Ion Addition by Electrolysis to Improve the Quantitative Analysis of Bacteria with MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is commonly applied to the identification of bacteria but rarely used for quantitative detection due to the inhomogeneous crystallization of the matrix leading to the unsatisfactory linear relationship between the sample amount and the mass spectrum signals. Herein, we proposed a noninterference ion addition (NIA) method by electrolysis to improve homogeneous crystallization during the evaporation progress of sample droplets on the target plates. The active metal wire was inserted in the droplet as the anode electrode, and metal ions were released through electrolysis. The directional migration of metal ions under the electric field can hinder the migration of matrix molecules to the boundary and homogenize the matrix crystals by forming spherical crystals. Simultaneously, trace cationic surfactant was added to the droplet for pinning the contact surface to define the circle crystallization region. The metal ions from the anode electrode wire were deposited on the surface of the target plates which served as the cathode. Therefore, ion addition has no interference effect on ionization during MALDI-MS detection. This NIA method benefits the homogeneous crystallization and so improves the quantitative analysis. NIA is suitable for biological samples with different matrices, and bacterial samples could be quantitatively analyzed.

Screening of Oral Potential Angiotensin-Converting Enzyme Inhibitory Peptides from Zizyphus jujuba Proteins Based on Gastrointestinal Digestion In Vivo.

Plant proteins are a good source of active peptides, which can exert physiological effects on the body. Predicting the possible activity of plant proteins and obtaining active peptides with oral potential are challenging. In this study, the potential activity of peptides from Zizyphus jujuba proteins after in silico simulated gastrointestinal digestion was predicted using the BIOPEP-UWM™ database. The ACE-inhibitory activity needs to be further investigated. The actual peptides in mouse intestines after the oral administration of Zizyphus jujuba protein were collected and analyzed, 113 Zizyphus jujuba peptides were identified, and 3D-QSAR models of the ACE-inhibitory activity were created and validated using a training set (34 peptides) and a test set (12 peptides). Three peptides, RLPHV, TVKPGL and KALVAP, were screened using the 3D-QSAR model and were found to bind to the active sites of the ACE enzyme, and their IC50 values were determined. Their values were 6.01, 3.81, and 17.06 µM, respectively. The in vitro digestion stabilities of the RLPHV, TVKPGL, and KALVAP peptides were 82%, 90%, and 78%. This article provides an integrated method for studying bioactive peptides derived from plant proteins.

A Comparative Study of Binding Interactions between Proteins and Flavonoids in Angelica Keiskei: Stability, α-Glucosidase Inhibition and Interaction Mechanisms.

Flavonoids are easily destroyed and their activity lost during gastrointestinal digestion. Protein-based nanocomplexes, a delivery system that promotes nutrient stability and bioactivity, have received increasing attention in recent years. This study investigated the stability, inhibitory activity against α-glucosidase and interaction mechanisms of protein-based nanocomplexes combining whey protein isolate (WPI), soybean protein isolate (SPI) and bovine serum albumin (BSA) with flavonoids (F) from A. keiskei using spectrophotometry, fluorescence spectra and molecular docking approaches. The results show that the flavonoid content of WPI-F (23.17 ± 0.86 mg/g) was higher than those of SPI-F (19.41 ± 0.56 mg/g) and BSA-F (20.15 ± 0.62 mg/g) after simulated digestion in vitro. Furthermore, the inhibition rate of WPI-F (23.63 ± 0.02%) against α-glucosidase was also better than those of SPI-F (18.56 ± 0.02%) and BSA-F (21.62 ± 0.02%). The inhibition rate of WPI-F increased to nearly double that of F alone (12.43 ± 0.02%) (p < 0.05). Molecular docking results indicated that the protein-flavonoids (P-F) binding occurs primarily through hydrophobic forces, hydrogen bonds and ionic bonds. Thermodynamic analysis (ΔH > 0, ΔS > 0) indicated that the P-F interactions are predominantly hydrophobic forces. In addition, the absolute value of ΔG for WPI-F is greater (-30.22 ± 2.69 kJ mol-1), indicating that WPI-F releases more heat energy when synthesized and is more conducive to combination. This paper serves as a valuable reference for the stability and bioactivity of flavonoids from A. keiskei.

