Amino Acid Analysis Research Articles

Characterization of Amino Acid Composition, Nutritional Value, and Taste of Fruits from Different Actinidia arguta Resources

The nutritional value and flavor and texture characteristics of fruits from different Actinidia argute resources were scientifically evaluated and compared. Using 35 A. arguta fruits as materials, the amino acid composition and content were determined by an automatic amino acid analyzer, and differentiation analysis, amino acid nutritional value evaluation, TAV flavor analysis, correlation analysis, PCA comprehensive evaluation, and cluster analysis were conducted to clarify the diversity of A. arguta resources in terms of amino acid content, composition, and flavor characteristics. Analysis of differential results showed that the A. arguta resource fruits contained 17 amino acids with a total amino acid content of 384.20∼2590.56 mg/100 g. The results of the nutritional value evaluation showed that the Leu of the fruits of the A. arguta resources all conformed to the ideal model proposed by FAO/WHO, and the Leu content of all the resources exceeded the human body’s needs, and it was also found that the first limiting amino acid of the Actinidia argute resources was Ile and the second limiting amino acid was Lys. TAV of the flavor-presenting amino acids was calculated to evaluate the flavor-presenting taste characteristics, and the amino acids that influenced the flavor of A. arguta fruit were Glu and Cys. PCA showed that the 2 principal components could better reflect the comprehensive information of amino acids in A. arguta, and the cumulative variance contribution rate was 87.88%, which could represent the main trend of amino acids in A. arguta. A comprehensive amino acid evaluation model was established, and the composite scores indicated that the top 5 excellent resources were S4, S10, S18, S25, and S30. Hierarchical cluster analysis classified the 35 A. arguta resources into 4 categories, which better reflected the differences in amino acid content and composition, nutritional value, and taste characteristics among A. arguta fruits from different collection sites.

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers.The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase.The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Omnivorous summer feeding by juvenile Antarctic krill in coastal waters

AbstractThe Antarctic krill Euphausia superba is often considered an herbivore but is notable for its trophic flexibility, which includes feeding on protistan and metazoan zooplankton. Characterizing krill trophic position (TP) is important for understanding carbon and energy flow from phytoplankton to vertebrate predators and to the deep ocean, especially as plankton composition is sensitive to changing climate. We used repeated field sampling and experiments to study feeding by juvenile krill during three austral summers in waters near Palmer Station, Antarctica. Our approach was to combine seasonal carbon budgets, gut fluorescence measurements, imaging flow cytometry, and compound‐specific isotope analysis of amino acids. Field measurements coupled to experimentally derived grazing functional response curves suggest that phytoplankton grazing alone was insufficient to support the growth and basal metabolism of juvenile krill. Phytoplankton consumption by juvenile krill was limited due to inefficient feeding on nanoplankton (2–20 μm), which constituted the majority of autotrophic prey. Mean krill TP and the metazoan dietary fraction increased in years with higher mesozooplankton biomass, which was not coupled to phytoplankton biomass. Comparing TP estimates using δ15N of different amino acids indicated a substantial and consistent food‐web contribution from heterotrophic protists. Phytoplankton, metazoans, and heterotrophic protists all were important contributors to a diverse krill diet that changed substantially among years. Juvenile krill fed mostly on heterotrophic prey during summer near Palmer Station, and this food web complexity should be considered more broadly throughout the changing Southern Ocean.

Development of Two-Dimensional LC-MS/MS Methods for Highly-Selective Analysis of Chiral Amino Acids and the Evaluation of Their Intrinsic Amounts in Mammals and Stereoinversion in Proteins

Development of Two-Dimensional LC-MS/MS Methods for Highly-Selective Analysis of Chiral Amino Acids and the Evaluation of Their Intrinsic Amounts in Mammals and Stereoinversion in Proteins

Identification of a leucine-mediated threshold effect governing macrophage mTOR signalling and cardiovascular risk.

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.

Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC–MS/MS) and gas chromatography with mass spectrometry (GC)–MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as “in-house” standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC–MS and LC–MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.Graphical abstract

Important nutrient sources and carbohydrate metabolism patterns in the growth and development of spargana

BackgroundSparganosis is a worldwide food-borne parasitic disease caused by spargana infection, which infects the muscle of frogs and snakes as well as many tissues and organs in humans. There are currently no viable treatments for sparganosis. Understanding spargana’s nutrition source and carbohydrate metabolism may be crucial for identifying its energy supply and establishing methods of treatment for sparganosis.MethodsUsing an amino acid analyzer and nutrient concentration detection kits, we assessed nutrient concentrations in the muscles of Fejervarya limnocharis and Pelophylax plancyi infected or not infected with spargana. Quantitative polymerase chain reaction (PCR) was used to quantify the major enzymes involved in five glucose metabolism pathways of spargana developing in vivo. We also used quantitative PCR to assess key enzymes and transcriptome sequencing to explore the regulation of carbohydrate metabolic pathways in vitro in response to different 24-h food treatments.ResultsInfected muscle tissues had considerably higher concentrations of glucogenic and/or ketogenic amino acids, glucose, and glycogen than non-infected muscle tissues. We discovered that the number of differentially expressed genes in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was larger in low-glucose than in other dietary groups. We examined differences in the expression of genes producing amino acid transporters, glucose transporters, and cathepsins in spargana grown in various nutritional environments. In the normal saline group, only the major enzymes in the tricarboxylic acid cycle (TCA), glycogenesis, and glycogenolysis pathways were expressed. The l-glutamine group had the greatest transcriptional levels of critical rate-limiting enzymes of gluconeogenesis and glycogenesis. Furthermore, the low-glucose group had the highest transcriptional levels of critical rate-limiting enzymes involved in the TCA, glycolytic, and glycogenolysis pathways. Surprisingly, when compared to the in vitro culturing groups, spargana developing in vivo exhibited higher expression of these critical rate-limiting enzymes in these pathways, with the exception of the pentose phosphate pathway.ConclusionsSpargana have a variety of nutritional sources, and there is a close relationship between nutrients and the carbohydrate metabolism pathways. It takes a multi-site approach to block nutrient absorption and carbohydrate metabolism pathways to provide energy to kill them.Graphical

Changes and correlation analysis of volatile flavor compounds, amino acids, and soluble sugars in durian during different drying processes

Durian contains rich flavor components that undergo complex changes during drying. In this study, durian was subjected to integrated freeze-drying (IFD), conventional freeze-drying (CFD), and hot air drying (AD). Compared with the fresh samples, those dried by IFD, CFD, and AD lost 11, 9, and 7 original volatile compounds, respectively, and generated 7, 6, and 8 new volatile compounds, respectively, and showed a rapid and then slow decreasing trend in the total content during drying. However, the types of amino acids and soluble sugars remained unchanged during each of the drying methods. Furthermore, volatile compounds showed a significant negative correlation with the majority of amino acids and a significant positive correlation with soluble sugars. The IFD samples had the highest content of volatile compounds, amino acids, and soluble sugars. Therefore, IFD is recommended as a preferable drying method for durian.

Structural and functional properties of protein hydrolysates from myofibrillar protein of crocodile (Crocodylus siamensis) meat

In this study, three proteases (papain, neutral protease, bromelain) were employed to hydrolyze crocodile (Crocodylus siamensis) myofibrillar protein (MP), effects of hydrolysis duration on functional and structural properties of MP hydrolysates was investigated. The findings revealed that solubility of all hydrolysates exceeded 60%, with the highest reaching 96.7%. The emulsifying activity of neutral protease hydrolysates exhibited a significant superiority over the other two hydrolysates (P < 0.05). The Fe2+ chelating and DPPH radical scavenging activity of hydrolysates ranged from 20% to 60%, and 60%–90%, respectively. Based on the experiment results, the hydrolysates of papain at 1h, neutral protease at 1h, and bromelain at 2h were used for structural analysis. Compared to MP, the secondary structure of hydrolysates showed a reduction of 9.6%, 15.2%, and 4.7% in α-helix and the increase of 42.4%, 29.6%, and 44.2% in β-fold content of papain, neutral protease and bromelain hydrolysates respectively, suggesting the transition from spiral to folding structure and the exposure of hydrophobic group, which was proved by free amino acid analysis. SEM results confirmed the alterations in MP structure. The molecular weight distributions of three hydrolysates were predominantly below 1000 Da, demonstrating that crocodile MP was effectively hydrolyzed into bioactive peptides.

