Towards prediction of maturation-dependent kokumi taste in cheese by comprehensive high throughput quantitation of glutamyl dipeptides.

The study focuses on the comprehensive analysis of glutamyl dipeptides in cheese, particularly their formation during the cheese ripening process and the influence of various factors, such as origin, the use of various mold cultures, and cheese types. For the first time, all three subgroups of glutamyl dipeptides, namely α-Glu-X, X-Glu, and γ-Glu-X, are covered in a comprehensive analytical LC-MS/MS method offering robust quantitation of all 56 glutamyl dipeptides. The workflow includes a simplified extraction protocol and an optimized separation of the analytes on the stationary phase. Validation experiments demonstrate the method's reliability, including repeatability, detection limits, and recovery. The comprehensive analysis of all glutamyl dipeptides in 122 cheese samples with ripening times between 2 weeks and 15 years shows a strong increase in all peptide classes with prolonged ripening and particularly in the presence of mold.

Molecular Changes during Germination of Cocoa Beans, Part 1.

Some germination is known to occur during the process of fermentation in cocoa beans. The impact of this biological process on the course of cocoa fermentation is not known and was thus investigated. In order to determine the impact of germination at the molecular level as well as on flavor, an untargeted metabolomics approach using Ultra Performance Liquid Chromatography-Electrospray Ionization-Time of Flight-Mass Spectrometry (UPLC-ESI-ToF-MS) with simultaneous acquisition of low- and high-collision energy mass spectra (MSe) was performed. Extracts of raw and germinated cocoa beans of the same origin were measured and compared for characteristic differences by unsupervised principal component analysis. OPLS-DA revealed 12-hydroxyjasmonic acid (HOJA) sulfate, (+)-catechin and (-)-epicatechin as most down-regulated compounds as well as two hydroxymethylglutaryl (HMG) glucosides A and B among others as decisive up-regulated compounds in the germinated material. Additionally, further HMG glucosides and 12-hydroxyjasmonic acid could be identified in cocoa for the first time by coelution with isolated and synthesized reference compounds. HOJA sulfate, which has been postulated in cocoa, and HOJA were revealed to impart bitter and astringent taste qualities.

UPLC-ESI-TOF MS Profiling Discriminates Biomarkers in Authentic and Adulterated Italian Samples of Saffron (Crocus sativus L.).

Italian saffron (Crocus sativus L.) is gaining visibility due to its high quality and difference in growing area. In this study, the metabolite composition and quality of Italian saffron samples purchased from local producers and supermarkets were investigated using an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectrometry (MSe). Unsupervised statistical method (PCA) highlighted significant differences in the metabolomes, even if not related to the geographical origin. OPLS-DA revealed 9(S)-,10-(S)-,13-(S)-tri-hydroxy-11-(E)-octadecenoic acid as the most decisive compound to distinguish supermarket saffron, while oxidized crocins represented the most valuable markers to further describe the quality of saffron, even in locally produced samples. Known adulterations with paprika and turmeric were detected at a limit of 10%, and the increasing signals of cyclocurcumin was a significant biomarker for turmeric contamination. The results were underlined with conventional and kinetic antioxidant assays.

Screening of a Microbial Culture Collection: Empowering Selection of Starters for Enhanced Sensory Attributes of Pea-Protein-Based Beverages.

Pea-protein-based ingredients are gaining attention in the food industry due to their nutritional benefits and versatility, but their bitter, astringent, green, and beany off-flavors pose challenges. This study applied fermentation using microbial cultures to enhance the sensory qualities of pea-protein-based beverages. Using UHPLC-TOF-MS analyses along with sensory profile comparisons, microbial species such as Limosilactobacillus fermentum, Lactococcus lactis, Lactobacillus johnsonii, Lacticaseibacillus rhamnosus, and Bifidobacterium longum were preselected from an entire culture collection and found to be effective in improving the overall flavor impression by reducing bitter off-notes and enhancing aroma profiles. Notably, L. johnsonii NCC533 and L. fermentum NCC660 exhibited controlled proteolytic activities after 48 h of fermentation, enriching the matrix with taste-active amino acids, nucleotides, and peptides and improving umami and salty flavors while mitigating bitterness. This study has extended traditional volatile analyses, including nonvolatile metabolomic, proteomic, and sensory analyses and offering a detailed view of fermentation-induced biotransformations in pea-protein-based food. The results highlight the importance of combining comprehensive screening approaches and sensoproteomic techniques in developing tastier and more palatable plant-based protein products.

