Tryptic Hydrolysis Research Articles

Myofibrillar protein hydrolysis under hydroxyl radical oxidative stress: Structural changes and their impacts on binding to selected aldehydes

In this study, a hydroxyl radical oxidation system was established to simulate the oxidation process in fermented meat products. This system was employed to examine the structural changes in myofibrillar proteins (MPs) resulting from tryptic hydrolysis after a hydroxyl radical oxidative regime. The effect of these changes on the ability of MPs to bind selected aldehydes (3-methyl butanal, pentanal, hexanal, and heptanal) was also investigated. Moderate oxidation (H2O2 ≤ 1.0 mM) unfolded the structure of MPs, facilitating trypsin-mediated hydrolysis and increasing their binding capacity for the four selected aldehydes. However, excessive oxidation (H2O2 ≥ 2.5 mM) led to cross-linking and aggregation of MPs, inhibiting trypsin-mediated hydrolysis. The oxidised MPs had the best binding capacity for heptanal. The interaction of the oxidised trypsin-hydrolysed MPs with heptanal was driven by hydrophobic interactions. The binding of heptanal affected the structure of the oxidised trypsin-hydrolysed MPs and reduced their α-helix content.

Influence of the enzymatic treatment and pH on the interfacial and emulsifying properties of sunflower and olive protein hydrolysates

This work investigates the influence of the enzymatic treatment (Alcalase or trypsin, degree of hydrolysis 5%) and pH (pH 7 or 4) on the interfacial and emulsifying properties of sunflower and olive protein hydrolysates. Independently of the enzymatic treatment, short peptides (1–3 kDa) were the most abundant in sunflower protein hydrolysates, whereas olive protein hydrolysates were richer in large peptides (>10 kDa). Peptides present in all hydrolysates gained in structure when adsorbing at the oil-water interface due to their facial amphiphilicity, with sunflower peptides presenting a more marked β-sheet conformation than olive peptides. Tryptic hydrolysates of both substrates showed higher interfacial adsorption compared to hydrolysates produced with Alcalase, especially at pH 4. All hydrolysates resulted in elastic interfaces, with generally higher values of dilatational complex modulus at pH 7 compared to pH 4. These findings correlated well with the higher emulsifying activity of all hydrolysates at pH 7 than pH 4. Particularly, sunflower protein hydrolysates led to stiffer and more solid-like viscoelastic interfacial layers than olive peptides due to increased interactions between β-sheet peptides at the interface. Indeed, the use of sunflower protein hydrolysates as emulsifiers resulted in 5 wt% oil-in-water emulsions with higher physical stability at both pH 7 and 4 when compared to olive protein hydrolysates.

The Shelf Life of Milk-A Novel Concept for the Identification of Marker Peptides Using Multivariate Analysis.

The quality of food is influenced by several factors during production and storage. When using marker compounds, different steps in the production chain, as well as during storage, can be monitored. This might enable an optimum prediction of food's shelf life and avoid food waste. Especially, proteoforms and peptides thereof can serve as indicators for exogenous influences. The development of a proteomics-based workflow for detecting and identifying differences in the proteome is complex and time-consuming. The aim of the study was to develop a fast and universal workflow with ultra-high temperature (UHT) milk as a proteinaceous model food with expectable changes in protein/peptide composition. To find an optimum shelf life without sticking to a theoretically fixed best-before date, new evaluation and analytical methods are needed. Consequently, a modeling approach was used to monitor the shelf life of the milk after it was treated thermally and stored. The different peptide profiles determined with high-resolution mass spectrometry (HRMS) showed a significant difference depending on the preparation method of the samples. Potential marker peptides were determined using orthogonal projections to latent structures discriminant analysis (OPLSDA) and principal component analysis (PCA) following a typical proteomics protocol with tryptic hydrolysis. An additional Python-based algorithm enabled the identification of eight potential tryptic marker peptides (with mass spectrometric structural indications m/z 885.4843, m/z 639.3500, m/z 635.8622, m/z 634.3570, m/z 412.7191, m/z 623.2967, m/z 880.4767, and m/z 692.4041), indicating the effect of the heat treatment. The developed workflow is flexible and can be easily adapted to different research questions in the field of peptide analysis. In particular, the process of feature identification can be carried out with significantly less effort than with conventional methods.