Extraction and Characterization of Fish Collagen for Enhanced Wound Healing Activity on A549 Cell Lines

Collagen is a proteinaceous material that is present abundantly in animal tissues and gives physical strength and stability to the tissues. Collagen extracted from animal tissues has been applied in the biomedical and healthcare sectors. In recent days, aquatic organisms have shown great potential for collagen extraction and purification. In the present study, we selected three fish species: Naucrates ductor, Oreochromis mossambicus and Cyprinus carpiofor collagen extraction from both their skin and scales. The biomass was pre-treated to remove any non-collagenous matter and fat. Acid-soluble collagen was extracted using acetic acid and a maximum extraction of 51.55±1.02% was reported. The total protein fractions and the denaturation temperatures of the collagen extractswere measured. The molecular weights and the subunit compositions of the collagen extracts were analyzed using SDS-PAGE and the presence of two α chains, one β, and one γ chain. Amino acid profiling was done in an amino acid analyzer after hydrolysis and about one third of the total amino acid content was glycine. The solubility characteristics of the collagenextracts were analyzed at different ranges of pH and salinity. The antimicrobial activities of the collagen extracts were examined against four bacterial species, and it was proven that skin-derived collagens had greater antibacterial potential than extracts of the scales. In vitro studies on the wound healing capacity of the extracted collagen were performed using A549 cell line cultured in F-12K medium, and 95% of cell regeneration was observed in collagen-supplemented dispersed cell lines.

Effect of laser power on Raman analyses of lipids and amino acids: Implications for extraterrestrial life exploration

One of the primary objectives of Mars exploration missions is to search for traces of past or present life. The search for bioorganic molecules is highly desirable because they have diagnostic features to indicate the presence of life. Lipids and amino acids are particularly favored because of their high preservation potential in the geological record. Raman spectroscopy has emerged as a promising tool for detecting and identifying organic molecules on extraterrestrial planets. However, cautions should be exercised in operating Raman instruments to detect organic samples, especially concerning laser power. Excessive laser power can potentially alter spectral characteristics and even damage samples, while low power levels suffer from attenuation of the measured Raman signal, making it challenging to interpret. Therefore, it is crucial to determine the optimal Raman laser power for organic detection and build a laser power-based spectral library to increase our capability to interpret the Martian spectroscopic data. As such, we performed a comprehensive Raman analysis of several lipids (decanol, hexadecane, and palmitic acid) and amino acids (alanine, aspartic acid, glycine, histidine, and tyrosine). The Raman laser power varies in two modes, i.e., increasing from 1% (0.63 ± 0.01 mW) to 100% (66.40 ± 1.85 mW) and vice versa, within the operational range of Raman payloads of Mars rovers. The results show that different spectra can be obtained for the organic samples when using different excitation powers. Using high-power lasers could cause damage to lipid samples, whereas amino acids exhibit stability even under the strong irradiation of 100% laser power. In addition, low-power lasers significantly reduce the Raman signal of lipids, while amino acids remain detectable at low laser powers even as low as 1%. Therefore, the most effective and secure operation is to gradually increase the laser power in the Raman analyses for detecting various organic compounds. Our study serves as a reference for operating Raman instruments and contributes to the accurate interpretation of spectral data returned from spacecraft. The supplementary material provides all the spectral data, facilitating further comparisons with future in situ and orbital measurements on Mars.