Sensoproteomic Characterization of Lactobacillus Johnsonii-Fermented Pea Protein-Based Beverage: A Promising Strategy for Enhancing Umami and Kokumi Sensations while Mitigating Bitterness.

This study investigated the mechanism underlying the flavor improvement observed during fermentation of a pea protein-based beverage using Lactobacillus johnsonii NCC533. A combination of sensomics and sensoproteomics approach revealed that the fermentation process enriched or generated well-known basic taste ingredients, such as amino acids, nucleotides, organic acids, and dipeptides, besides six new taste-active peptide sequences that enhance kokumi and umami notes. The six new umami and kokumi enhancing peptides, with human recognition thresholds ranging from 0.046 to 0.555 mM, are produced through the degradation of Pisum sativum's storage protein. Our findings suggest that compounds derived from fermentation enhance umami and kokumi sensations and reduce bitterness, thus improving the overall flavor perception of pea proteins. In addition, the analysis of intraspecific variations in the proteolytic activity of L. johnsonii and the genome-peptidome correlation analysis performed in this study point at cell-wall-bound proteinases such as PrtP and PrtM as the key genes necessary to initiate the flavor improving proteolytic cascade. This study provides valuable insights into the molecular mechanisms underlying the flavor improvement of pea protein during fermentation and identifies potential future research directions. The results highlight the importance of combining fermentation and senso(proteo)mics techniques in developing tastier and more palatable plant-based protein products.

Quantitation of Key Antioxidants and Their Contribution to the Oxidative Stability of Beer.

A sensitive high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MSMRM) method, leveraging both technique and internal calibration, was developed for the simultaneous and comprehensive quantitative analysis of 46 antioxidants and antioxidant precursors in different beer types without any cleanup procedure. Combined with their in vitro antioxidant activity, a dose-activity estimation exposed a group of 10 key antioxidants, namely, tryptophan, tyrosine, hordatine A, hordatine B, procyanidin B3, prodelphinidin B3, tachioside (3-methoxy-4-hydroxyphenyl-ß-d-glucopyranoside), (+)-catechin, tyrosol, and ferulic acid. To study the effect of antioxidants in spiking and aging studies, another liquid chromatography-MS (LC-MS)-based method was developed, monitoring markers for oxidation in beer. A positive effect of the antioxidants on the flavor stability at naturally relevant concentrations was shown by a slowing of oxygen-dependent aging reactions highlighted in beer storage trials under oxygen atmosphere. Thereby, a doubling of the natural concentration of all investigated antioxidants in beer revealed a limit inhibition of 67% on the degradation of cis-isocohumulone to hydroxy-cis-alloisocohumulone.

Development of a Global Metabo-Lipid-Prote-omics Workflow to Compare Healthy Proximal and Distal Colonic Epithelium in Mice.

A multimetabo-lipid-prote-omics workflow was developed to characterize the molecular interplay within proximal (PC) and distal (DC) colonic epithelium of healthy mice. This multiomics data set lays the foundation to better understand the two tissue types and can be used to study, for example, colon-related diseases like colorectal cancer or inflammatory bowel disease. First, the methyl tert-butyl ether extraction method was optimized, so that from a single tissue biopsy >350 reference-matched metabolites, >1850 reference-matched lipids, and >4500 proteins were detected by using targeted and untargeted metabolomics, untargeted lipidomics, and proteomics. Next, each omics-data set was analyzed individually and then merged with the additional omics disciplines to generate a deep understanding of the underlying complex regulatory network within the colon. Our data demonstrates, for example, differences in mucin formation, detected on substrate level as well as on enzyme level, and altered lipid metabolism by the detection of phospholipases hydrolyzing sphingomyelins to ceramides. In conclusion, the combination of the three mass spectrometry-based omics techniques can better entangle the functional and regional differences between PC and DC tissue compared to each single omics technique.

Molecular Changes during Germination of Cocoa Beans, Part 2.

