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The effect of the released amount and bitterness threshold of bitter peptides on the sensory properties of different wheat gluten hydrolysates (WGHs) after hydrolysis was investigated. The results showed that the endo-activity of the enzyme promoted the release of bitter peptides, leading to enhanced bitterness intensity in WGHs. With the increase in degree of hydrolysis (DH), the bitter threshold of bitter peptides became the main reason affecting bitterness of the WGHs. Proteax exerted the strong exo-activity at the DH of 20%, which could reduce bitterness of Pro-16 hydrolysates. The reason for debittering was the reduction in the content with molecular weights (MWs) of 500-1000 Da and the decrease of surface hydrophobicity (SH) in the Pro-20 M hydrolysates, which led to the increase of the bitterness threshold of bitter peptide. Meanwhile, HPLC-MS/MS analysis demonstrated the reduced proportion of C-terminal hydrophobic amino acids (HAAs) in Pro-20 M extracts verifying the cause of debittering.

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About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

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In view of potential future changes of German food legislation with regard to cheese product quality parameters, this study aimed to evaluate the quality of whey protein-enriched semihard cheese (WPEC). Model WPEC was produced in a pilot plant and on an industrial scale by adding defined amounts of high-heat (HH) milk to the cheese milk and comprehensively analyzed during cheese processing. The dry matter, total protein, pure protein, fat, and sodium chloride content of six-week ripened cheese samples were not significantly different (p < 0.05) when the technologically necessary heating of the curd was adapted to the amount of HH milk. However, the ripening, firmness, and melting behavior of WPEC was different compared to cheese without HH milk. During ripening, no formation of whey protein peptides was observed, but differences in the amount of some bitter peptides deriving from the casein fraction were found. Sensory data suggested a slightly more bitter taste perception by the panelists for the WPEC. Further technological adjustments are recommended to obtain marketable WPEC.

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Peptides in cheese flavoring produced through proteolysis plus fermentation generated bitterness. Bitterness of individual peptide can be quantified using quantitative structure-activity relationship, where molecular mass (M), hydrophobicity, residues, C-terminal hydrophobic amino acids (C-HAAs), and N-terminal basic ones (N-BAAs) are crucial. However, their accumulative influence on the overall bitterness of peptide mixture remains unknown. This study delved into extensive proteolysis to debitter and to correlate the multi-influencing factors of peptides and the collective bitterness. As hydrolysis increased from 7.5 % to 28.0 %, bitterness reduced from 5.0 to 0.3-2.7 scores, contingent on proteases used, in which FU was optimal. The overall bitterness cannot be predicted through the summation of individual peptide bitterness, which depended on M (0.5-3 kDa) and 5-23 residues, followed by N-BAAs and C-HAAs. Analysis of enzymatic cleavage sites and substrate characteristics revealed, to more effectively debitter bovine milk protein hydrolysates, proteases specifically cleaving Pro, Leu, Phe, and Val were desired.

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Fresh leaves of Echa 1 were fixed by roller, steam/hot air and light-wave, and the effects of the three fixation methods on the chemical characteristics of straight-shaped green teas (GTs) were studied by widely targeted metabolomic analysis. 1001 non-volatile substances was identified, from which 97 differential metabolites were selected by the criteria of variable importance in projection (VIP) > 1, p < 0.05, and |log2(fold change)| > 1. Correlation analysis indicated that 14 taste-active metabolites were the major contributors to the taste differences between differently processed GTs. High-temperature fixation induces protein oxidation or degradation, γ-glutamyl peptide transpeptidation, degradation of flavonoid glycosides and epimerization of cis-catechins, resulting in the accumulation of amino acids, peptides, flavonoids and trans-catechins, which have flavor characteristics such as umami, sweetness, kokumi, bitterness and astringency, thereby affecting the overall taste of GTs. These findings provided a scientific basis for the directional processing technology of high-quality green tea.