Rapid screening based on machine learning and molecular docking of umami peptides from porcine bone.

BACKGROUND: The traditional screening method for umami peptide, extracted from porcine bone, was labor-intensive and time-consuming. In this study, the rapid screening method and molecular mechanism of umami peptide was investigated. RESULTS: This article showed that a more precisely rapid screening method with composite machine learning and molecular docking was used to screen the potential umami peptide from porcine bone. As reference, 24 reported umami peptides were predicated by composite machine learning, with the accuracy of 86.7%. In this study, potential umami peptide sequences from porcine bone were screened by UMPred-FRL, Umami-MRNN Demo, and molecular docking was used to provide further screening. Finally, nine peptides were screened and verified as umami peptides by this method: LREY, HEAL, LAKVH, FQKVVA, HVKELE, AEVKKAP, EAVEKPQS, KALSEEL and KKMFETES. The hydrogen bonding was deemed to be the main interaction force with receptor T1R3, and domain binding sites were Ser146, His121 and Glu277. The result demonstrated the feasibility of machine learning assisted T1R1/T1R3 receptor for rapid screening umami peptides. The screening method would not only adapt to screen umami peptides from porcine bone but possibly applied for other sources. It also provided a reference for rapid screening of umami peptides. CONCLUSION: The manuscript lays a rapid screening method in screening umami peptide, and nine umami peptides from porcine bone were screened and identified. © 2022 Society of Chemical Industry.

Uncovering the Metabolic Mechanism of Salidroside Alleviating Microglial Hypoxia Inflammation Based on Microfluidic Chip-Mass Spectrometry.

Microglia are the main immune cells in the brain playing a critical role in neuroinflammation, and numerous pieces of evidence have proved that energy metabolism is closely associated with inflammation in activated microglia. Salidroside (Sal) isolated from Tibetan medicine Rhodiola crenulate can inhibit microglial hypoxia inflammation (HI). However, whether the inhibition is due to the intervening energy metabolic process in microglia is not clear. In this work, the hypoxic microenvironment of BV2 microglial cells was simulated using deferoxamine (DFO) in vitro and the change of cell metabolites (lactate, succinate, malate, and fumarate) was real-time online investigated based on a cell microfluidic chip-mass spectrometry (CM-MS) system. Meanwhile, for confirming the metabolic mechanism of BV2 cells under hypoxia, the level of HI-related factors (LDH, ROS, HIF-1α, NF-κB p65, TNF-α, IL-1ß, and IL-6) was detected by molecular biotechnology. Integration of the detected results revealed that DFO-induced BV2 cell HI was associated with the process of energy metabolism, in which cell energy metabolism changed from oxidative phosphorylation to glycolysis. Furthermore, administration of Sal treatment could effectively invert this change, and two metabolites of Sal were identified: tyrosol and 4-hydroxyphenylacetic acid. In general, we illustrated a new mechanism of Sal for reducing BV2 cell HI injury and presented a novel analysis strategy that opened a way for real-time online monitoring of the energy metabolic mechanism of the effect of drugs on cells and further provided a superior strategy to screen natural drug candidates for HI-related brain disease treatment.

Isomer-specific biomarker discovery in multiple myeloma with dual-derivatized N-glycans.

As one of the most important post-translational modifications, protein glycosylation plays vital role in various physiological processes. With multitudinous glycosyltransferases, N-glycans present structural diversity in linkages and branching styles. Structure-specific glycan profiling may provide more potential biological information than compositional profiling. In this work, N-glycans released from human serum samples were derivatized with reduction and methylamination prior to profiling using nanoLC-ESI-MS with PGC as stationary phase. In addition, α 2-3 neuraminidase was also applied for distinguishing the linkage types of sialic acid corresponding to different isomers. Relative abundances of 280 isomeric N-glycans were compared and 20 isomers showed significant difference between multiple myeloma cases and healthy controls. ROC was performed to assess the significantly altered isomeric glycans and 6 AUCs have exceeded 0.80, providing high diagnostic accuracy for MM. PCA is also employed to establish the differences among sample sets. Furthermore, these specific isomers have also been used for early detection of multiple myeloma, presenting important clinical application value. Isomer-specific biomarker discovery in multiple myeloma with dual-derivatized N-glycans.