A recently published untargeted metabolomics approach toward marker compounds of cocoa germination revealed and identified 12-hydroxyjasmonic acid sulfate, (+)-catechin, and (-)-epicatechin as the most downregulated compounds and two hydroxymethylglutaryl glucosides (HMG gluc) A and B, among others, as the decisive upregulated compounds in the germinated material. These findings were quantitatively evaluated using ultrahigh-performance liquid chromatography-tandem mass spectrometry not only in previously examined sample material but also in a vastly expanded array of cocoa samples of different provenience and process and in cocoa products such as cocoa liquor and chocolate. Hereby, yields of newly identified HMG gluc derivatives could be determined in raw, fermented, germinated, and alternatively processed cocoa, and isomers of HMG gluc A and B could be established as key process indicators. Based on unsupervised clustering and supervised classification, models could identify germinated samples in testing sets consisting of raw, fermented, and germinated samples.

Isolation and Identification of Novel Taste-Modulating N2-Guanosine 5'-Monophosphate Derivatives Generated by Maillard-Type Reactions.

Several compounds with taste-modulating properties have been investigated, improving the taste impression without having a pronounced intrinsic taste. The best-known representatives of umami taste-modulating compounds are ribonucleotides and their derivatives. Especially the thio derivatives showed high taste-modulating potential in structure-activity relationship investigations. Therefore, this study focuses on the formation of guanosine 5'-monophosphate derivatives consisting of Maillard-type generated compounds like the aroma-active thiols (2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-furfurylthiol) and formaldehyde to gain insights into the potential of combinations of taste and aroma-active compounds. One literature-known (N2-(furfurylthiomethyl)-guanosine 5'-monophosphate) and three new derivatives (N2-(2-methyl-1-furylthiomethyl)-guanosine 5'-monophosphate, N2-((5-hydroxymethyl)-2-methyl-1-furylthiomethyl)-guanosine 5'-monophosphate, N2-((2-pentanon-1-yl)thiomethyl)-guanosine 5'-monophosphate) were successfully produced using green natural deep eutectic solvents and isolated, and their structures were completely elucidated. Besides the intrinsic taste properties, the kokumi and umami taste-modulating effects of the four derivatives were evaluated via psychophysical investigations, ranging from 19 to 22 µmol/L.

Quantitative Antioxidant Profiling Throughout Beer Brewing Followed by Discovery and Isolation of Precursors from Barley (Hordeum vulgare L.).

The application of high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) revealed the origin and evolution of antioxidants during the brewing process of hopped and unhopped reference beer. As tachioside (3-methoxy-4-hydroxyphenyl-ß-d-glucopyranoside), arbutin (4-hydroxyphenyl-ß-d-glucopyranoside), and hordatines clearly increased during the fermentation step, the raw material barley was investigated as a source of the corresponding precursors. Therefore, 4-hydroxyphenyl-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside, 4-hydroxy-3-methoxyphenyl-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside, 4-hydroxy-3-methoxyphenyl-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside, and 4-hydroxy-2-methoxyphenyl-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside were isolated from barley for the first time, and identified using liquid chromatography-mass spectrometry (LC-MS) and one-dimensional/two-dimensional-nuclear magnetic resonance (1D/2D-NMR) experiments. Moreover, hordatine glucosides A, B, and C were isolated and identified from barley, and hordatine C glucoside was characterized for the first time. A fermentation model followed by HPLC-MS/MS analysis substantiated the release of tachioside from 4-hydroxy-3-methoxyphenyl-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside by Saccharomyces cerevisiae. Quantitation experiments monitoring the content in wheat, barley, and different barley malt types demonstrated a wide range of concentrations, providing a basis for further comprehensive investigations to optimize the antioxidant yield in beer to contribute to improved flavor stability.

Human Sensory, Taste Receptor, and Quantitation Studies on Kaempferol Glycosides Derived from Rapeseed/Canola Protein Isolates.