Sour cherry seed proteins devoted to covalently bonded carbohydrate moieties: efficacy of transaction and carbohydrate type on amino acid distribution and emulsifier behavior

This paper handled the Maillard reaction under controlled conditions to conjugate sour cherry seed protein isolate (SCPI) with gum Arabic (GA), carboxymethyl cellulose (CMC), and pectin (P) polysaccharides. Characteristic investigations for conjugates were conducted by browning intensity, glycation degree, amino acid, FTIR, SEM, and TGA analyses. After glycation, the level of lysine and arginine declined. Conjugation process displayed beneficial efficacy on techno-functional attributes, namely solubility, water holding capacity, oil holding capacity, foaming capacity/stability and emulsion activity/stability of SCPI but maximum values in terms of these attributes were variable according to conjugate type. Stability index of emulsions stabilized by using conjugates including SCPI-GA (0.87 R), SCPI-CMC (0.94 R), and SCPI-P (0.95 R) was superior than that of SCPI alone (0.60 R). Centrifugal precipitation rate was 59.17, 36.04, 32.61, and 30.92% for emulsions prepared in the presence of SCPI, SCPI-GA, SCPI-CMC, and SCPI-P, respectively. Resilience to freeze-thawed, pH-shifting, various salt concentrations, and different temperature applications of emulsions were investigated to evaluate emulsifier behavior of protein and conjugates in food systems. Findings showed that emulsifying properties of the conjugates prepared using P and CMC came to the fore compared to SCPI alone and SCPI-GA.

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids.

The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water-acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

Amino acid distribution in blood following high-intensity interval exercise: a preliminary study.

This study investigated the effect of high-intensity interval exercise on total and individual amino acid concentrations in red blood cells (RBCs) and plasma. Seven males (31 ± 13yr) provided venous blood samples at rest, immediately and 15min and 30min following an 8-min high-intensity exercise bout. The exercise bout was 16 × 15s cycle efforts at 0.4N/kg of body mass and 90rpm, interspersed with 15s passive recovery. Total and individual amino acid concentrations of RBC and plasma and blood cell parameters were analysed. No significant differences for total amino acid concentrations between RBC and plasma were found. Individual amino acid analyses showed significant interaction effects for alanine and α-aminoadipic acid (P < 0.05), with plasma alanine significantly increased from baseline across the recovery period (P < 0.001). Blood fraction (group) effects showed greater concentrations of glycine, serine, asparagine, aspartic acid, glutamic acid, α-aminoadipic acid and ornithine in RBC, while greater concentrations of alanine, α-aminobutyric acid, valine, leucine, isoleucine, threonine, proline, phenylalanine, glutamine, tryptophan and cystine were found in plasma (P < 0.05). Comparable levels of histidine, lysine and tyrosine were observed between blood fractions. Significant differences in the variation of total amino acids in RBC were reported with higher variance at rest compared to following exercise (P = 0.01). Haemoglobin, pack cell volume and white blood cell count significantly increased immediately following exercise (P < 0.05) but returned to baseline after 15min recovery. These results support the notion of individualised amino acid transportation roles for RBC and plasma during exercise.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Incorporation of Partial Least Squares-Discriminant Analysis with Ultra-High-Performance Liquid Chromatography Diode-Array Detector for Authentication of Skin Gelatine Sources

This research seeks to (1) authenticate sources of skin gelatine by combining putative 17 aminoacids (AAs) analysis with chemometrics by Ultra-High-Performance Liquid ChromatographyDiode-Array Detector (UHPLC-DAD) and (2) create AA profiles in skin gelatines. Theclassification capability of partial least square-discriminant analysis (PLS-DA) models wasassessed to determine the most effective discriminant model. Principal component analysis(PCA) with quartimax rotation was utilised to accurately organise gelatine clusters and assign thesignificantly contributing AAs to each cluster. The PLS-DA model with 13 AAs (PLS-DAVIPAA)outperformed the PLS-DA model with 17 AAs (PLS-DAAA) because its R2Y (0.938), R2X (0.881),and Q2 (0.929) values were greater. With 13 significant AAs, the PLS-DAVIPAA model obtainedcluster classification accuracy of 100% on training and cross-validation datasets and 93.3% ontesting and verification datasets. The chemical structure of gelatines may shed light on theinteractions between AAs. Following six quartimax rotations, the gelatines were groupedcorrectly. The PCA showed the dominant presence of these AAs: L-Valine, L-Phenylalanine andL-Tyrosine in porcine gelatine; Glycine, L-Threonine, L-Arginine, L-Methionine, L-Histidine andL-Serine in fish gelatine; and L-Hydroxyproline, L-Leucine and L-Proline in bovine gelatine. Theauthority could use this technique to set a standard for authenticating skin gelatine samples.