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Fermented soybean foods with a long history are popular worldwide because of rich nutrition. However, many traditional fermented soybean foods have unacceptable bitterness, which mostly comes from the bitter peptides produced from the hydrolysis of soybean proteins. In this review, the bitter peptides in fermented soybean foods is briefly reviewed. The structural properties of bitter receptors and bitter peptides were reviewed. Bitterness is perceived through the binding between bitter compounds and specific sites of bitter receptors (25 hTAS2Rs), which further activate the downstream signal pathway mediated by G-protein. And it converts chemical signals into electrical signals, and transmit them to the brain. In addition, the influencing factors of bitter peptides in fermented soybean foods were summarized. The bitterness of fermented soybean foods primarily results from the raw materials, microbial metabolism during fermentation, unique techniques, and interactions of various flavor compounds. Moreover, the structure-bitterness relationship of bitter peptides was also discussed in this review. The bitterness degree of the bitter peptide is related to the polypeptide hydrophobicity, amino acids in the peptide, peptide molecular weight and polypeptide spatial structure. Studying the bitter peptides and their bitter characteristics in fermented soybean foods is beneficial for improving the sensory quality of fermented soybean foods and prompting more consumers accept them.

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The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D-spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC-tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D-spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.

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Bitterness is a common flavor attribute of aged cheese associated with the peptide fraction, but excessive levels are a defect leading to consumer rejection. Bitterness in cheese has been primarily associated with peptides that arise from the breakdown of casein. The last review of bitter peptides was published in 1992. This updated review compiled information about the bitter peptides published up to 2022. Our comprehensive search of the literature compiled 226 peptides associated with bitterness and cheese protein origins into a database (Supplemental Materials). The influences of a peptide's physical properties, such as molecular weight, average hydrophobicity, peptide length, number of prolines and the presence of hydrophobic amino acids in the peptide's terminus, were assessed for correlation with bitterness threshold values this assessment found that, among variables considered, higher molecular weight had the strongest correlation with higher bitterness among known peptides. Heatmaps of bitter peptides and their bitterness threshold values highlight ß-casein as the primary source of known bitter peptides in cheese. This comprehensive database of cheese protein-derived bitter peptides and this discovery of the correlation of a peptide's physical properties to bitterness will aid future researchers in the identification and discovery of contributors to cheese bitterness.

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Casein hydrolysates are important additives to foods for elderly and sports nutrition. However, due to the enzymatic generation of so-called bitter peptides, their application is limited. Therefore, the procedure needs to be optimized in order to restrict their occurrence. For this, extensive sensory evaluations are necessary. By combining two separation techniques using comprehensive two-dimensional liquid chromatography, we present a novel method for estimating the bitter taste of hydrolysate samples on the basis of their elution pattern. Using a size exclusion column in the first and a reversed phase column in the second dimension allows for a detailed sample evaluation regarding peptide size and relative hydrophobicity. The results obtained for different casein hydrolysates were correlated with the sensory evaluation. We found that hydrolysates with increasing bitterness contain a higher amount of peptides of high hydrophobicity and a molecular weight less than 6.5 kDa.

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Bitter peptides in the enzymatic hydrolysates were prepared and purified from wheat gluten using aqueous ethanol solutions and macroporous resin, which has opened a new road for the extraction and separation of bitter peptides. This report contains the release regularity of bitter peptides and the factors affecting the change of bitter intensity during enzymatic hydrolysis, providing a scientific basis for the research on debitterizing method. In this study, the effects of different degrees of hydrolysis (DH) and enzyme active sites on the bitter peptide content and bitter taste thresholds were discussed. The relationship between amino acid composition, molecular weight distribution, surface hydrophobicity and bitter taste thresholds was extensively researched. The results showed the exposure of hydrophobic amino acids and the bitterness intensity of the hydrolysates increased as the DH increased, and the bitterness of wheat gluten hydrolysates (WGHs) hydrolyzed by Alcalase was stronger than that of Trypsin. According to correlation analysis, the proportion of total hydrophobic amino acid is the first factor that affects the sensory properties of bitter peptide, and the release content of bitter peptides and the content of total bitter amino acids are the second, following by the content of peptide in the molecular weight range of 500-1,000 Da and the surface hydrophobicity. The amino acid sequence of bitter peptides from WGHs were identified and predicted using high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and bioinformatics. It was found that the molecular weight of most of the peptides was below 1,500 Da, and the Q value was higher than 5.86 kJ/mol.