Research progress in the screening and evaluation of umami peptides.

Umami is an important element affecting food taste, and the development of umami peptides is a topic of interest in food-flavoring research. The existing technology used for traditional screening of umami peptides is time-consuming and labor-intensive, making it difficult to meet the requirements of high-throughput screening, which limits the rapid development of umami peptides. The difficulty in performing a standard measurement of umami intensity is another problem that restricts the development of umami peptides. The existing methods are not sensitive and specific, making it difficult to achieve a standard evaluation of umami taste. This review summarizes the umami receptors and umami peptides, focusing on the problems restricting the development of umami peptides, high-throughput screening, and establishment of evaluation standards. The rapid screening of umami peptides was realized based on molecular docking technology and a machine learning method, and the standard evaluation of umami could be realized with a bionic taste sensor. The progress of rapid screening and evaluation methods significantly promotes the study of umami peptides and increases its application in the seasoning industry.

The evaluation of sea cucumber (Acaudina leucoprocta) peptide on sex hormone regulation in normal and premature ovarian failure female mice.

Sea cucumber peptides (SCPs) have various functional activities. However, studies to evaluate the efficacy and safety of SCPs from the perspective of sex hormones are still lacking. In this study, normal and premature ovarian failure (POF) female mice were used to assess the effect of SCPs on the sex hormones. The ovarian and uterine indices were not influenced by SCP both in normal and POF mice. In normal mice, SCP showed no significant impact on the estrous cycle, ovarian, uterine morphology, sex hormone levels, and sex hormone synthesis-related genes of the ovary. However, 0.6 mg per g bw dosage of SCP (SCPH) statistically increased mapk1 expression on normal mice hypothalamus. In POF mice, SCPH played a more positive role than a low dosage of SCP (0.2 mg per g bw). SCP ameliorated POF-induced estrous cycle disturbances and significantly increased serum estradiol, testosterone, and AMH levels. Moreover, SCP increased the synthesis of the sex hormone by upregulating the expression of StAR, Fshr, and Cyp19a1 in the ovary, which might be due to the activation of the cAMP-related signaling pathways. The upregulation of mapk1, Esr1, and Gnrh was also observed in the hypothalamus. Together, SCP is safe for normal female mice and seems to have positive effects on POF mice from sex hormone regulation. However, the risk of excessive supplementation of sex hormones induced by the SCP intake in POF mice needs to be further explored.

The enhancement and mechanism of the perception of saltiness by umami peptide from Ruditapes philippinarum and ham.

To explore the saltiness enhancement effect and mechanism of umami peptides, umami peptides from Ruditapes philippinarum and ham were mixed with NaCl and determined using electronic tongue, sensory evaluation, and the aroma chicken model (ACM), then transmembrane channel-like protein 4(TMC4) receptor was constructed for molecular docking. The results showed that KEMQKN, NGKET, RGEPNND, AHSVRFY, LSERYP, NRTF, TYLPVH, EV, AGAGPTP, and GPAGPAGPR had saltiness enhancement effect, which could be increased to 0.4-0.6 % NaCl salty taste in 0.3 % NaCl. Under neutral conditions (pH6.5), most umami peptides were in negative ion state which may be the main reason that umami peptides could bind to the TMC4 receptor and enhance saltiness. The lowest docking energy was -113.325 kcal/mol among 10 peptides and the active sites Lys568, Trp145, Tyr565, Arg151, and Gln155 in TMC4 may play a key role. Thus, this study provides basic theory and data for salt-reduction strategies.

A rapid selection strategy for umami peptide screening based on machine learning and molecular docking.

Umami peptides have been the focus of umami studies in recent years because of their high nutritional value and flavor activity. However, the existing screening methods of umami peptides were cumbersome, complex, time-consuming and laborious, and it was difficult to achieve high-throughput screening. In this study, a novel umami peptide rapid screening model was designed and by using lamb bone aqueous extract as raw material, through the step-by-step screening of peptidomics, machine learning methods, and molecular docking technology. Results showed that six novel peptides about lamb bones were obtained, which verified the feasibility of the model and could be used for high-throughput screening of umami peptides. Results of molecular docking between umami peptide and T1R3 subunit revealed that the main interaction forces were hydrogen bonding and electrostatic interaction, and the key binding sites were GLU277 and SER146. It provides the basis for studying the binding mechanism of umami peptide.

