid in both free and bound form as a result of oxidative subsequent reactions in presence of iron (III).

This opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) presents arguments for an updated risk assessment of diet-related exposure to acrylamide (AA), based on a critical review of scientific evidence relevant to low dose exposure. The SKLM arrives at the conclusion that as long as an appropriate exposure limit for AA is not exceeded, genotoxic effects resulting in carcinogenicity are unlikely to occur. Based on the totality of the evidence, the SKLM considers it scientifically justified to derive a tolerable daily intake (TDI) as a health-based guidance value. published

The Maillard reaction is traditionally subdivided into three stages that start consecutively and run in parallel. Here, we show that N-ε-carboxymethyllysine (CML), a compound formed in the late stage of the reaction, can undergo a second glycation event at its secondary amino group leading to a new class of Amadori rearrangement products. When N-α-hippuryl-CML was incubated in the presence of reducing sugars such as glucose, galactose, ribose, xylose, maltose, or lactose in solution for 1 h at 75 °C, the compound was degraded by 6–21%, and N-ε-carboxymethyl-N-ε-deoxyketosyl lysine derivatives were formed. Under the same conditions, lysine was 5–10 times more reactive than CML. N-α-hippuryl-N-ε-carboxymethyl-N-ε-(1-deoxyfructosyl)-l-lysine (hippuryl-CMFL) and N-ε-carboxymethyl-N-ε-(1-deoxyfructosyl)-l-lysine (CMFL) were synthesized, isolated and characterized by MS/MS and NMR experiments. Depending on the reaction conditions, up to 21% of CMFL can be converted to the furosine analogue N-ε-carboxymethyl-N-ε-furoylmethyl-l-lysine (CM-Fur) during standard acid protein hydrolysis with hydrochloric acid. Incubation of bovine serum albumin (BSA) with glucose for up to 9 weeks at 37 °C revealed the formation of CMFL in the protein as assessed by HPLC–MS/MS in the MRM mode. Under these conditions, ca. 13% of lysine residues had been converted to fructosyllysine, and 0.03% had been converted to CMFL. The detection of glycation products of glycated amino acids (heterogeneous multiple glycation) reveals a novel pathway in the Maillard reaction.

The reactive 1,2-dicarbonyl compound methylglyoxal (MGO) is consumed with food and its concentrations decrease during digestion. In the present paper, the reaction of MGO with creatine, arginine, and lysine during simulated digestion, and its reaction with creatine during the digestion in human volunteers, was studied. Therefore, simulated digestion experiments with a gastric and an intestinal phase were performed. Additionally, an intervention study with 12 subjects consuming MGO-containing Manuka honey and creatine simultaneously or separately was conducted. Derivatization with o-phenylenediamine and HPLC–UV was used to measure MGO, while creatine and glycated amino compounds were analyzed via HPLC–MS/MS. We show that MGO quickly reacts with creatine and arginine, but not lysine, during simulated digestion. Creatine reacts with 56% of MGO to form the hydroimidazolone MG-HCr, and arginine reacted with 4% of MGO to form the hydroimidazolone MG-H1. In the intervention study, urinary MG-HCr excretion is higher in subjects who consumed MGO and creatine simultaneously compared to subjects who ingested the substances separately. This demonstrates that the 1,2-dicarbonyl compound MGO reacts with amino compounds during human digestion, and glycated adducts are formed. These contribute to dietary glycation products consumed, and should be considered in studies investigating their physiological consequences.

Protein-polyphenol interactions affect the structure, stability, and functional properties of proteins and polyphenols. Oxidized polyphenols (o-quinones) react rapidly with the sulfhydryl group of cysteine (Cys) residues, inducing covalent bonding between proteins and polyphenols. However, quantitative data on such reactions remain elusive, despite the importance of depicting the significance of such interactions on food structure and function. This work reports the synthesis, purification, and characterization of caffeic acid-cysteine (CA-Cys) and chlorogenic acid-cysteine (CGA-Cys) adducts and their stable isotope analogs, CA-[13C3,15N]Cys and CGA-[13C3,15N]Cys. A sensitive LC-MS/MS isotope dilution method was developed to simultaneously quantify these adducts in foods and beverages. Protein-bound CA-Cys and CGA-Cys were detected in the micro-molar range in milk samples with added CA and CGA, confirming covalent bonding between milk proteins and CA/CGA. These adducts were detected in commercial coffee-containing beverages but not in cocoa-containing drinks. Furthermore, the adducts were found to be partially stable during enzymatic protein hydrolysis.

Manuka honey is known for its unique antibacterial activity, which is due to methylglyoxal (MGO). After establishing a suitable assay for measuring the bacteriostatic effect in a liquid culture with a time dependent and continuous measurement of the optical density, we were able to show that honey differs in its growth retardingeffect on Bacillus subtilis despite the same content of MGO, indicating the presence of potentially synergistic compounds. In model studies using artificial honey with varying amounts of MGO and 3-phenyllactic acid (3-PLA), it was shown that 3-PLA in concentrations above 500 mg/kg enhances the bacteriostatic effect of the model honeys containing 250 mg/kg MGO or more. It has been shown that the effect correlates with the contents of 3-PLA and polyphenols in commercial manuka honey samples. Additionally, yet unknown substances further enhance the antibacterial effect of MGO in manuka honey. The results contribute to the understanding of the antibacterial effect of MGO in honey.

The adsorption of bovine milk caseins on the tooth surface might have a positive impact on the prevention of dental diseases. Therefore, the present study aimed to investigate the efficacy of mouthrinses with different types of bovine milk and milk protein isolates to accumulate caseins in the pellicle. An indirect enzyme-linked immunosorbent assay (ELISA) was established to quantify the amount of caseins adsorbed into the in situ pellicle. In situ pellicle samples were collected from 2 volunteers on ceramic specimens (A = 8 cm2). After 10 min of pellicle formation, different types of bovine milk, 3% micellar casein in synthetic milk ultrafiltrate (SMUF) or 3% non-micellar caseinate in SMUF, were used as mouthrinses for 10 min. The pellicle material was harvested after 30 min in situ and examined for caseins by the indirect ELISA. Selected pellicle samples were subjected to TEM analysis. All mouthrinses accumulated caseins in the in situ pellicle (2.0 ± 0.7–20 ± 1.7 μg/ml) that, under native conditions, expressed no casein signal. Micellar protein association increased the adsorption of casein into the pellicle. Milk homogenization also had an influence on the casein accumulation in the pellicle. TEM analysis confirmed the integration of micellar casein into the pellicle. The mouthrinses altered the protein composition and the ultrastructure of the in situ pellicle to a different extent: bovine milk with 3.8% fat content and 3% micellar casein in SMUF being particularly effective. The study provides interesting perspectives for innovative prevention strategies in dentistry.

