CmBr confers fruit bitterness under CPPU treatment in melon.

Many biotic or abiotic factors such as CPPU (N-(2-chloro-pyridin-4-yl)-N'-phenylurea), a growth regulator of numerous crops, can induce bitterness in cucurbits. In melon, cucurbitacin B is the major compound leading to bitterness. However, the molecular mechanism underlying CuB biosynthesis in response to different conditions remains unclear. Here, we identified a set of genes involved in CPPU-induced CuB biosynthesis in melon fruit and proposed CmBr gene as the major regulator. Using CRISPR/Cas9 gene editing, we confirmed CmBr's role in regulating CuB biosynthesis under CPPU treatment. We further discovered a CPPU-induced MYB-related transcription factor, CmRSM1, which specifically binds to the Myb motif within the CmBr promoter and activates its expression. Moreover, we developed an introgression line by introducing the mutated Cmbr gene into an elite variety and eliminated CPPU-induced bitterness, demonstrating its potential application in breeding. This study offers a valuable tool for breeding high-quality non-bitter melon varieties and provides new insights into the regulation of secondary metabolites under environmental stresses.

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands.

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and through chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. 9% of the ~ 1800 pharmaceutical drugs here tested activate TAS2R14, nine of them at sub-micromolar concentrations. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures.

Taste Sensor Assessment of Bitterness in Medicines: Overview and Recent Topics.

In recent decades, taste sensors have been increasingly utilized to assess the taste of oral medicines, particularly focusing on bitterness, a major obstacle to patient acceptance and adherence. This objective and safe method holds promise for enhancing the development of patient-friendly medicines in pharmaceutical companies. This review article introduces its application in measuring the intensity of bitterness in medicine, confirming the achievement of taste masking, distinguishing taste differences between branded and generic medicines, and identifying substances to suppress bitterness in target medicines. Another application of the sensor is to predict a significant increase in bitterness when medicine is taken with certain foods/beverages or concomitant medication. Additionally, to verify the sensor's predictability, a significant correlation has been demonstrated between the output of a bitter-sensitive sensor designed for drug bitterness (BT0) and the bitterness responses of the human taste receptor hT2R14 from BitterDB (huji.ac.il). As a recent advancement, a novel taste sensor equipped with lipid/polymer membranes modified by 3-Br-2,6-dihydroxybenzoic acid (2,6-DHBA), based on the concept of allostery, is introduced. This sensor successfully predicts the bitterness of non-charged pharmaceuticals with xanthine skeletons, such as caffeine or related compounds. Finally, the future prospects of taste sensors are discussed.

Genetic variation in TAS2R38 bitterness receptor is associated with body composition in Korean females.

Bitterness-receptor gene TAS2R38 is associated with taste sensitivity and dietary behaviour, and is known to play a critical role in adiposity. However, evidence regarding body composition from a large cohort is lacking. This study aimed to ascertain whether TAS2R38 rs10246939 C > T bitterness genetic variation is associated with body composition in Korean individuals. The TAS2R38 rs10246939 genotypes, anthropometric measurements, and body composition of 1,843 males and 1,801 females from the Korean Genome and Epidemiology Study were analysed. Findings suggested that there was a significant difference in body fat components by TAS2R38 genotype. Furthermore, the bitterness genotype exhibited a positive association with adiposity markers in females. The TT genotype showed greater body mass index, body fat percentage, and degree of obesity than those with the C allele. However, such an association was not observed in males. In conclusion, this study suggests that TAS2R38 rs10246939 is associated with fat tissue markers in Korean females.

Enhancing the catalytic efficiency of M32 carboxypeptidase by semi-rational design and its applications in food taste improvement.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

Short-term high-light intensity and low temperature improve the quality and flavor of lettuce grown in plant factory.