Transport, Stability, and In Vivo Hypoglycemic Effect of a Broccoli-Derived DPP-IV Inhibitory Peptide VPLVM.

Diabetes is a major metabolic disease that requires long-term pharmacotherapy. Bioactive peptides have unique advantages such as higher potency, selectivity, and safety over small molecules and have achieved great success in the treatment of diabetes. We previously isolated a dipeptidyl peptidase-IV (DPP-IV) inhibitory peptide VPLVM with IC50 = 99.68 µM from the protein hydrolysates of broccoli stems and leaves. Here, we evaluated the interaction with DPP-IV, transport, stability, and in vivo hypoglycemic effects of VPLVM. VPLVM interacted closely and steadily with DPP-IV at S1 and S2 pockets. VPLVM had a good gastrointestinal enzyme resistance and was transported through the Caco-2 cell monolayer via paracellular diffusion and by the PepT1 with a Papp of 6.96 × 10-7 cm/s. VPLVM has a t1/2 of 12.56 ± 0.41 min in vitro plasma stability. In the oral glucose tolerance test, VPLVM showed an excellent hypoglycemic effect at 30 min after administration. VPLVM has potential as a candidate for the treatment of hyperglycemia.

A strategy for screening novel umami dipeptides based on common feature pharmacophore and molecular docking.

To shorten the complex and tedious process of traditional umami peptide identification, a novel model based on common feature pharmacophore (HipHop, a ligand molecule-based screening method) and molecular docking (a receptor protein-based screening method) was established for umami peptide screening. In this study, HipHop was used to perform a preliminary screening of peptides. Dipeptides with potential umami activity were docked into the umami taste receptor T1R1/T1R3 for a second screening. Twenty previously unreported umami dipeptides identified through virtual screening were validated using sensory evaluation and electronic tongue analysis. All 20 dipeptides (HE, HD, KE, EH, ET, EQ, DH, DR, DQ, DN, DY, DM, DI, DV, QE, QD, NE, ND, CE, and SE) had umami taste with umami threshold values ranging from 0.094 to 1.517 mmol/L. Therefore, when we increased the screening criteria for docking energy to -60 kcal/mol, the virtual screening results had 100% accuracy. The T1R1-peptide complexes of the four dipeptides with the lowest umami threshold values were subjected to molecular dynamics (MD) simulations for 100 ns, and the results showed that the four umami dipeptides remained in the starting active cavity. Overall, this screening strategy could be applied to the rapid screening of umami peptides in food products.

Advances in the stability challenges of bioactive peptides and improvement strategies.

Bioactive peptides are widely used in functional foods due to their remarkable efficacy, selectivity, and low toxicity. However, commercially produced bioactive peptides lack quality stability between batches. Furthermore, the efficacies of bioactive peptides cannot be guaranteed in vivo due to gastrointestinal digestion and rapid plasma, liver, and kidney metabolism. The problem of poor stability has restricted the development of peptides. Bioactive peptide stability assessments use different stability assays, so the results of different studies are not always comparable. This review summarizes the quality stability challenges in the enzymatic hydrolysis production of bioactive peptides and the metabolism stability challenges after oral administration. Future directions on the strategies for improving their stability are provided. It was proposed that we use fingerprinting as a quality control measure using qualitative and quantitative characteristic functional peptide sequences. The chemical modification and encapsulation of bioactive peptides in microcapsules and liposomes are widely used to improve the digestive and metabolic stability of bioactive peptides. Additionally, the establishment of a universal stability test and a unified index would greatly improve uniformity and comparability in research into bioactive peptides. In summary, the reliable evaluation of stability is an essential component of peptide characterization, and these ideas may facilitate further development and utilization of bioactive peptides.

Identification and virtual screening of novel anti-inflammatory peptides from broccoli fermented by Lactobacillus strains.