Acetaldehyd kommt natürlicherweise in zahlreichen Lebensmitteln vor und wird aufgrund seines fruchtigen Aromas auch als Aromastoff eingesetzt. Die Internationale Agentur für Krebsforschung (IARC) stufte Acetaldehyd als möglicherweise krebserregend sowie in Verbindung mit der oralen Aufnahme über alkoholhaltige Getränke als humanes Kanzerogen ein. Vor diesem Hintergrund stellt sich die Frage, ob die Verwendung von Acetaldehyd als Aromastoff weiterhin vertretbar ist. Die Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) hat die aktuelle Datenlage zur Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff geprüft und hierzu eine Stellungnahme verabschiedet. Demnach kann die Frage, ob Acetaldehyd nach oraler Exposition in vivo gentoxisch und mutagen wirkt, derzeit nicht abschließend beantwortet werden. Weiterhin ist auch unklar, welchen Beitrag die Verwendung von Acetaldehyd als Aromastoff zur Gesamtexposition des Verbrauchers gegenüber Acetaldehyd leistet. Eine wissenschaftliche Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff ist daher weiterhin nicht möglich. Die SKLM weist darauf hin, dass aufgrund des gentoxischen Gefährdungspotenzials sowie zahlreicher Datenlücken, die für eine vollständige Risikobewertung geschlossen werden müssen, Zweifel an der Sicherheit von Acetaldehyd als Aromastoff bestehen. Nach Ansicht der SKLM sollte der gezielte Zusatz von Acetaldehyd als Aromastoff aus Gründen des vorsorgenden Verbraucherschutzes neu beurteilt werden. Acetaldehyde occurs naturally in many foods and is also used as a flavouring due to its fruity aroma. The International Agency for Research on Cancer (IARC) classified acetaldehyde as possibly carcinogenic to humans and, in combination with oral intake via alcoholic beverages, as carcinogenic to humans. Therefore, the question arises whether the use of acetaldehyde as a flavouring agent is still justifiable. The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) reviewed the scientific basis for health risk assessment of the use of acetaldehyde as a flavouring substance and adopted an opinion. Based on the available data, it is at present not possible to conclude if acetaldehyde is genotoxic and mutagenic in vivo after oral exposure. There is also uncertainty regarding the contribution of acetaldehyde as a flavouring substance to the overall exposure to acetaldehyde. Therefore, a science-based assessment on health risk related to the use of acetaldehyde as a flavouring is not possible at present. Considering the genotoxic potential as well as numerous data gaps that need to be closed for a full risk assessment, the SKLM is concerned about the safety of acetaldehyde as a flavouring substance. For reasons of precautionary consumer protection, the SKLM considers that the use of acetaldehyde as a food additive should be re-evaluated.

Flavor instability of beer is affected by the rise of aroma-active aldehydes during aging. Aldehydes can be either released from bound-state forms or formed de novo. This second part of our study focused on the de novo formation of aldehydes during the Maillard reaction, Strecker degradation, and oxidation reactions. Key precursor compounds for de novo pathways are free amino acids. This study varied the potential for reactions by varying free amino acid content in fresh beer using different proteolytic malt modification levels (569–731 mg/100 g d. m. of soluble nitrogen) of the used malt in brewing trials. Overall, six pale lager beers were produced from three malts (different malt modification levels), each was made from two different barley varieties and was naturally and forcibly aged. It was found that higher malt modification levels in fresh beer and during beer aging increased amino acid and dicarbonyl concentrations as aging precursors and Strecker aldehyde contents as aging indicators. Dicarbonyls were degraded during aging. Advanced glycation end products as possible degradation products showed no consistent formation during aging. Therefore, Strecker reactions were favored during beer aging. No alternative oxidative formation of Strecker aldehydes from their corresponding alcohols could be confirmed. Along with the preceding part one of our investigation, the results of this study showed that de novo formation and release occur simultaneously. After 4 months of natural aging, aldehyde rise is mainly accounted for by de novo formation.

Individual Maillard reaction products (MRPs), namely furosine, which is formed from Amadori product of lysine during acid hydrolysis, as well as N-ε-(carboxymethyl)lysine (CML), pyrraline and the arginine derivative MG-H1 (methylglyoxal-derived hydroimidazolone 1) were quantified in 78 samples of animal feed, belonging to 17 different feed types. The concentrations of the MRPs were dependent on the heat treatment during processing. Within similar feed types, significant differences in concentrations could be observed. MRPs can be suitable indicators to evaluate the impact of technological processing on the nutritional quality of animal feed.

Casein is a group of milk proteins with high nutritional value, and the exploitation of its techno-functional potentials has been investigated for decades. In this study, acid casein powder was dissolved in 0.1 mol/L phosphate buffers with different pH, resulting in casein solutions with pH 5.9, 6.6 and 7.3. During preparation and storage (40 °C) of the samples, casein hydrolysis was observed in size exclusion chromatography and gel electrophoresis. The degree of hydrolysis increased with increasing pH, and treatment of casein with commercial plasmin resulted in similar polypeptides, suggesting that the hydrolysis was caused by residual indigenous plasmin present in the acid casein powder. Most polypeptides could be cross-linked by microbial transglutaminase, except for one particular fraction which appeared at constant intensity in the chromatograms. The stiffness of acid-induced gels as determined in small amplitude oscillatory shear rheology decreased with increasing degree of hydrolysis, and was also lower for cross-linked samples when the preceding casein hydrolysis was more pronounced. Enzymatic cross-linking increased the resistance of casein against plasmin-related hydrolysis, presumably because of the resulting lysine modification. However, one particular fraction of polypeptides was released by hydrolysis in spite of cross-linking, suggesting that they did not contain lysine residues that are susceptible for mTGase. The results indicate that plasmin-related hydrolysis should be taken into account for the application of acid casein or sodium caseinate as additive in food design.