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Recent scientific evidence indicates that protein hydrolysates contain bioactive peptides that have potential benefits for human health. However, the bitter-tasting hydrophobic peptides in protein hydrolysates negatively affect the sensory quality of resulting products and limit their utilization in food and pharmaceutical industries. The approaches to reduce, mask, and remove bitter taste from protein hydrolysates have been extensively reported. This review paper focuses on the advances in the knowledge regarding the structure-bitterness relationship of peptides, the release mechanism of bitter peptides, and the debittering methods for protein hydrolysates. Bitter tastes generating with enzymatic hydrolysis of protein is influenced by the type, concentration, and bitter taste threshold of bitterness peptides. A "bell-shaped curve" is used to describe the relationship between the bitterness intensity of the hydrolysates and the degree of hydrolysis. The bitter receptor perceives bitter potencies of bitter peptides by the hydrophobicity recognition zone. The intensity of bitterness is influenced by hydrophobic and electronic properties of amino acids and the critical spatial structure of peptides. Compared to physicochemical debittering (i.e., selective separation, masking of bitter taste, encapsulation, Maillard reaction, and encapsulation) and other biological debittering (i.e., enzymatic hydrolysis, enzymatic deamidation, plastein reaction), enzymatic hydrolysis is a promising debittering approach as it combines protein hydrolyzation and debittering into a one-step process, but more work should be done to advance the knowledge on debittering mechanism of enzymatic hydrolysis and screening of suitable proteases. Further study can focus on combining physicochemical and biological approaches to achieve high debittering efficiency and produce high-quality products.

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Eating satiating, protein-rich foods is one of the key aspects of modern diet, although a bitter off-taste often limits the application of some proteins and protein hydrolysates, especially in processed foods. Previous studies of our group demonstrated that bitter-tasting food constituents, such as caffeine, stimulate mechanisms of gastric acid secretion as a signal of gastric satiation and a key process of gastric protein digestion via activation of bitter taste receptors (TAS2Rs). Here, we tried to elucidate whether dietary non-bitter-tasting casein is intra-gastrically degraded into bitter peptides that stimulate mechanisms of gastric acid secretion in physiologically achievable concentrations. An in vitro model of gastric digestion was verified by casein-fed pigs, and the peptides resulting from gastric digestion were identified by liquid chromatography-time-of-flight-mass spectrometry. The bitterness of five selected casein-derived peptides was validated by sensory analyses and by an in vitro screening approach based on human gastric parietal cells (HGT-1). For three of these peptides (YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV), an upregulation of gene expression of TAS2R16 and TAS2R38 was observed. The functional involvement of these TAS2Rs was verified by siRNA knock-down (kd) experiments in HGT-1 cells. This resulted in a reduction of the mean proton secretion promoted by the peptides by up to 86.3 ± 9.9% for TAS2R16kd (p < 0.0001) cells and by up to 62.8 ± 7.0% for TAS2R38kd (p < 0.0001) cells compared with mock-transfected cells.

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Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.

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During the last few years, key taste-active compounds have been isolated and identified by means of a combination of a time- and lab-consuming successive fractionation and sensory characterization. Because the peptidome of fermented, protein-rich food is very complex, new strategies are necessary to accelerate the identification of taste-active peptides. In this study, two advanced mass spectrometric approaches were developed to comprehensively map the bitter tasting peptidome of fermented foods by data-independent acquisition (DIA) using sequential window acquisition of all theoretical fragment ion-mass spectrometry (SWATH-MS) and an in silico-assisted triple quadrupole (QQQ)-based targeted proteomics approach, separately. Application of both techniques on two fresh cheese samples as well as on crude medium-pressure liquid chromatography fractions exhibiting intense bitter taste, followed by filtering the hydrophobic target peptides (Q value of ≥1200 cal/mol) showing a signal-to-noise ratio of ≥10 and a fold change of ≥3 when comparing the less bitter to the more bitter cheese sample, revealed the candidate bitter peptides, which were then validated by means of synthetic reference peptides and human sensory evaluation. The bitter peptides were then quantitated in the fresh cheese samples as well as in a series of dairy products by means of QQQ-MS and SWATH-MS, separately. Although the QQQ-MS method showed 2-80-fold lower limits of quantitation (LOQ), the SWATH-MS method could be shown for the first time to enable the comprehensive quantitation of all sensorially relevant key bitter peptides with LOQs far below the bitter taste recognition concentration of each peptide.