Interspecies metabolite transfer fuels the methionine metabolism of Fusobacterium nucleatum to stimulate volatile methyl mercaptan production.

The major oral odor compound methyl mercaptan (CH3SH) is strongly associated with halitosis and periodontitis. CH3SH production stems from the metabolism of polymicrobial communities in periodontal pockets and on the tongue dorsum. However, understanding of CH3SH-producing oral bacteria and their interactions is limited. This study aimed to investigate CH3SH production by major oral bacteria and the impact of interspecies interactions on its generation. Using a newly constructed large-volume anaerobic noncontact coculture system, Fusobacterium nucleatum was found to be a potent producer of CH3SH, with that production stimulated by metabolic interactions with Streptococcus gordonii, an early dental plaque colonizer. Furthermore, analysis of extracellular amino acids using an S. gordonii arginine-ornithine antiporter (ArcD) mutant demonstrated that ornithine excreted from S. gordonii is a key contributor to increased CH3SH production by F. nucleatum. Further study with 13C, 15N-methionine, as well as gene expression analysis, revealed that ornithine secreted by S. gordonii increased the demand for methionine through accelerated polyamine synthesis by F. nucleatum, leading to elevated methionine pathway activity and CH3SH production. Collectively, these findings suggest that interaction between S. gordonii and F. nucleatum plays a key role in CH3SH production, providing a new insight into the mechanism of CH3SH generation in oral microbial communities. A better understanding of the underlying interactions among oral bacteria involved in CH3SH generation can lead to the development of more appropriate prophylactic approaches to treat halitosis and periodontitis. An intervention approach like selectively disrupting this interspecies network could also offer a powerful therapeutic strategy.IMPORTANCEHalitosis can have a significant impact on the social life of affected individuals. Among oral odor compounds, CH3SH has a low olfactory threshold and halitosis is a result of its production. Recently, there has been a growing interest in the collective properties of oral polymicrobial communities, regarded as important for the development of oral diseases, which are shaped by physical and metabolic interactions among community participants. However, it has yet to be investigated whether interspecies interactions have an impact on the production of volatile compounds, leading to the development of halitosis. The present findings provide mechanistic insights indicating that ornithine, a metabolite excreted by Streptococcus gordonii, promotes polyamine synthesis by Fusobacterium nucleatum, resulting in a compensatory increase in demand for methionine, which results in elevated methionine pathway activity and CH3SH production. Elucidation of the mechanisms related to CH3SH production is expected to lead to the development of new strategies for managing halitosis.

Formulation and organoleptic characteristics of flavor enhancer from shrimp head protein hydrolysate

Protein hydrolysate can be produced from by-products of the fishing industry, such as shrimp heads. This product is made by enzyme hydrolysis through the breakdown of shrimp head protein into short-chain peptides and amino acids. Enzymatic hydrolysis produces protein hydrolysate, which has savory, umami, and water-soluble characteristics. The savory and umami taste results from the high content of glutamic acid and other free amino acids. This study aimed to determine the best formulation and organoleptic characteristics of the shrimp head protein hydrolysate flavor enhancer. Production of shrimp head protein hydrolysate (SPH) using alkalase enzyme at a temperature of 55°C with an enzyme concentration of 20,000 units/kg substrate for 7 hrs. Parameters tested were yield, proximate and amino acid analysis, and organoleptic test for SPH flavor enhancer. SPH has an amino acid composition dominated by arginine, alanine, glycine, glutamic acid, leucine and lysine. The flavor enhancer with 40% SPH composition was chosen as the best formula because it has sensory characteristics close to P0 commercial flavoring. The dominant sensory attributes were spicy, salty, umami and sweet. Overall, protein hydrolysate-based flavor enhancers' solubility and color properties are close to commercial flavor enhancers. The flavor enhancer made from SPH has the potential to be used as a substitute for commercial flavor enhancers which are rich in essential amino acids.