3-Deoxyglucosone (3-DG) is a metabolite from sugar degradation obtained by the Maillard reaction. It is an important precursor compound in Strecker reactionism that directly leads to known beer aging indicators and can influence the final sensory beer quality. However, the conditions of 3-DG formation in the malting process have not yet been described. To investigate the reaction pathways of 3-DG formation, we varied the composition of reactants (sugars, amino acids) by using different malting modification levels (germination time 5–7 d; steeping degree 42–45%; germination temperature 12–14 °C); final kilning temperature (60 °C to 100 °C). After its derivatization with ortho-phenylenediamine, we analyzed 3-DG with HPLC-UV. 3-DG concentration was between 5 and 120 µmol/100 g dry weight. The formation of 3-DG increased for high malt modification levels and high final kilning temperature. The abundant formation of 3-DG in the malting process is already comparable to the occurred brewing process concentration.

Objectives The present study aimed to investigate if bovine milk or milk protein isolates, respectively, alter the ultrastructure of thein situ pellicle and might therefore have an influence on oral health. Methods In situ pellicle samples were formed on bovine enamel slabs exposed in the oral cavity of three subjects for 6, 30, 60 or 120 min. After 3 min of pellicle formation, mouthrinses were performed for 3 min with (non-)homogenized UHT- or fresh milk (0.3% or 3.8% fat), 30% UHT-treated cream or different types of casein- or milk protein isolates containing preparations. The specimens were removed after the exposure times and transmission electron microscopy (TEM) was performed. Native pellicle samples served as controls. Results Topical ultrastructural pellicle modifications were detected after mouthrinses with all types of homogenized UHT- or fresh milk and after the application of a 3% native casein micelles containing experimental solution. Atypical globular protein structures, identified as casein micelles, were temporarily adsorbed onto the pellicle. They were closely associated with lipid droplets. Furthermore, the mouthrinses occasionally affected the morphology of salivary bacteria. However, no notable ultrastructural alterations remained after 120 min of pellicle formation. Conclusion For the first time, bovine milk- and micellar casein-induced pellicle modifications were revealed by TEM. The adsorption of micellar casein is possibly due to its molecular interactions. Clinical significance Bovine milk or micellar caseins provide some potential for the development of preventive strategies against bacterial biofilm formation or erosive processes at the tooth surface.

Sodium caseinate and β-casein self-associate to casein particles in solution because of hydrophobic interactions. Microbial transglutaminase (mTGase) presumably preferentially cross-links molecules that are located within the same particles, meaning that polymer size is limited to the number of monomers initially present in the particles. The aim of this study was to affect the self-association of casein by varying the temperature and thereby controlling the maximum size of casein polymers formed using mTGase. Activity and stability of mTGase were determined at different temperatures, showing that about 10 times more enzyme had to be added at 10 than at 40 °C to compensate for differences in enzyme activity. Analysis with gel electrophoresis showed that incubation temperature had no effect on maximum polymer size at the selected protein concentration (27 g/kg), resulting in similar stiffness of acid-induced gels after incubation for 24 h. For a more detailed characterization of casein polymers, size exclusion chromatography was coupled to multi-angle light scattering (MALS). Estimated molar mass distributions of casein polymers were similar at both incubation temperatures and the increase in molar mass leveled off after moderate incubation time. This underlines the idea of a maximum polymer size and suggests no cross-linking between existing polymers. Besides that, the MALS detector showed a contaminant of low concentration but large size which co-eluted with casein polymers and possibly led to overestimation of their molar masses.

In the Maillard reaction (non-enzymatic browning), reducing carbohydrates react to 1,2-dicarbonyl compounds that may combine with the e-amino group of lysine to form characteristic pyrrole compounds such as pyrraline and formyline in a Paal–Knorr reaction. In the present study, the Maillard reaction of the rare sugar l-rhamnose was studied. The l-rhamnose-derived dehydration product 3,6-dideoxy-l-mannosone (3,6-DDM) was synthesized and structurally characterized via NMR. Mainly the α-pyranose is formed in aqueous solution. 2-(2′(S),3′(S)-Dihydroxybutyl)quinoxaline was synthesized for the first time and used as a standard for quantification of 3,6-DDM in food. Analysis of 3,6-DDM after conversion to the respective quinoxaline via RP-HPLC–MS/MS revealed concentrations not exceeding 23 µg/kg in food items such as coffee and brewing malt. Moreover, the Paal–Knorr reaction product of 3,6-DDM with lysine, namely 6-(2-formyl-5-methylpyrrol-1-yl)-l-norleucine (rhamnolysine) was synthesized for the first time by reaction of N-α-Boc-lysine and 3,6-DDM and purified by semi-preparative ion-exchange chromatography followed by characterization by NMR and HPLC–MS/MS. Rhamnolysine was analysed in 42 food items by RP-HPLC–MS/MS in the MRM mode after enzymatic hydrolysis. The highest concentration of the compound was found in rye bread crust (4.9 mg/kg). It appears mainly in foods that are highly heat-treated, roasted or fried.

Glycation reactions play a key role in post-translational modifications of amino acids in food proteins. Questions have arisen about a possible pathophysiological role of dietary glycation compounds. Several studies assessed the metabolic fate of dietary glycation compounds into blood and urine, but studies about saliva are rare. We investigated here the dietary impact on salivary concentrations of the individual Maillard reaction products (MRPs) N-ε-fructosyllysine, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), pyrraline (Pyr), and methylglyoxal-derived hydroimidazolone 1 (MG-H1). Quantitation was performed using stable isotope dilution analysis (LC-MS/MS). We describe here, that a low MRP diet causes a significant lowering of salivary levels of Pyr from 1.9 ± 0.4 ng/mL to below the LOD and MG-H1 from 2.5 ± 1.5 ng/mL to 0.7 ± 1.8 ng/mL. An impact on the salivary protein fraction was not observed. Furthermore, salivary Pyr and MG-H1 levels are modified in a time-dependent manner after a dietary intervention containing 1.2 mg Pyr and 4.7 mg MG-H1. An increase in mean salivary concentrations to 1.4 ng/mL Pyr and 4.2 ng/mL MG-H1 was observed within 30–210 min. In conclusion, saliva may be a useful tool for monitoring glycation compound levels by using Pyr and MG-H1 as biomarkers for intake of heated food.