A novel and intelligent molecularly imprinted enzymatic biosensor for biosensing of human serum albumin in the presence of gamma-globulin, and glucose as uncalibrated interference

Human serum albumin (HSA) is not electroactive, because the electroactive amino acids have been buried in its structure. Therefore, direct electrochemical determination of the HSA as the most abundant protein in human plasma cannot be performed. In this work, a novel electrochemical biosensor has been developed based on synthesis of dual templates molecularly imprinted polymers (DTMIPs) having trypsin (TRP) and HSA as template molecules onto the surface of a glassy carbon electrode (GCE) modified with graphene-ionic liquid (Gr-IL). By incubation of the biosensor with TRP, and HSA, tryptic hydrolysis of the HSA is occurred which breaks the HSA down into free amino acids. The HSA involves five electroactive amino acids including cysteine, tryptophan, tyrosine, methionine and histidine whose differential normal pulse voltammetric (DNPV) responses are overlapped and generated a single peak. In order to increase the sensitivity of the DTMIP/Gr-IL/GCE for determination of the HSA, hydrodynamic DNPV (HDNPV) data were generated, and used for analytical purposes. The second-order HDNPV data were generated at different pulse amplitudes and used to develop four second-order calibration models by multivariate curve resolution-alternating least squares (MCR-ALS), parallel factor analysis2 (PARAFAC2), multi-way partial least squares/residual bilinearization (N-PLS/RBL), and unfolded partial least squares/residual bilinearization (U-PLS/RBL) to select the best procedure for determination of the HSA in the presence of gamma-globulin and glucose as uncalibrated interferences. The results confirmed the best performance for the biosensor assisted by MCR-ALS for ultrasensitive and selective determination of the HSA in both synthetic and real matrices which was comparable with HPLC-UV as a reference method.

Synthesis and Characterisation of Chickpea Peptides-Zinc Chelates Having ACE2 Inhibitory Activity.

Tryptic hydrolysates of protein fractions obtained by the Osborne method from chickpea (Cicer arietinum L.) seeds interacted with zinc ions and the results of chelation were monitored by the Energy Dispersive X-Ray (EDX) technique. The glutelin hydrolysate (GluHyd) reacted with zinc ions and depicted a relatively higher zinc content. For this reason, the zinc complex of the glutelin hydrolysate (GluHyd-Zn) was studied deeper, and 11 peptides were identified in its more zinc-containing second fraction obtained after gel filtration. The peptide HKERVQLHIIPTAVGK showed a relatively higher chelating capacity (57.86 ± 2.14%). According to the result of the ICP-OS analysis, 1mg peptide could chelate 381.61 ± 133.39µg zinc, and the molar ratio of peptide-zinc was about 1:4. Spectral methods proved that side chain and C-termini carboxyl groups of the peptide mostly were involved in chelation and N atoms of amino side chains, imidazole group of histidine, and N-termini at some extents were occupied by the metal ions. Modeling of zinc-peptide interaction was done using Molecular Operating Environment (MOE) software. The results of the docking correlate with the experimental data.ACE2 inhibitory effect of HKERVQLHIIPTAVGK-Zn complex (IC50 = 1.5mg/mL) was better than that of HKERVQLHIIPTAVGK (IC50 = 2.2mg/mL).

Identification of Marker Peptides for the Whey Protein Quantification in Edam-Type Cheese.

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides ('bottom-up' proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and β-lactoglobulin (β-LG). Based on the findings, α-LA and β-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for β-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

Hydrogel formation from peptides of a β-lactoglobulin tryptic hydrolysate: Contribution of self-assembling peptide β-Lg f1-8

Peptide-based hydrogels, typically mediated by non-covalent interactions, are becoming increasingly attractive due to their many biotechnological applications such as drug and antimicrobial delivery, as well as tissue engineering. In our previous work, we demonstrated that specific peptide fractions from beta-lactoglobulin (β-Lg) could form hydrogels either by peptide-peptide interaction or through peptide self-assembly of β-Lg f1-8, depending on the purity of β-Lg f1-8 in peptide fractions. However, mechanisms that govern hydrogel formation were not investigated. Consequently, this study focused on the impact of peptide fraction (pellet) concentration, temperature, pH and β-Lg f1-8 concentration on hydrogel formation. Moreover, contribution of peptides β-Lg f15-20 and β-Lg f41-60 to hydrogel formation was determined. We hypothesized that hydrogel formation results from a balance between hydrogen, hydrophobic and electrostatic interactions during gelation at a concentration of 50 mg/mL, 20 °C and pH close to 11. The peptide β-Lg f1-8 was shown to initiate the gelation by increasing the density of nanofibers in a concentration-dependent manner which promoted peptide-peptide interactions. Finally, interactions between both β-Lg f15-20 and β-Lg f41-60 peptides and β-Lg f1-8 occurred during gel formation negatively impacted the self-assembly process. The proposed mechanism for β-Lg f1-8 gelation would facilitate the development of functional soft biomaterials and bioactive encapsulation systems.