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Multiple linear regression (MLR) models were constructed to explain the bitter taste of di- and tripeptides based on their chemical nature (structure). Sequences (51 di- and 51 tripeptides) were derived from the BIOPEP-UWM database of sensory peptides and amino acids. The measure of their bitterness was Rcaf. , that is, bitterness relative to that of 1 mM caffeine solution (Rcaf. = 1.0). The variables were the indices describing properties of a single residue forming a peptide structure taken from ProtScale and Biological Magnetic Resonance Data Bank. MLR was made for two separate data sets by use of Statistica 13.1. We found that the presence of branched side residues or ring in a di- or tripeptide sequence (as in L, I, V, Y, F) affected its bitterness. Another variable affecting the bitter taste of di- and tripeptides was the hydrophobicity of amino acids. Using the commonly available statistical tools as well as chemical information reflecting the nature of peptides may be helpful in understanding the structure-taste relationship in food peptides. PRACTICAL APPLICATIONS: Our approach takes account of bioinformatic and cheminformatic techniques of data mining to analyze structure-bitterness of di- and tripeptides derived from food protein sources. Data on bitter peptides available in databases of biological and chemical information can be useful in creating models which help understanding the relationship between the role of structural properties of a molecule (e.g., peptide) and its function (e.g., taste). The bitterness of a peptide resulting from the presence of specific residues in its sequence, which represent different physicochemical properties may contribute to extending the knowledge about their taste-forming role in food systems. Such knowledge may be useful in designing food products with improved properties like taste which can be either enhanced or masked (considered as unwanted when thinking about the sensory value of foods). Our research strategy is universal and can also be applied to study structure-function relationships of peptides with other activities.

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BACKGROUND: Many studies have been performed over the past four decades to identify and quantify the odor-active key volatiles in yeast extract (YE) but knowledge of the nonvolatile taste compounds is still rather fragmentary. In particular, research on bitter peptides with various structures during the thermal treatment of YE is still scarce. RESULTS: Compounds imparting a bitter taste to thermally treated YE were investigated using sensory-guided fractionation. This research found that when the treatment temperature reached 130 °C, bitter peptides were generated. Sensory evaluation of the purified, synthesized peptides revealed that four of these peptides showed a pronounced bitter taste with a taste dilution (TD) factor from 5 to 9. Guidance is provided for the production of bitter peptides in the flavor industry. CONCLUSION: Based on results from previous work on umami peptides, and this study, keeping the thermal reaction temperature under 120 °C could maximize the umami flavor and control bitterness so that it remains in an acceptable range. © 2019 Society of Chemical Industry.

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Chickpeas are inexpensive, protein rich (approximately 20% dry mass) pulses available worldwide whose consumption has been correlated with positive health outcomes. Dietary peptides are important molecules derived from dietary proteins, but a comprehensive analysis of the peptides that can be produced from chickpea proteins is missing in the literature. This review provides information from the past 20 years on the enzymatic production of peptides from chickpea proteins, the reported bioactivities of chickpea protein hydrolysates and peptides, and the potential bitterness of chickpea peptides in food products. Chickpea peptides have been enzymatically produced with pepsin, trypsin, chymotrypsin, alcalase, flavorzyme, and papain either alone or in combination, but the sequences of many of the peptides in chickpea protein hydrolysates remain unknown. In addition, a theoretical hydrolysis of chickpea legumin by stem bromelain and ficin was performed by the authors to highlight the potential use of these enzymes to produce bioactive chickpea peptides. Antioxidant activity, hypocholesterolemic, and angiotensin 1-converting enzyme inhibition are the most studied bioactivities of chickpea protein hydrolysates and peptides, but anticarcinogenic, antimicrobial, and anti-inflammatory effects have also been reported for chickpea protein hydrolysates and peptides. Chickpea bioactive peptides are not currently commercialized, but their bitterness could be a major impediment to their incorporation in food products. Use of flavorzyme in the production of chickpea protein hydrolysates has been proposed to decrease their bitterness. Future research should focus on the optimization of chickpea bioactive peptide enzymatic production, studying the bioactivity of chickpea peptides in humans, and systematically analyzing chickpea peptide bitterness.