Sterols, especially cholesterol and phytosterols, are important components of food lipids. During food processing, such as heating, sterols, like unsaturated fatty acids, can be oxidized. Protein modification by secondary products of lipid peroxidation has recently been demonstrated in food through a process called lipation. Similarly, this study was performed to assess, for the first time, the possibility of reactions between food proteins and sterol oxidation products in conditions relevant for food processing. Therefore, reaction models consisting of oxysterol (cholesterol 5&alpha, 6&alpha, epoxide) and reactive amino acids (arginine, lysine, and methionine) were incubated in various conditions of concentration (0&ndash, 8 mM), time (0&ndash, 120 min), and temperature (30&ndash, 180 &deg, C). The identification of lysine adducts through thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with a diode array detector (DAD), and electrospray ionization (ESI) mass spectrometry (MS) evidenced a reaction with lysine. Moreover, the HPLC-ESI with tandem mass spectrometry (MS/MS) analyses allowed observation of the compound, whose mass to charge ratio m/z 710.5 and fragmentation patterns corresponded to the reaction product [M + H]+ between cholesterol-5&alpha, epoxide and the &epsilon, amino-group of N&alpha, benzoylglycyl-l-lysine. Moreover, kinetic studies between N&alpha, benzoylglycyl-l-lysine as a model for protein-bound lysine and cholesterol 5&alpha, epoxide were performed, showing that the formation of lysine adducts strongly increases with time, temperature, and oxysterol level. This preliminary study suggests that in conditions commonly reached during food processing, sterol oxidation products could react covalently with protein-bound lysine, causing protein modifications.

Heat-processed diets contain high amounts of advanced glycation end products (AGEs). Here we explore the impact of an AGE-enriched diet on markers of metabolic and inflammatory disorders as well as on gut microbiota composition and plasma proteins glycosylation pattern. C57BL/6 mice were allocated into control diet (CD, n = 15) and AGE-enriched diet (AGE-D, n = 15) for 22 weeks. AGE-D was prepared replacing casein by methylglyoxal hydroimidazolone-modified casein. AGE-D evoked increased insulin and a significant reduction of GIP/GLP-1 incretins and ghrelin plasma levels, altered glucose tolerance, and impaired insulin signaling transduction in the skeletal muscle. Moreover, AGE-D modified the systemic glycosylation profile, as analyzed by lectin microarray, and increased N&epsilon, carboxymethyllysine immunoreactivity and AGEs receptor levels in ileum and submandibular glands. These effects were associated to increased systemic levels of cytokines and impaired gut microbial composition and homeostasis. Significant correlations were recorded between changes in bacterial population and in incretins and inflammatory markers levels. Overall, our data indicates that chronic exposure to dietary AGEs lead to a significant unbalance in incretins axis, markers of metabolic inflammation, and a reshape of both the intestinal microbiota and plasma protein glycosylation profile, suggesting intriguing pathological mechanisms underlying AGEs-induced metabolic derangements.

In the course of the Maillard reaction in vivo or in food, creatine reacts with the 1,2-dicarbonyl compound methylglyoxal to N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr). We studied whether the urinary excretion of MG-HCr is affected by its intake with meat or by the intake of creatine and subsequent in vivo formation of MG-HCr. Therefore, 24 h urine of 30 subjects with different dietary habits was analyzed with HPLC-MS/MS. The daily MG-HCr excretion via urine varied between omnivores (0.39-9.67 μmol/day, n = 24), vegetarians (0.18-0.97 μmol/day, n = 19), and vegans (0.10-0.27 μmol/day, n = 8). An intervention study with 18 subjects demonstrated the bioavailability of MG-HCr (ca. 54%) from 200 g of heated meat and its quick excretion with urine. A creatine intervention of 0.44 g did not increase MG-HCr excretion. Thus, the differences in MG-HCr excretion between different diets are mainly caused by the dietary uptake of MG-HCr. We additionally found MG-HCr in milk and egg products, where it is formed during heat treatment. This partly explains differences in MG-HCr excretion of vegetarians and vegans. Hence, MG-HCr in urine is a short-term marker for the intake of heat-processed animal source food.

Hydrolysates of plant or whey proteins, respectively, contain a variety of bioactive peptides which are promising ingredients of functional foods. The application of hydrolysates in foods, however, is limited due to their notable bitter taste, which is attributed to aromatic and hydrophobic amino acid residues. In the present study, NMR and UV-/fluorescence measurements were performed to investigate interactions between cyclodextrins and individual peptides originating from enzymatically hydrolyzed food proteins, namely isoleucyl-tryptophan and tryptophyl-leucine. It was found that the hydrophobic side chain of aromatic amino acids penetrates directly into the cavity of the cyclodextrins. For tryptophan, tyrosine and phenylalanine, the calculated association constants were less than 600 M−1 (1H-NMR), indicating weak–moderate interactions. Tryptophan and β-cyclodextrin formed the most stable complexes. The strength of these complexes increased for tryptophan within the sequence of dipeptides, especially in C-terminal position. Initial investigations on the taste of rice protein hydrolysate have shown that the addition of cyclodextrins except of α-cyclodextrin significantly reduced its bitterness. Especially, the use of β-cyclodextrin was particularly effective. For the first time, we could determine the bitter taste-lowering potential of γ-cyclodextrin, however, less pronounced compared to β-cyclodextrin. Therefore, β-cyclodextrin is the most suitable additive for improving the sensory quality of hydrolysate-containing functional foods.

3-Deoxyglucosone (3-DG) is an important intermediate of the Maillard reaction in food and in vivo. Such 1,2-dicarbonyl compounds shall play a role for the development of age-related diseases as well as diabetes-related complications. As food may contain 3-DG in considerable concentrations, knowledge about the absorption and metabolism of 3-DG is required. The aim of the study was to analyze the dietary influence of 3-DG on the urinary excretion of two of its metabolites, namely 3-deoxyfructose (3-DF) and 2-keto-3-deoxygluconic acid (3-DGA). 18 participants followed a dietary protocol and collected their 24 h urine, which was subsequently analyzed with GC-MS. Urinary excretion of 3-DG, 3-DF and 3-DGA decreased by 60, 57 and 28%, respectively, when the participants changed their conventional diet to a raw-food diet virtually free of dicarbonyl compounds. Four subjects participated in a dietary intervention with honey containing 3-DG, which led to a significant increase of 3-DG, 3-DF and 3-DGA in the urine of the subjects. In comparison to 3-DG, amounts of 3-DF and 3-DGA excreted per day were 50 and 5 times higher, respectively. From the ingested amount of 3-DG, around 1.1% were excreted as 3-DG, 23.4% were excreted as 3-DF and 1.6% as 3-DGA. This indicates that 3-DF is by far the main product of the metabolism of dietary 3-DG.