Characterization of active peptides derived from three leeches and comparison of their anti-thrombotic mechanisms using the tail vein thrombosis model in mice and metabonomics

Background and aims: The increasing incidence of cardiovascular diseases has created an urgent need for safe and effective anti-thrombotic agents. Leech, as a traditional Chinese medicine, has the effect of promoting blood circulation and removing blood stasis, but its real material basis and mechanism of action for the treatment of diseases such as blood stasis and thrombosis have not been reported.Methods: In this study, Whitmania Pigra Whitman (WPW), Hirudo nipponica Whitman (HNW) and Whitmania acranutata Whitman (WAW) were hydrolyzed by biomimetic enzymatic hydrolysis to obtain the active peptides of WPW (APP), the active peptides of HNW (APH) and the active peptides of WAW (APA), respectively. Then their structures were characterized by sykam amino acid analyzer, fourier transform infrared spectrometer (FT-IR), circular dichroism (CD) spectrometer and LC-MS. Next, the anti-thrombotic activities of APP, APH and APA were determined by carrageenan-induced tail vein thrombosis model in mice, and the anti-thrombotic mechanisms of high-dose APP group (HAPP), high-dose APH group (HAPH) and high-dose APA group (HAPA) were explored based on UHPLC-Q-Exactive Orbitrap mass spectrometry.Results: The results showed that the amino acid composition of APP, APH and APA was consistent, and the proportion of each amino acid was few different. The results of FT-IR and CD showed that there were no significant differences in the proportion of secondary structures (such as β-sheet and random coil) and infrared absorption peaks between APP, APH and APA. Mass spectrometry data showed that there were 43 common peptides in APP, APH and APA, indicating that the three have common material basis. APP, APH and APA could significantly inhibit platelet aggregation, reduce black-tail length, whole blood viscosity (WBV), plasma viscosity (PV), and Fibrinogen (FIB), and prolong coagulation time, including activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT). In addition, 24 metabolites were identified as potential biomarkers associated with thrombosis development. Among these, 19, 23, and 20 metabolites were significantly normalized after administration of HAPP, HAPH, and HAPA in the mice, respectively. Furthermore, the intervention mechanism of HAPP, HAPH and HAPA on tail vein thrombosis mainly involved in linoleic acid metabolism, primary bile acid biosynthesis and ether lipid metabolism.Conclusion: Our findings suggest that APP, APH and APA can exert their anti-blood stasis and anti-thrombotic activities by interfering with disordered metabolic pathways in vivo, and there is no significant difference in their efficacies.

Effects of canagliflozin on amino acid metabolism in atherosclerotic mice

Objective: To explore the possible anti-atherosclerotic mechanisms of glucose co-transporter-2 inhibitor canagliflozin. Methods: ApoE-/-mice fed on Western diet were randomly assigned into the model group (n=10) and the canagliflozin group (n=10). C57BL/6J mice fed on normal diet were chosen as the control group (n=10). Mice in the canagliflozin group were gavaged with canagliflozin for 14 weeks. The presence and severity of atherosclerosis were evaluated with HE and oil red O stainings in aortic root section slices. PCR assay was performed to determine the mRNA expression levels of nitric oxide synthase. Hepatic transcriptome analysis and hepatic amino acid detection were conducted using RNA-seq and targeted LC-MS, respectively. Results: HE staining and oil red O staining of the aortic root showed that AS models were successfully established in ApoE-/-mice fed on Western diet for 14 weeks. Canagliflozin alleviated the severity of atherosclerosis in pathology. Hepatic transcriptome analysis indicated that canagliflozin impacted on amino acid metabolism, especially arginine synthesis in ApoE-/-mice. Targeted metabolomics analysis of amino acids showed that canagliflozin reduced hepatic levels of L-serine, L-aspartic acid, tyrosine, L-hydroxyproline, and L-citrulline, but raised the hepatic level of L-arginine. Compared to the model group, the canagliflozin group exhibited higher serum arginine and nitric oxide levels as well as elevated nitric oxide mRNA expression in aortic tissues (P<0.05). Conclusion: Canagliflozin regulated the amino acid metabolism, reduced the levels of glucogenic amino acids,and promoted the synthesis of arginine in atherosclerotic mice.