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Aiming at the identification of the key bitter peptides in fermented foods, a new approach, coined "sensoproteomics", was developed and applied to fresh cheese samples differing in bitter taste intensity. By means of MPLC fractionation of the water-soluble cheese extracts in combination with taste dilution analysis, complex fractions with intense bitter taste were located and then screened by UPLC-MS/MS for the entire repertoire of ∼1600 candidate peptides, extracted from a literature meta-analysis on dairy products, by using a total of 120 selected reaction monitoring methods computed in silico. A total of 340 out of the 1600 peptides were found in the cheese samples, among which 17 peptides were identified as candidate bitter peptides by considering only peptides that were located in the bitter-tasting MPLC fractions (signal-to-noise ratio: ≥10) with a fold-change of ≥3 when comparing the less bitter to the more bitter cheese sample and that were validated by comparison with the synthetic reference peptides. While EIVPNS[phos]VEQK (αs1-CN70-78) and INTIASGEPT (κ-CN122-131) did not exhibit any bitter taste up to 2000 µmol/L, 15 of the 17 target peptides showed bitter taste thresholds ranging from 30 (ARHPHPHLSFM, κ-CN96-106) to 690 µmol/L (IQKEDVPS, αs1-CN81-88). Finally, quantitative peptide analysis followed by calculation of dose-overthreshold factors revealed a primary contribution of MAPKHKEMPFPKYPVEPF (ß-CN102-119) and ARHPHPHLSFM (κ-CN96-106) to the perceived bitter taste of the fresh cheese samples. Finally, the evolution of the bitter peptides throughout two different fresh cheese manufacturing processes was quantitatively recorded.

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The large yellow croakers (Larimichthys crocea) are mainly present in the Chinese coast and near seas with high economic importance, but vulnerable to many diseases, especially in the breeding and aquaculture. The purpose of this research was to boost the innate immune system of the large yellow croaker by administering bitter peptides into their peritoneal cavity. Total 120 Juvenile of large yellow croakers in very even weight of 60 g were divided into 4 different groups in 200/300 L of water tank, respectively. Fish growth were observed for 3 months before and after different treatments. The bitter peptides from pepsin hydrolysis were applied because they possess the highest bitter sensory scores. The blood of fish from the different groups was collected and tested for different immune parameters to evaluate the effectiveness of bitter peptides as immune stimulants after administration for 8 weeks. The average ratio of leukocytes/total blood cells (%) for control was found at 14.6%, for the low dose of bitter peptides 0.6 mg/fish was at 29.3%, for middle dose of 1.2 mg/fish was at 35%, and high dose of 2.4 mg/fish was at 30%. The lysozyme assay showed that the OD (optical density) units of relative progress lysis activity at 60 min were 0.17, 0.101, 0.307 and 0.198, respectively. Similarly in the same order as in phagocyte assay, most importantly the middle dose (1.2mg/fish) gave the highest survival rate throughout the assay. The results showed that bitter peptides can be used as immune boosters for the yellow croakers and the optimum dose was 1.2 mg/fish due to both leukocytes and lysozyme activity in the treated samples increased significantly compared with the control group. According to the results obtained, we suggest that the incorporation of middle dose of bitter peptides into fish feeds may reduce the fish diseases in aquaculture, at least for large yellow croakers.

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