Manuka honey from New Zealand is derived from the nectar of Leptospermum scoparium and contains high amounts of methylglyoxal (MGO), which is responsible for its extraordinary antibacterial properties and its high market value. It was shown recently, that honeys from Australian Leptospermum species, often referred to as “jellybush honey”, also contain MGO. Recently, an official method has been published by the Ministry of Primary Industries (MPI) New Zealand to characterize monofloral and multifloral manuka honey from New Zealand, based on the concentration of the phytochemical compounds 2′-methoxyacetophenone (2-MAP), 2-methoxybenzoic acid (2-MB), 3-phenyllactic acid (3-PLA) and 4-hydroxyphenyllactic acid (4-HPLA). Here we show that these phytochemical markers are suitable to differentiate honeys originating from Leptospermum species from New Zealand or Australia, respectively. Three non-manuka honeys and eight manuka honeys from New Zealand as well as four non-Leptospermum honeys and 15 Leptospermum honeys from Australia were analysed for their MGO content via RP-HPLC-UV and their 2-MAP, 2-MB, 4-HPLA and 3-PLA concentration via RP-HPLC–MS/MS. All non-manuka honeys contained only negligible amounts of MGO, whereas honeys from Leptospermum species contained up to 1100 mg/kg, independent of the geographical origin. No significant amounts of 3-PLA, 4-HPLA, 2-MB and 2-MAP were found in the non-manuka honeys. In contrast, all marker compounds were abundantly present in manuka honeys from New Zealand, thus enabling a characterization of monofloral or multifloral quality. All honey samples from Australian Leptospermum species contained 2-MAP in concentrations below the limit of detection. The concentrations of the other marker compounds ranged from 58 to 357 mg/kg for 3-PLA, from 1 to 3 mg/kg for 4-HPLA and from 26 to 175 mg/kg for 2-MB and were significantly different when compared to New Zealand manuka honeys. Based on the results of this study, the marker compounds suggested by the MPI might be useful to distinguish honeys derived from Leptospermum species of different geographical origins.

Fructose and dicarbonyl compounds resulting from fructose in heated foods have been linked to pathophysiological pathways of several metabolic disorders. Up to now, very little has been known about the Maillard reaction of fructose in food. Heyns rearrangement compounds (HRCs), the first stable intermediates of the Maillard reaction between amino components and fructose, have not yet been quantitated as protein-bound products in food. Therefore, the HRCs glucosyllysine and mannosyllysine were synthesized and characterized by NMR. Protein-bound HRCs in cookies containing various sugars and in commercial bakery products were quantitated after enzymatic hydrolysis by RP-HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Protein-bound HRCs were quantitated for the first time in model cookies and in commercial bakery products containing honey, banana, and invert sugar syrup. Concentrations of HRCs from 19 to 287 mg/kg were found, which were similar to or exceeded the content of other frequently analyzed Maillard reaction products, such as N-ε-carboxymethyllysine (10-76 mg/kg), N-ε-carboxyethyllysine (2.5-53 mg/kg), and methylglyoxal-derived hydroimidazolone 1 (10-218 mg/kg) in the analyzed cookies. These results show that substantial amounts of HRCs form during food processing. Analysis of protein-bound HRCs in cookies is therefore useful to evaluate the Maillard reaction of fructose.

Manuka honey (Leptospermum scoparium) exerts a strong antibacterial effect. Bacterial enzymes are an important target for antibacterial compounds. The enzyme urease produces ammonia and enables bacteria to adapt to an acidic environment. A new enzymatic assay, based on photometric detection of ammonia with ninhydrin, was developed to study urease activity. Methylglyoxal (MGO) and its precursor dihydroxyacetone (DHA), which are naturally present in manuka honey, were identified as jack bean urease inhibitors with IC50 values of 2.8 and 5.0mM, respectively. Urease inhibition of manuka honey correlates with its MGO and DHA content. Non-manuka honeys, which lack MGO and DHA, showed significantly less urease inhibition. MGO depletion from manuka honey with glyoxalase reduced urease inhibition. Therefore, urease inhibition by manuka honey is mainly due to MGO and DHA. The results obtained with jack bean urease as a model urease, may contribute to the understanding of bacterial inhibition by manuka honey.

This study compared the fluorescence properties (λex/em=350/450nm) and molecular size of proteins from manuka and non-manuka honey. The fluorescence characteristics of non-manuka and manuka proteins differ markedly, whereby manuka honey protein fluorescence increases with increasing methylglyoxal (MGO) content of the honey. It was concluded that manuka honey proteins are modified due to MGO-derived glycation and crosslinking reactions, thus resulting in fluorescent structures. The molecular size of honey proteins was studied using size exclusion chromatography. Manuka honey proteins contain a significantly higher amount of high molecular weight (HMW) fraction compared to non-manuka honey proteins. Moreover, HMW fraction of manuka honey proteins was stable against reducing agents such as dithiothreitol, whereas HMW fraction of non-manuka honey proteins was significantly decreased. Thus, the chemical nature of manuka honey HMW fraction is probably covalent MGO crosslinking, whereas non-manuka HMW fraction is caused by disulfide bonds. Storage of a non-manuka honey, which was artificially spiked with MGO and DHA, did not induce above mentioned fluorescence properties of proteins during 84days of storage. Hence, MGO-derived fluorescence and crosslinking of honey proteins can be useful parameters to characterize manuka honey.

To investigate the influence of high hydrostatic pressure on the reactions of glyoxal with lysine residues, the formation of end-products, namely N-e-(carboxymethyl)lysine (CML) and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), was studied in model systems via LC–MS. Following a kinetic interpretation, reaction volumes were calculated. Corresponding values of +5.4 (CML) and +9.9 cm³/mol (GOLD) indicate a suppressive effect of pressure on the formation of these glycation compounds. Above this, several other known end-products, namely N(6)-glycoloyllysine (GALA), N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA), and reaction intermediates, were qualitatively monitored and proofed a similar behavior compared to the GOLD formation pathway. The reasons for the influence of pressure are discussed in the context of the reaction mechanism.