Beyond the key bitter compound kaempferol 3-O-(2‴-O-sinapoyl-ß-d-sophoroside) previously described in the literature (1), eight further bitter and astringent-tasting kaempferol glucosides (2-9) have been identified in rapeseed protein isolates (Brassica napus L.). The bitterness and astringency of these taste-active substances have been described with taste threshold concentrations ranging from 3.3 to 531.7 and 0.3 to 66.4 µmol/L, respectively, as determined by human sensory experiments. In this study, the impact of 1 and kaempferol 3-O-ß-d-glucopyranoside (8) on TAS2R-linked proton secretion by HGT-1 cells was analyzed by quantification of the intracellular proton index. mRNA levels of bitter receptors TAS2R3, 4, 5, 13, 30, 31, 39, 40, 43, 45, 46, 50 and TAS2R8 were increased after treatment with compounds 1 and 8. Using quantitative UHPLC-MS/MSMRM measurements, the concentrations of 1-9 were determined in rapeseed/canola seeds and their corresponding protein isolates. Depending on the sample material, compounds 1, 3, and 5-9 exceeded dose over threshold (DoT) factors above one for both bitterness and astringency in selected protein isolates. In addition, an increase in the key bitter compound 1 during industrial protein production (apart from enrichment) was observed, allowing the identification of the potential precursor of 1 to be kaempferol 3-O-(2‴-O-sinapoyl-ß-d-sophoroside)-7-O-ß-d-glucopyranoside (3). These results may contribute to the production of less bitter and astringent rapeseed protein isolates through the optimization of breeding and postharvest downstream processing.

Enrichment and Quantitation of Dipeptidyl Peptidase IV Inhibitory Peptides in Quinoa upon Systematic Malting.

Food-derived peptides with an inhibitory effect on dipeptidyl peptidase IV (DPP-IV) can be used as an additive treatment for type 2 diabetes. The inhibitory potential of food depends on technological protein hydrolysis and gastrointestinal digestion, as the peptides only act after intestinal resorption. The effect of malting as a hydrolytic step on the availability of these peptides in grains has yet to be investigated. In this study, quinoa was malted under systematic temperature, moisture, and time variations. In the resulting malts, the DPP-IV inhibition reached a maximum of 45.02 (±10.28) %, whereas the highest overall concentration of literature-known inhibitory peptides was 4.07 µmol/L, depending on the malting parameters. After in vitro gastrointestinal digest, the inhibition of most malts, as well as the overall concentration of inhibitory peptides, could be increased significantly. Additionally, the digested malts showed higher values in both the inhibition and the peptide concentration than the unmalted quinoa. Concerning the malting parameters, germination time had the highest impact on the inhibition and the peptide concentration after digest. An analysis of the protein sizes before and after malting gave first hints toward the origin of these peptides, or their precursors, in quinoa.

Exploring the Relationships Between Bacterial Community, Taste-Enhancing Peptides and Aroma in Thai Fermented Fish (Pla-ra).

As a traditional Thai condiment, Pla-ra is used to add flavor and richness to dishes. Nine treatment combinations of Pla-ra formulations created from 3 types of fish (Mor fish, Kradee fish, and Mor + Kradee fish) and 4 different carbohydrate sources (none, rice bran, roasted rice, and rice bran─roasted rice mixture) were studied through a 12 month fermentation period (1, 3, 5, 7, 8, 9, 10, 11, and 12 months). 16S rRNA Next Generation Sequencing (NGS) and LC-MS/MS techniques were used to analyze the microbial diversity and identify taste-enhancing peptides. Descriptive sensory analysis was performed on the extracts of the 108 Pla-ra samples mixed in a model broth. Koku perception and saltiness-enhancing attributes were clearly perceived and dominant in all samples, even though glutamyl peptides, including γ-Glu-Val-Gly, were found at subthreshold levels. The samples from mixed fish and Mor fish fermented with roasted ground rice and rice bran for 12 months had the most typical Pla-ra odors and tastes and had high taste-enhancing activities. NGS analysis revealed the presence of bacteria containing a large number of protease and aminopeptidase genes in the samples. Bacillus spp., Gallicola spp., and Proteiniclasticum spp. correlated well with the generation of glutamyl and arginyl peptides and typical odors in the samples. These results confirmed the typical sensory quality of Pla-ra depended on protein sources, carbohydrate sources, and bacteria communities. Further optimization of the microbial composition found could lead to the development of starter cultures to control and promote flavor development in fermented fish products.

Curry-Odorants and Their Metabolites Transfer into Human Milk and Urine.