Lactobacillus strains fermentation of broccoli as a good source of bioactive peptides has not been fully elucidated. In this work, the peptide composition of broccoli fermented by L. plantarum A3 and L. rhamnosus ATCC7469 was analyzed by peptidomics to study the protein digestion patterns after fermentation by different strains. Results showed that water-soluble proteins such as rubisco were abundant sources of peptides, which triggered the sustained release of peptides as the main target of hydrolysis. In addition, 17 novel anti-inflammatory peptides were identified by virtual screening. Among them, SIWYGPDRP had the strongest ability to inhibit the release of NO from inflammatory cells at a concentration of 25 µM with an inhibition rate of 52.32 ± 1.48%. RFR and KASFAFAGL had the strongest inhibitory effects on the secretion of TNF-α and IL-6, respectively. At a concentration of 25 µM, the corresponding inhibition rates were 74.61 ± 1.68% and 29.84 ± 0.63%, respectively. Molecular docking results showed that 17 peptides formed hydrogen bonds and hydrophobic interactions with inducible nitric oxide synthase (iNOS). This study is conducive to the high-value utilization of broccoli and reduction of the antibiotic use.

Molecular docking and antihypertensive effects of a novel angiotensin-I converting enzyme inhibitory peptide from yak bone.

A novel angiotensin-converting enzyme (ACE) inhibitory peptide ser-ala-ser-val-ile-pro-val-ser-ala-val-arg-ala (SASVIPVSAVRA) was purified and identified from yak bone by Electrospray Ionization-Time of Flight-Mass Spectrometry (ESI-TOF-MS). Results in vitro showed that the peptide exhibited strong ACE inhibition activities with an IC50 of 54.22 µM. Molecular docking results showed the binding between the peptide SASVIPVSAVRA and ACE mainly driven by van der Waals forces, hydrogen bonds and metal receptor. Interestingly, the ACE inhibition activities of the peptide increased about 19% after digestion, but none of its metabolites showed stronger activity than it. The in vivo experiment showed that the antihypertensive effect of peptide SASVIPVSAVRA at dose of 30 mg/kg is nearly equal to Captopril at dose of 10 mg/kg to spontaneously hypertensive rats (SHRs). The antihypertensive effect mechanism of SASVIPVSAVRA should be further studied through plasma metabolomics and bioanalysis. Structure analysis of amino acids and peptides produced during digestion may help better understand the antihypertensive effect of peptides.

Novel Umami Peptide IPIPATKT with Dual Dipeptidyl Peptidase-IV and Angiotensin I-Converting Enzyme Inhibitory Activities.

A novel umami peptide, IPIPATKT, showed excellent dual dipeptidyl peptidase-IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory activities, the IC50 values were 64 and 265 µM, respectively. Molecular docking displayed that IPIPATKT was docked into the S1 and S2 pockets of ACE, and it was close to the active site pocket of DPP-IV. The insulin-resistant-HepG2 (IR-HepG2) cell model and human umbilical vein endothelial cell (HUVEC) model showed that the peptide significantly increased the content of glucose, the activities of hexokinase, pyruvate kinase, and the concentration of nitric oxide (p < 0.01), while it reduced the content of endothelin-1 (ET-1). IPIPATKT exhibited a hypotensive effect (-23.5 ± 2.2 mmHg) and attenuated the increase in glucose levels in vivo, as demonstrated using spontaneous hypertensive rats (SHRs) and C57BL/6N mice. We reported the in vivo activities of the umami peptide with dual hypertensive and hypoglycemic effects for the first time.

Isolation, characterization and molecular docking of novel umami and umami-enhancing peptides from Ruditapes philippinarum.

To understand the taste of the Ruditapes philippinarum, 14 novel umami peptides were isolated and identified by gel chromatography, HPLC and UPLC-ESI-QTOF-MS/MS. Separations were combined with sensory evaluations and electronic tongue determinations. The peptide sequences were GRVSNCAA, SEEK, KEMQKN, KSAEN, QIEELEGK, TDVEQEGD, HNESQN, RGEPNND, TGDPEK, KGGGGP, TYLPVH, PAATIPE, GPAGPAGPR and AGAGPTP. All peptides had umami and umami-enhancing qualities, KSAEN and QIEELEGK had higher sensory evaluation than the others, while PAATIPE and HNESQN had the best umami-enhancing taste in a 0.35% MSG solution. Molecular docking of the peptides with T1R1/T1R3 indicated that Ser123, Ser146 and Tyr143 may be important in the interaction of the peptides with T1R3. Arg303, Ser123 and Asp166 appear to be involved in the synergistic effect of umami peptides combined with monosodium glutamate. The omission test and the addition test confirmed that the 14 umami peptides contributed to the umami taste of R. philippinarum.