In contrast to most European countries, raw and processed insects are a part of the traditional diet in Africa, especially among poor people in rural areas. This paper presents a first nutritional analysis of the caterpillar species Imbrasia epimethea (Drury), which is widely collected and eaten in the Uige Province, northern Angola. Major nutrients were analysed with focus on the impact of different traditional preparation techniques (evisceration, cooking and/or drying). The native larvae revealed similar nutritional values (protein and fat content, amino and fatty acid composition) as Gonimbrasia belina, another member of Lepidoptera, and a higher quality than mealworms and crickets. With respect to more “Western” diets, the protein content of I. epimethea was found to be similar to tuna, chicken and beef, while essential amino acid were lower, resulting in lower essential amino acid indices. Fat contents were lower than in chicken, tuna and beef. Its fatty acid profile, with high percentages of polyunsaturated fatty acids, makes it a valuable contribution to human nutrition. The different preparation methods had only limited impact on the nutritional composition of the caterpillars. The removal of the gut reduced carbohydrates originating from the leaves of the host plant, and thus significantly raised protein and (non-significantly) fat contents. Thermal processing had no significant negative effect on the nutritional value except for the decrease of monounsaturated fatty acids. Thus, I. epimethea represents a valuable traditional alternative to food from other animal sources, especially with regard to its fatty acid composition.

Ile–Trp (IW) and Trp–Leu (WL) are present in enzymatic hydrolysates of whey proteins and act as efficient inhibitors for angiotensin-converting enzyme (ACE) in vitro. Baseline plasma concentrations of IW and WL were analysed to 0.9 ± 0.3 n m for IW and 8.3 ± 1.3 n m for WL. Following administration of 50 mg IW or 100 mg WL to human volunteers (n = 3 for IW and n = 2 for WL, respectively), a significant increase in plasma concentrations to a maximum of 2.4 ± 0.5 n m for IW and 29–36 n m for WL after 0.5 h was observed, followed by rapid elimination. ACE activity in plasma decreased by 32% ± 8% following 50 mg IW administration. Administration of the corresponding amino acids had no effect on ACE activity. Thus, these tryptophan-containing dipeptides may serve as bioactive food additives with beneficial effects for the cardiovascular system.

During food processing or storage, milk proteins can react with reducing sugars via the Maillard reaction (glycation), which may alter their techno-functional properties. The aim of this study was to investigate the relationship between molecular changes of casein occurring during different stages of the Maillard reaction and its acid-induced gelling properties. Therefore, sodium caseinate was heated in a dry state at 60 °C in the presence of lactose and analyzed for structural modifications by determining Amadori compounds (glycoconjugation) indirectly as furosine, the total lysine modification, and the extent of protein cross-linking. For techno-functional characterization, acid-induced gels were prepared by the addition of glucono-δ-lactone and evaluated by measuring pH kinetics during gel formation, gel strength, and water holding capacity. The time to reach pH 4.6 during the gelation process was significantly delayed with increasing extent of the Maillard reaction. Glycation with lactose also led to a significant increase in gel strength and water holding capacity. The increase in gel stability was rather independent from the amount of sugars covalently bound to the proteins during the early phase of the Maillard reaction but strongly correlated to the degree of protein polymerization. Small- and medium-sized casein oligomers, formed during advanced stages of the Maillard reaction, contributed considerably to the formation of stronger gels with higher water holding capacity, whereas a sharp increase in the relative amount of the polymer fraction observed during prolonged cross-linking processes caused a spontaneous destabilization of the gel network. Knowledge about structure-function relationships on a molecular level can provide useful information to control food texture by raw material quality.

Maltosine (6-(3-Hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine) is formed in the late stage of the Maillard reaction. Quantitative data on the occurrence in food of this metal-chelating 3-hydroxy-4(1H)pyridinone are not available. In the present study, the formation of the compound was studied first in model systems simulating bread crust and crumb. Maltosine was formed predominantly in the presence of di- and oligosaccharides and glycosylated isomaltol derivatives under conditions resembling crust formation. Disaccharide derivatives with β-glycosidic bonds were less potent precursors than derivatives with α-glycosidic bonds. Moreover, a method was developed for the quantification of maltosine in food. The compound was released from proteins by enzymatic hydrolysis due to artefact formation during acid hydrolysis. [13C6,15N2] labelled maltosine was synthesized and utilized as an internal standard, and the analysis of 62 commercial food samples was performed by HPLC–MS/MS in the multiple reaction monitoring mode. Maltosine was present predominantly in bread samples (0.1–4.2 mg/kg). The highest concentrations of the compound were found in the crust of wheat bread (up to 19.3 mg/kg), representing a maximal lysine modification of 0.4 %. The concentrations of maltosine in most food samples reached approximately 10 % of the concentrations of the advanced glycation end product pyrraline. From these data, an intake of maltosine of 1–2 mg per day can be expected from the diet.

The influence of the addition of a flavour enhancer solution (FES) (d-glucose, d-ribose, l-cysteine and thiamin) and of sous-vide cooking or roasting on moisture, cooking loss, instrumental colour, sensory characteristics and formation of Maillard reaction (MR) compounds in lamb loins was studied. FES reduced cooking loss and increased water content in sous-vide samples. FES and cooking method showed a marked effect on browning development, both on the meat surface and within. FES led to tougher and chewier texture in sous-vide cooked lamb, and enhanced flavour scores of sous-vide samples more markedly than in roasted ones. FES added meat showed higher contents of furosine; 1,2-dicarbonyl compounds and 5-hydroxymethylfurfural did not reach detectable levels. N-ε-carboxymethyllysine amounts were rather low and not influenced by the studied factors. Cooked meat seems to be a minor dietary source of MR products, regardless the presence of reducing sugars and the cooking method.

Somatic angiotensin-converting enzyme (ACE) contains two active sites, the C- and N-domain, from which the C-domain is supposed to play a major role in blood pressure regulation and is therefore a promising pharmacological target to reduce blood pressure without side-effects. We report for the first time that tryptophan-containing dipeptides such as Ile-Trp or Val-Trp, which were recently found in food protein hydrolysates, are selective and competitive inhibitors for the C-domain with a selectivity factor of 40 and 70, respectively. Structure-activity studies showed that an N-terminal aliphatic amino acid and a tryptophan moiety in the P2' position are favourable structures for C-domain inhibition in dipeptides. In contrast, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro, which were widely used as ingredients for hypotensive food, showed a slight selectivity for the N-domain. Hence, tryptophan containing dipeptides are interesting ingredients for functional foods as a natural prevention for hypertension with reduced side effects due to its selective inhibition of the C-domain.