BACKGROUND: Lettuce holds a prominent position in the year-round supply of vegetables, offering a rich array of health-beneficial substances, such as dietary fiber, phenolic compounds, lactucopicrin and lactucin. As such, its flavor has garnered increasing attention. Balancing the enhancement of beneficial compounds with the reduction of undesirable taste is a key focus of scientific research. To investigate short-term management to improve the nutritional quality and flavor of lettuce, combinations of different light intensities (200, 500 and 800 µm ol m-2 s-1) and temperatures (10 and 22 °C) were applied separately to 'Lollo Rosso' and 'Little Butter Lettuce' for 7 days before harvest. RESULTS: The results obtained showed that increasing light intensity at low temperatures decreased nitrate content and increased soluble sugar, soluble protein, anthocyanin and phenolic compound content. In the case of lettuce flavor, the bitterness-related metabolites such as lactucin and lactucopicrin were reduced with high light intensity at a low temperature of 10 °C. With this combination, the fructose and glucose contents increased, significantly improving lettuce flavor. CONCLUSION: Higher light intensity combined with low temperature for 7 days before harvest effectively improved the nutritional quality and flavor of lettuce, suggesting its great potential for use in horticultural practices. © 2024 Society of Chemical Industry.

Purification and characterization of limonin D-ring lactone hydrolase from sweet orange (Citrus sinensis (L.) Osbeck) seeds.

BACKGROUND: Citrus products often suffer from delayed bitterness, which is generated from the conversion of non-bitter precursors (limonoate A-ring lactone, LARL) to limonin under the catalysis of limonin D-ring lactone hydrolase (LDLH). In this study, LDLH was isolated and purified from sweet orange seeds, and a rapid and accurate high-performance liquid chromatography method to quantify LARL was developed and applied to analyze the activity and enzymatic properties of purified LDLH. RESULTS: Purified LDLH (25.22 U mg-1) showed bands of 245 kDa and 17.5 kDa molecular weights in native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE analysis respectively. After a 24 h incubation under strongly acidic (pH 3) or strongly alkaline (pH 9) conditions, LDLH still retained approximately 100% activity. Moreover, LDLH activity was not impaired by thermal treatment at 50 °C for 120 min. Enzyme inhibition assays showed that LDLH was inactivated only after ethylenediaminetetraacetic acid treatment, and other enzyme inhibitors showed no significant effect on its activity. In addition, the LDLH activity of calcium ion (Ca2+) intervention was 108% of that in the blank group, and that of zinc ion (Zn2+) intervention was 71%. CONCLUSION: LDLH purified in this study was a multimer containing 17.5 kDa monomer with a wide pH tolerance range (pH 3-9) and excellent thermal stability. Moreover, LDLH might be a metallopeptidase, and its activity was stimulated by Ca2+ and significantly inhibited by Zn2+. These findings improve our understanding of LDLH and provide some important implications for reducing the bitterness in citrus products in the future. © 2024 Society of Chemical Industry.

[Research on bitter taste and efficacy of Ginkgo biloba extract based on UPLC-Q-TOF-MS~E integrated with molecular docking].

Based on high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS~E) and molecular docking technique, bitter compounds of Ginkgo biloba extract(GBE) were characterized, and their relationship with bitter efficacy was investigated. Firstly, UPLC-Q-TOF-MS~E was used for qualitative analysis of GBE components, and 60 chemical components were identified. These chemical components were molecular-docked with bitter receptors, and 26 bitter substances were selected, mainly flavonoids. Secondly, sensory and electronic tongue bitterness evaluation techniques were used to verify that total flavones of GBE were the main bitter substances, which was consistent with the molecular docking results. Finally, network pharmacology was used to predict and analyze bitter substances. The relationship between the target of bitter substance and bitter effect was explored. The key targets of bitter substances are CYP2B6, ALOX15, and PTGS2, etc., and bitter substances may exert a bitter efficacy by ac-ting on related disease targets, indicating that bitter substances of GBE are the material basis of the bitter effect. In summary, the study indicated that the molecular docking technique had a guiding effect on the screening of bitter substances in traditianal Chinese medicine(TCM), and bitter substances of GBE had a bitter efficacy. It provides ideas and references for the study of the "taste-efficacy relationship" of TCM in the future.

A comprehensive database of cheese-derived bitter peptides and correlation to their physical properties.