Novel Broccoli-Derived Peptides Hydrolyzed by Trypsin with Dual-Angiotensin I-Converting Enzymes and Dipeptidyl Peptidase-IV-Inhibitory Activities.

Broccoli-derived peptides show beneficial metabolic effects, and it is necessary to examine their exact functional sequences. First, peptides from the trypsin hydrolysate of broccoli proteins were isolated and identified using column chromatography and quadrupole time-of-flight mass spectrometry. After that, their functions were verified by oral administration. The results identified two novel peptides as Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (LPGVLPVA) and Tyr-Leu-Tyr-Ser-Pro-Ala-Tyr (YLYSPAY). LPGVLPVA exhibited an ACE IC50 value of 0.776 ± 0.03 µM and a DPP-IV IC50 value of 392 ± 24 µM; YLYSPAY showed an ACE IC50 value of 8.52 ± 0.63 µM and a DPP-IV IC50 value of 181 ± 4 µM. Administration of the peptides reduced the blood pressure of spontaneously hypertensive rats and reduced blood glucose levels in the oral glucose tolerance test in mice. The results indicated that LPGVLPVA and YLYSPAY could be potential nutritional candidates for hypertensive and diabetic people, especially for those with diabetes associated with hypertension.

Transport, In Vivo Antihypertensive Effect, and Pharmacokinetics of an Angiotensin-Converting Enzyme (ACE) Inhibitory Peptide LVLPGE.

The angiotensin-converting enzyme (ACE) inhibitory peptide LVLPGE provides outstanding antihypertensive effects in vivo, with a maximum systolic blood pressure (SBP) drop of 39 mmHg at a dose of 10 mg/kg. We evaluated the gastrointestinal digestion, transport, and in vivo antihypertensive effects of LVLPGE at different doses. LVLPGE was resistant to gastrointestinal enzymes with a stability of 97.8% and a permeability Papp of (5.09 ± 0.94) × 10-7 cm/s. LVLPGE was mainly transported through the Caco-2 cell monolayer by the peptide transporter PepT 1 and passive-mediated transport. LVLPGE at doses of 30 and 50 mg/kg had a positive antihypertensive effect in vivo; 30 mg/kg had a more significant effect than 50 mg/kg. After oral administration, the pharmacokinetics of LVLPGE showed that the Cmax was 4.65 ng/mL at 2 min. The blood pressure-lowering effect increased as the concentration of LVLPGE increased in the plasma of spontaneous hypertensive rats (SHRs).

Angiotensin I-Converting Enzyme (ACE) Inhibitory and Antioxidant Activity of Umami Peptides after In Vitro Gastrointestinal Digestion.

Umami peptides can help reduce the salt content in foods while still maintaining a savory taste. Few studies have reported the bioactivity of umami peptides after consumption. We studied the bioactivities of 12 umami peptides after gastrointestinal digestion. Three umami peptides exhibited angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activity after digestion. Six novel peptides were identified from digestion solutions of the peptides by HPLC-MS/MS. Among them, CC, CCNK, and HCHT had both ACE inhibitory activity (IC50 values were 9.81, 9.00, and 114.99 µM, respectively) and antioxidant activity (strong 1,1-Diphenyl-2-pycryl-hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging activities). AHSVRF had strong ACE inhibitory activity. These peptides increased the nitric oxide concentration and decreased the content of endothelin-1 in a medium of human umbilical vein endothelial cells in a dose-dependent manner. Experiments with damaged HepG2 cells showed that peptides CC, CCNK, and HCHT had antioxidant activity through their cytoprotective effects and by reducing the reactive oxygen species content. The results indicated that umami peptides may provide many health benefits after consumption.