Protein isolate was hydrolysed by Alcalase, thermolysin and trypsin. BBPH produced by Alcalase showed highest angiotensin-converting enzyme (ACE) inhibitory properties (IC50: 52 µg/mL). Hydrolysates produced by Alcalase and thermolysin exhibited similar dipeptidyl peptidase-IV (DPP-IV) inhibitory activity (IC50: 1.73 mg/mL), while low inhibitory activity was observed for hydrolysate produced by trypsin. BBPH also showed protective effect against oxidative stress with significant 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferrous chelating activity. Bioactive peptides of BBPH produced by thermolysin showed better resistance to simulated gastrointestinal digestion (SGID), while the DPP-IV and ACE inhibitory properties were significantly reduced. Molecular weight distribution showed significant reduction in peptides of the molecular weight range 200–400 Da in BBPH produced by Alcalase, after SGID. LC-ESI-TOF-MS and in silico analysis showed the presence of potential peptides with both ACE and DPP-IV inhibitory properties in BBPH produced by thermolysin.

Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial increase of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that cross-linking must occur between and/or within already existing aggregates, which become larger and less flexible, and supports the hypothesis that, starting with a critical aggregate size the network formation is somewhat impaired by the size and reduced flexibility of the proteins.

cope Heated foods often present low allergenicity, and have recently been used in specific oral immunotherapy for food allergies. However, the influence of heating on tolerogenicity of food allergens is not well elucidated. Here, we investigated biochemical, allergenic, and tolerogenic properties of heated egg white (EW) using a murine model of food allergy. Methods and results Raw EWs were treated at 80°C for 15 min (80EW, mild heating condition), 100°C for 5 min (100EW, cooking condition), or 121°C for 40 min (121EW, retort pouch condition), and freeze-dried. A transgenic OVA23–3 mice model expressing T-cell receptor specific for ovalbumin (OVA, a major EW allergen) induced Th2 cells and IgE production, and presented intestinal inflammation when fed untreated EW diet. 80EW-fed mice presented only moderate inflammation but high Th2 responses. 100EW-fed mice did not present inflammation but induced tolerance as seen in reduced T-cell responses and IgE levels. 100EW demonstrated higher digestive stability and slower absorption in intestine, compared with untreated EW and 80EW. 121EW was strongly aggregated, was not absorbed well, and developed Th1 responses without tolerance induction. Conclusion OVA in EW treated only under a particular heat condition (e.g. 100°C for 5 min) lost allergenicity, but possessed tolerogenicity.

The glycation product 6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine (pyrraline), which is formed in the final stage of the Maillard reaction, is taken up in significant amounts with the daily diet. The high bioavailability of pyrraline known from human balance studies can be partly explained by the fact that pyrraline-containing dipeptides are substrates of the human peptide transporter 1 (PEPT1). In the present study we assessed whether pyrraline-containing peptides are formed during luminal digestion of glycated proteins. For this, casein and β-casein, respectively, were enriched with peptide-bound pyrraline by incubation with 3-deoxyglucosone (3-DG), and the modified casein samples were subjected to simulated gastrointestinal digestion. The digestibility of modified casein decreased with increasing pyrraline concentration as measured by analytical size-exclusion chromatography of the digested peptide mixtures. After digestion, 50–60 % of pyrraline was bound in peptides smaller than 1,000 Da isolated by ultrafiltration. Only

Enzymatic hydrolysis of hen egg white lysozyme led to hydrolysates with pronounced inhibition of angiotensin I-converting enzyme (ACE). Two dipeptides, alanyl-tryptophan (AW) and tryptophanyl-tryptophan (WW), were identified for the first time in lysozyme hydrolysates by LC–ESI-TOF-MS and quantified by RP-HPLC-UV/fluorescence. Hydrolysis conditions were established for enhanced release of ACE-inhibiting tryptophan-containing dipeptides from alpha-lactalbumin and hen egg white lysozyme. The highest ACE inhibition potency and release of tryptophan-containing dipeptides were observed for the hydrolysates prepared by chymotrypsin followed by thermolysin. All identified dipeptides showed a significant and competitive inhibitory effect on ACE activity (AW: IC50 = 20 μmol/L; WW: IC50 = 68 μmol/L, IW: IC50 = 0.7 μmol/L, WL: IC50 = 10 μmol/L) in vitro and proved to be moderate resistant towards simulated gastrointestinal digestion. Protein hydrolysates containing tryptophan-containing dipeptides are interesting candidates as ingredients for functional foods with possible blood pressure-lowering effect, but also as a good source for the amino acid tryptophan.

Methylglyoxal (MGO) is responsible for the pronounced antibacterial activity of manuka honey, in which it may reach concentrations up to 800 mg/kg. As MGO formed in vivo is discussed to play a role in diabetic complications, the metabolic transit of dietary MGO was studied within a 3 day dietary recall with four healthy volunteers. Determination of MGO in 24 h urine was performed with GC-MS after derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, and D-lactate was quantified enzymatically. Following a diet virtually free from MGO and other glycation compounds, a defined amount of MGO (500 μmol in manuka honey) was administered in the morning of day 2. Renal excretion was between 0.1 and 0.4 μmol/day for MGO and between 50 and 220 μmol/day for D-lactate. No influence on excretion of both compounds was observed following administration of MGO. To investigate the stability of MGO under physiological conditions, a simulated in vitro gastrointestinal digestion was performed with MGO-containing honey. After 8 h of in vitro digestion, only 5-20% of the initial methylglyoxal was recovered. This indicates that dietary MGO is rapidly degraded during the digestion process in the intestine and, therefore, exerts no influence on the MGO level in vivo.

The Maillard reaction is important for beer color and flavor, but little is known about the occurrence of individual glycated amino acids in beer. Therefore, seven Maillard reaction products (MRPs), namely, fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine, were synthesized and quantitated in different types of beer (Pilsner, dark, bock, wheat, and nonalcoholic beers) by HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Free MRPs were analyzed directly. A high molecular weight fraction was isolated by dialysis and hydrolyzed enzymatically prior to analysis. Maltulosyllysine was quantitated for the first time in food. The most important free MRPs in beer are fructosyllysine (6.8-27.0 mg/L) and maltulosyllysine (3.7-21.8 mg/L). Beer contains comparatively high amounts of late-stage free MRPs such as pyrraline (0.2-1.6 mg/L) and MG-H1 (0.3-2.5 mg/L). Minor amounts of formyline (4-230 μg/L), maltosine (6-56 μg/L), and argpyrimidine (0.1-4.1 μg/L) were quantitated. Maltulosyllysine was the most significant protein-bound MRP, but both maltulosyllysine and fructosyllysine represent only 15-60% of the total protein-bound lysine-derived Amadori products. Differences in the patterns of protein-bound and free individual MRPs and the ratios between them were identified, which indicate differences in their chemical, biochemical, and microbiological stabilities during the brewing process.