SCOPE: The excretion of dietary odorants into urine and milk is evaluated and the impact of possible influencing factors determined. Furthermore, the metabolic relevance of conjugates for the excretion into milk is investigated. METHODS AND RESULTS: Lactating mothers (n = 20) are given a standardized curry dish and donated one milk and urine sample each before and 1, 2, 3, 4.5, 6, and 8 h after the intervention. The concentrations of nine target odorants in these samples are determined. A significant transition is observed for linalool into milk, as well as for linalool, cuminaldehyde, cinnamaldehyde, and eugenol into urine. Maximum concentrations are reached within 1 h after the intervention in the case of milk and within 2-3 h in the case of urine. In addition, the impact of glucuronidase treatment on odorant concentrations is evaluated in a sample subset of twelve mothers. Linalool, eugenol, and vanillin concentrations increased 3-77-fold in milk samples after treatment with ß-glucuronidase. CONCLUSION: The transfer profiles of odorants into milk and urine differ qualitatively, quantitatively, and in temporal aspects. More substances are transferred into urine and the transfer needs a longer period compared with milk. Phase II metabolites are transferred into urine and milk.

Chemical Characterization of Red Wine Polymers and Their Interaction Affinity with Odorants.

In order to characterize red wine polymers with regard to their binding properties to aroma compounds (odorants), a qualitative and quantitative analysis of chemical degradation products after different chemical treatments (thiolytic, acidic, and alkaline depolymerization) of high -molecular-weight (HMW) fractions of red wine was performed. Using 1H NMR, LC-ToF-MS, LC-MS/MS, and HPIC revealed key structural features such as carbohydrates, organic acids, phenolic compounds, anthocyanins, anthocyanidins, amino acids, and flavan-3-ols responsible for odorant-polymer interactions. Further, NMR-based interaction studies of the selected aroma compounds 3-methylbutanol, cis-whisky lactone, 3-methylbutanoic acid, and 3-isobutyl-2-methoxypyrazine with HMW polymers after chemical treatment demonstrated a reduced interaction affinity of the polymer compared to the native HMW fractions, and further, the importance of aromatic compounds such as flavan-3-ols for the formation of odorant polymer interactions. In addition, these observations could be verified by human sensory experiments. For the first time, the combination of a compositional analysis of red wine polymers and NMR-based interaction studies with chemically treated HMW fractions enabled the direct analysis of the correlation of the polymer's structure and its interaction affinity with key odorants in red wine.

Effect of peptide formation during rapeseed fermentation on meat analogue structure and sensory properties at different pH conditions.

This study aimed to modify the sensory properties of rapeseed protein concentrate using a combination of fermentation and high-moisture extrusion processing for producing meat analogues. The fermentation was carried out with Lactiplantibacillus plantarum and Weissella confusa strains, known for their flavour and structure-enhancing properties. Contrary to expectations, the sensory evaluation revealed that the fermentation induced bitterness and disrupted the fibrous structure formation ability due to the generation of short peptides. On the other hand, fermentation removed the intensive off-odour and flavour notes present in the native raw material. Several control treatments were produced to understand the reasons behind the hindered fibrous structure formation and induced bitterness. The results obtained from peptidomics, free amino ends, and solubility analyses strongly indicated that the proteins were hydrolysed by endoproteases activated during the fermentation process. Furthermore, it was suspected that the proteins and/or peptides formed complexes with other components, such as hydrolysis products of glucosinolates and polysaccharides.

A 14-Day Double-Blind, Randomized, Controlled Crossover Intervention Study with Anti-Bacterial Benzyl Isothiocyanate from Nasturtium (Tropaeolum majus) on Human Gut Microbiome and Host Defense.

Despite substantial heterogeneity of studies, there is evidence that antibiotics commonly used in primary care influence the composition of the gastrointestinal microbiota in terms of changing their composition and/or diversity. Benzyl isothiocyanate (BITC) from the food and medicinal plant nasturtium (Tropaeolum majus) is known for its antimicrobial activity and is used for the treatment of infections of the draining urinary tract and upper respiratory tract. Against this background, we raised the question of whether a 14 d nasturtium intervention (3 g daily, N = 30 healthy females) could also impact the normal gut microbiota composition. Spot urinary BITC excretion highly correlated with a weak but significant antibacterial effect against Escherichia coli. A significant increase in human beta defensin 1 as a parameter for host defense was seen in urine and exhaled breath condensate (EBC) upon verum intervention. Pre-to-post analysis revealed that mean gut microbiome composition did not significantly differ between groups, nor did the circulating serum metabolome. On an individual level, some large changes were observed between sampling points, however. Explorative Spearman rank correlation analysis in subgroups revealed associations between gut microbiota and the circulating metabolome, as well as between changes in blood markers and bacterial gut species.

Combinatorial biosynthesis in yeast leads to over 200 diterpenoids.

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.