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.

Avoidance behavior and experience-dependent tolerance in response to bitter compounds in Caenorhabditis elegans.

This study investigates the mechanisms governing experience-dependent tolerance of bitter compounds in Caenorhabditis elegans. The nematodes showed an aversion to nicotinamide, MgCl2, isoleucine, and arginine in a Gα-dependent manner. Worms furthermore displayed diminished avoidance of MgCl2 upon MgCl2-preconditioning at the larval stages. AIY interneurons have been suggested to be involved in experience-dependent behavioral plasticity.

The Inhibitory Effect of Adenylic Acid on the Bitterness of the Antibacterial Combination Drug Trimethoprim/Sulfamethoxazole.

The purpose of the present study was to evaluate bitterness suppression effect of adenylic acid (AMP) as a nucleotide-derived nutrient enhancer on a bitter commercial drug. In the present study, we evaluated peripheral bitterness inhibition effect of AMP on the trimethoprim (TMP) and sulfamethoxazole (SMZ) combination formulation based on taste sensor. The taste sensor values of TMP solutions with different concentrations show large sensor output in correlation with the concentration of TMP, whereas no sensor output in shown for the SMZ solutions. Therefore, the bitterness of this combination formulation is mainly due to TMP. We evaluated the TMP bitterness inhibitory effects of AMP, sodium salt of AMP (AMP Na; sodium adenylate), sodium salt of GMP (GMP Na; sodium guanylate), and sodium salt of inosine monophosphate (IMP Na; sodium inosinate), and found that only AMP displayed very effective bitterness inhibition. MarvinSketch analysis revealed that potential electrostatic interaction between cationized TMP and anionized forms (II and III) of AMP may cause bitterness suppression. 1H-NMR study suggested an interaction of TMP and AMP molecules based on chemical shift perturbations and an interaction between the phosphate group of AMP and amino group of TMP. Lastly, conventional elution analysis simulating oral cavity capacity for up to one minute were performed using commercial TMP/SMZ combination granules. The sensor output gradually increased up to 60 s. The addition of AMP solution to the eluted sample at 60 s significantly decreased the bitterness sensor output of the eluted sample.

Masking the Taste of Fixed-Dose Combination Drugs: Particular NSAIDs Can Efficiently Mask the Bitterness of Famotidine.

This study aimed to evaluate the bitterness of famotidine (FAM) combined with each of three non-steroidal anti-inflammatory drugs (NSAIDs): ibuprofen (IBU), flurbiprofen (FLU), and naproxen (NAP), which have potential as fixed-dose combination (FDC) drugs. We evaluated the bitterness of FAM and each NSAID by taste sensor AN0 and C00, respectively. FAM showed high sensor output representing sensitivity to bitterness, whereas three NSAIDs did not show large sensor output, suggesting that the bitterness intensities of three NSAIDs were lower than that of FAM. The bitterness of FAM on sensor AN0 was suppressed in a concentration-dependent manner when mixed with IBU, FLU, or NAP. Among three NSAIDs, IBU most effectively inhibited bitterness on sensor output, and the gustatory sensation test confirmed that adding IBU to FAM reduced the bitterness of FAM in a concentration-dependent manner. MarvinSketch confirmed that the drugs were mostly present in an ionic solution when FAM was mixed with NSAIDs. The 1H-NMR spectroscopy analysis also revealed the presence of electrostatic interactions between FAM and NSAIDs, suggesting that the electrostatic interaction between FAM and NSAIDs might inhibit the adsorption of FAM on the bitter taste sensor membrane, thereby masking the bitter taste.

Salivary flow and turbidity development inconsistently associated with lower taste intensity of vegetables and juices.