Enzymatic hydrolysis of proteins from rice, soy, pea and wheat, with both chymotrypsin and thermolysin, resulted in hydrolysates, which are efficient inhibitors of the angiotensin-converting enzyme (ACE). IC50 values of the hydrolysates were between 27 and 39mg/l, which is comparable to enzymatically hydrolysed whey protein. A significant increase of the ACE-inhibiting effect was observed following butanol extraction due to accumulation of hydrophobic peptides (IC50 between 12 and 21mg/l). Based on the identification and quantification of individual tryptophan-, tyrosine- and phenylalanine-containing dipeptides, 50-80% of the total ACE-inhibiting potential of butanol extracts from plant protein hydrolysates could be explained. Compared to hydrolysates from whey protein, where the inhibitory effect can almost exclusively be attributed to Ile-Trp, the ACE inhibition by plant protein hydrolysates is caused by a variety of peptides, in particular tyrosine-containing peptides. Hydrolysates of plant proteins are promising ingredients for the development of functional foods.

Using LC-MS/MS and isotopically labeled standard substances, quantitation of free Maillard reaction products (MRPs), namely, N(ε)-(carboxymethyl)lysine (CML), 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (pyrraline, PYR), N(δ)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H), and N(ε)-fructosyllysine (FL), in bovine milk was achieved. Considerable variations in the amounts of the individual MRPs were found, most likely as a consequence of the nutritional uptake of glycated proteins. When comparing commercial milk samples labeled as originating from "organic" or "conventional" farming, respectively, significant differences in the content of free PYR (organic milk, 20-300 pmol/mL; conventional milk, 400-1000 pmol/mL) were observed. An analysis of feed samples indicated that rapeseed and sugar beet are the main sources for MRPs in conventional farming. Furthermore, milk of different dairy animals (cow, buffalo, donkey, goat, ewe, mare, camel) as well as for the first time human milk was analyzed for free MRPs. The distribution of their concentrations, with FL and PYR as the most abundant in human milk and with a high individual variability, also points to a nutritional influence. As the components of concentrated feed do not belong to the natural food sources of ruminants and equidae, free MRPs in milk might serve as indicators for an adequate animal feeding in near-natural farming and can be suitable parameters to distinguish between an "organic" and "conventional" production method of milk.

1,2-Dicarbonyl compounds, formed from carbohydrates during thermal processing in the course of caramelization and Maillard reactions, are intensively discussed as precursors for advanced glycation endproducts in foods and in vivo. To obtain information about the uptake of individual compounds with commonly consumed foods, a comprehensive analysis of the content of 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), and methylglyoxal (MGO) together with 5-hydroxymethylfurfural (HMF) in 173 food items like bakery products, pasta, nonalcoholic and alcoholic beverages, sweet spreads, and condiments was performed. Following suitable cleanup procedures, 1,2-dicarbonyl compounds were quantitated after derivatization with o-phenylenediamine via RP-HPLC with UV detection. 3-DG proved to be the predominant 1,2-dicarbonyl compound with concentrations up to 410 mg/L in fruit juices, 2622 mg/L in balsamic vinegars, and 385 mg/kg in cookies, thus exceeding the corresponding concentrations of HMF. 3-DGal was found to be of relevance in many foods even in the absence of galactose. MGO was only of minor quantitative importance in all foods studied, except for manuka honey. Dietary intake was estimated to range between 20 and 160 mg/day for 3-DG and 5 and 20 mg/day for MGO, respectively.

Caseins – the main constituents of bovine milk proteins – self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state 31 P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state 31 P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state 31 P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state 31 P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state 31 P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state 31 P HR NMR in combination with dynamic light scattering (DLS). Solid-state 31 P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.

The Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) was recently identified as a new pyrrole amino acid resulting from the reaction of free and protein-bound lysine residues with the 1,2-dicarbonyl compound 3-deoxypentosone, pentose sugars, and degradation products of disaccharides. In the present work, simultaneous analysis of formyline and its structural analog, pyrraline, in enzymatically hydrolyzed food matrices was achieved on an RP-phenyl column by high-pressure liquid chromatography (HPLC) with UV detection. The presence of formyline was for the first time unequivocally verified in the hydrolyzates and standard samples by HPLC coupled to triple quad mass spectrometry due to identity of product ion spectra after targeted fragmentation of the molecular ion of formyline. A total of 68 food samples were analyzed. Low concentrations of formyline were detected in milk and whey products. In breakfast cereals, pasta, and bakery products, formyline was quantified in concentrations up to 34.8 mg/kg. In the outmost bread crust, 0.6 % of lysine residues can be modified to formyline. Moreover, it was shown by analysis of concentrated beer samples that both formyline and pyrraline are constituents of beer proteins. From our data, we conclude that at least 2–3 mg of formyline and 20–40 mg of pyrraline are ingested with the daily diet.

In the present study, the extent of the Maillard reaction in peanuts was investigated, using N-e-fructosyllysine (e-Fru-Lys, determined via furosine) as an indicator for the early stage, and pyrraline and N-e-carboxymethyllysine (CML) as representatives for advanced glycation. In commercial samples, e-Fru-Lys ranged between 1.5 and 13.3 mmol/mol lysine. Pyrraline was found in amounts between not detectable (below 0.3 mmol/mol lysine) and 9.0 mmol/mol lysine, and CML between 0.8 and 2.7 mmol/mol lysine. Lysine modification by glycation products was very low in cooked and fried peanuts (below 1%). In laboratory-scale roasting experiments, the amount of e-Fru-Lys reached maximum values of 12.0 mmol/mol lysine after 20 min at 160 °C, whereas pyrraline increased up to 38.5 mmol/mol lysine after 25 min at 170 °C. Amount of CML was up to 1.8 mmol/mol lysine in peanuts roasted for 25 min at 170 °C. Such high amounts of pyrraline have not yet been described for any other food item. Only about one tenth of the totally observed lysine modification of up to 50% can be explained by the three glycation compounds, indicating that currently unknown reactions occur during peanut roasting. Reactions between proteins and carbonyl compounds, most likely originating from oxidative degradation of lipids, may play an important role for lysine derivatization in peanuts and should be analyzed more in detail.