The same phytochemicals that stimulate aversive sensations are often also responsible for purported health benefits in fruits and vegetables. Prior work indicates that some salivary proteins may reduce aversiveness of phytochemicals. In rodents, the salivary binding proteins have been shown to reduce bitter taste of polyphenols and alkaloids, but work in humans has focused primarily on polyphenol astringency (dry, rough, or puckery sensations). In this study, we tested if tastes of vegetable products might correlate to either salivary flow rate or the polyphenol binding capability of saliva, as measured by turbidity development when saliva is mixed with tannic acid. Participants (N=26) provided chewing-stimulated saliva samples and rated five juices and two chopped vegetables for bitterness, sourness, and sweetness intensity. Saliva was mixed with tannic acid and light absorbance was measured for quantification of haze development. Greater absorbance corresponded to less bitterness for one green vegetable juice blend, less sweetness for two green vegetable juices and chopped kale, and less sourness from cranberry juice. Greater salivary flow corresponded to less bitterness from chopped brussels sprouts, and less sweetness from one green vegetable juice blend and chopped kale. These findings indicate that greater salivary flow rate and presence of certain salivary binding proteins is not universally associated with lower aversive tastes from phytochemical-containing foods. Whether associations between these salivary properties are ingredient specific or simply not robustly related to taste in commercial products should be further investigated.

Interactions between Beer Compounds and Human Salivary Proteins: Insights toward Astringency and Bitterness Perception.

Beer is one of the most consumed beverages worldwide with unique organoleptic properties. Bitterness and astringency are well-known key features and, when perceived with high intensity, could lead to beer rejection. Most studies on beer astringency and bitterness use sensory assays and fail to study the molecular events that occur inside the oral cavity responsible for those perceptions. This work focused on deepening this knowledge based on the interaction of salivary proteins (SP) and beer phenolic compounds (PCs) and their effect toward these two sensory attributes. The astringency and bitterness of four different beers were assessed by a sensory panel and were coupled to the study of the SP changes and PC profile characterization of beers. The human SP content was measured before (basal) and after each beer intake using HPLC analysis. The beers' PC content and profile were determined using Folin-Ciocalteu and LC-MS spectrometry, respectively. The results revealed a positive correlation between PCs and astringency and bitterness and a negative correlation between SP changes and these taste modalities. Overall, the results revealed that beers with higher PC content (AAL and IPA) are more astringent and bitter than beers with a lower PC content (HL and SBO). The correlation results suggested that an increase in whole SP content, under stimulation, should decrease astringency and bitterness perception. No correlation was found between the changes in specific families of SP and astringency and bitterness perception.

Research on the Effect of Oriental Fruit Moth Feeding on the Quality Degradation of Chestnut Rose Juice Based on Metabolomics.

As a native fruit of China, chestnut rose (Rosa roxburghii Tratt) juice is rich in bioactive ingredients. Oriental fruit moth (OFM), Grapholita molesta (Busck), attacks the fruits and shoots of Rosaceae plants, and its feeding affects the quality and yield of chestnut rose. To investigate the effects of OFM feeding on the quality of chestnut rose juice, the bioactive compounds in chestnut rose juice produced from fruits eaten by OFM were measured. The electronic tongue senses, amino acid profile, and untargeted metabolomics assessments were performed to explore changes in the flavour and metabolites. The results showed that OFM feeding reduced the levels of superoxide dismutase (SOD), tannin, vitamin C, flavonoid, and condensed tannin; increased those of polyphenols, soluble solids, total protein, bitterness, and amounts of bitter amino acids; and decreased the total amino acid and umami amino acid levels. Furthermore, untargeted metabolomics annotated a total of 426 differential metabolites (including 55 bitter metabolites), which were mainly enriched in 14 metabolic pathways, such as flavonoid biosynthesis, tryptophan metabolism, tyrosine metabolism, and diterpenoid biosynthesis. In conclusion, the quality of chestnut rose juice deteriorated under OFM feeding stress, the levels of bitter substances were significantly increased, and the bitter taste was subsequently enhanced.

Non-Volatile Compounds Involved in Bitterness and Astringency of Pulses: A Review.

Despite the many advantages of pulses, they are characterised by off-flavours that limit their consumption. Off-notes, bitterness and astringency contribute to negative perceptions of pulses. Several hypotheses have assumed that non-volatile compounds, including saponins, phenolic compounds, and alkaloids, are responsible for pulse bitterness and astringency. This review aims to provide an overview highlighting the non-volatile compounds identified in pulses and their bitter and/or astringent characteristics to suggest their potential involvement in pulse off-flavours. Sensorial analyses are mainly used to describe the bitterness and astringency of molecules. However, in vitro cellular assays have shown the activation of bitter taste receptors by many phenolic compounds, suggesting their potential involvement in pulse bitterness. A better knowledge of the non-volatile compounds involved in the off-flavours should enable the creation of efficient strategies to limit their impact on overall perception and increase consumer acceptability.

Bitter Peptides in Fermented Soybean Foods - A Review.

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.

The effect of carboxymethyl cellulose and ß-cyclodextrin as debittering agents on bitterness and physicochemical properties of bitter gourd extract.

Bitter gourd extract (BGE) is rich in antioxidants and anti-diabetic components that promote good human health; however, its bitter taste makes it challenging to use in food. In this study, the effect of carboxymethyl cellulose and ß-cyclodextrin (ß-CD) on the bitterness and properties of BGE were investigated. The bitterness intensity was evaluated by the trained sensory panel, and the physicochemical properties were also determined, including viscosity, total saponin, polyphenol content, antioxidant capacity, and α-amylase inhibition activity. It was found that the bitterness of BGE with 0.75%, w/v ß-cyclodextrin decreased significantly by more than 90%. Additionally, FTIR, 1 H-NMR, and thermogravimetric analysis of BGE supplemented with ß-CD confirmed the formation of a complex between ß-CD and components of BGE. The findings of the current study also reveal that debittering agents did not inhibit the bioactivities of BGE.

Strategies for Taste Masking of Orodispersible Dosage Forms: Time, Concentration, and Perception.

Orodispersible dosage forms, characterized as quick dissolving and swallowing without water, have recently gained great attention from the pharmaceutical industry, as these forms can satisfy the needs of children, the elderly, and patients suffering from mental illnesses. However, poor taste by thorough exposure of the drugs' dissolution in the oral cavity hinders the effectiveness of the orodispersible dosage forms. To bridge this gap, we put forward three taste-masking strategies with respect to the intensity of time, concentration, and perception. We further investigated the raw material processing, the composition of auxiliary material, formulation techniques, and process control in each strategy and drew conclusions about their effects on taste masking.

Characterization of bitter-tasting and antioxidant activity of dry-hopped beers.

BACKGROUND: Bitter flavors and antioxidant activities are critical characteristics and indicators, respectively, of beer quality. To gain a better understanding of dry-hopped beer's bitterness, this work comprehensively evaluated the perceived bitterness intensity and bitterness attributes from aspects of beer aroma and non-volatile bitter compounds using sensory analysis under two conditions: (i) with and (ii) without nose clips. To quantify bitter and volatile compounds, the work conducted chromatographic analyses: high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Simultaneously, this work assessed the antioxidant activity of commercially dry-hopped beers. RESULTS: First, dry-hopped beer in this study contained abundant non-volatile bitter compounds (hop bitter acids, polyphenols and flavonoids), and aroma was validated using HPLC, UPLC-MS and GC-MS methods. Moreover, the bitter-tasting perception test findings demonstrated that many dry-hopped beers had a higher bitterness intensity when evaluated without a nose clip, whereas this phenomenon was adverse in several ale beers. Additionally, the 'lingering' and 'harsh' characteristics were diminished when beer aroma was blocked out (with nose clip) for dry-hopped beer. Meanwhile, most dry-hopped beers had greater antioxidant activity than ale beers (P < 0.05). CONCLUSION: This work revealed the bitterness complexity of dry-hopped beer; besides non-volatile bitter compounds, beer aroma had an impact on the perceived bitterness intensity and attributes, and dry-hopped beer had a relatively intense antioxidant capacity. This study facilitated the development of a detailed knowledge about the assessment of bitter-tasting perceptions in dry-hopped beers and provided a basis for the development of functional beer benefiting human health. © 2022 Society of Chemical Industry.