Proteome of monocled cobra (Naja kaouthia) venom and potent anti breast cancer peptide from trypsin hydrolyzate of the venom protein

Anticancer peptide is one of the target in the development of new anticancer drug. Bioactive peptide can be originated from isolated free peptide or produced by hydrolysis of protein. Protein is the main component of Naja kaouthia venom, and due to the toxicity of the venom, it can be assessed as the source of anticancer peptides. This study aims to characterize the venom protein and to identify peptides from the snake venom of N. kaouthia as anticancer. Proteome analysis was employed trypsin hydrolysis of N. kaouthia venom protein completed with HRMS analysis protein database query. Preparative tryptic hydrolysis of the protein followed by reverse-phased fractionation and anti breast cancer activity testing were performed to identify the potent anticancer from the hydrolysate. Proteomic analysis by high-resolution mass spectrometry revealed that there are 20 enzymatic and non-enzymatic proteins in N. kaouthia venom. The 25% methanol peptide fraction had the most active anticancer activity against MCF-7 breast cancer cells and showed promising selectivity (selectivity index = 12.87). Amino acid sequences of eight peptides were identified as potentially providing anticancer compounds. Molecular docking analysis showed that WWSDHR and IWDTIEK peptides gave specific interactions and better binding affinity energy with values of −9.3 kcal/mol and −8.4 kcal/mol, respectively. This study revealed peptides from the snake venom of N. kaouthia became a potent source of new anticancer agents.

Impact of UV-C pretreatment on β-lactoglobulin hydrolysis by trypsin: Production and bioavailability of bioactive peptides

The effects of UV-C light irradiation and low-temperature long-time (LTLT) pasteurization on protein structural changes, degree of hydrolysis (DH) by trypsin, peptide profile of tryptic hydrolysates by MALDI-TOF/TOF-MS, and bioavailability of β-lactoglobulin were compared. Compared with native or LTLT pasteurised samples, the hydrolysis rate constant of β-lactoglobulin treated with UV-C increased significantly, implying that the protein backbone cleavage sites became more accessible, whereas thermal treatment produced aggregates that impede trypsin activity. Tryptic hydrolyses of UV-C light treated β-lactoglobulin yielded more peptides and a more diverse peptide mass profile. Six bioactive peptides were revealed in β-LG tryptic hydrolysates of UV-C-treated protein; transepithelial transport in Caco-2 cell monolayers showed intermediate in vivo transport and absorption for three (β-LG f87–91, β-LG f91–99, and β-LG f158–164). The moderate allergen peptide LSFNPTQLEEQCHI β-LG was absent after tryptic hydrolysis of UV-C-treated protein, indicating that UV-C photolysis may be a useful tool for allergenicity reduction.

Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations.

Tryptic proteolysis of protein micelles was studied using β-casein (β-CN) as an example. Hydrolysis of specific peptide bonds in β-CN leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. Samples of these nanoparticles dried on a mica surface were characterized by atomic force microscopy (AFM) when the proteolytic reaction had been stopped by tryptic inhibitor or by heating. The changes in the content of β-sheets, α-helices, and hydrolysis products during proteolysis were estimated by using Fourier-transform infrared (FTIR) spectroscopy. In the current study, a simple kinetic model with three successive stages is proposed to predict the rearrangement of nanoparticles and the formation of proteolysis products, as well as changes in the secondary structure during proteolysis at various enzyme concentrations. The model determines for which steps the rate constants are proportional to the enzyme concentration, and in which intermediate nano-components the protein secondary structure is retained and in which it is reduced. The model predictions were in agreement with the FTIR results for tryptic hydrolysis of β-CN at different concentrations of the enzyme.

Interfacial Properties of Pea Protein Hydrolysate: The Effect of Ionic Strength

The effect of a tryptic hydrolysis as well as the effect of ionic strength (0–0.4 M NaCl) was investigated on the oil/water interfacial properties of soluble pea protein hydrolysate (SPPH) at neutral pH and room temperature (20 ± 0.01 °C). SEC-MALS and SDS-Page analysis showed that tryptic hydrolysis created a lower molecular weight polypeptide mixture, whereas FTIR analysis and DSC thermograms demonstrated a more disordered and flexible structure. The bulk properties of SPPH were studied in terms of hydrodynamic diameter and turbidity, where higher particle size (+ ~13 nm) and turbidity were observed at 0.4 M NaCl. Regarding the interfacial properties, the surface activity of SPPH improved by increasing ionic strength, with maximum interfacial pressure (14.28 mN/m) at 0.4 M NaCl. Nevertheless, the addition of NaCl negatively affected the elasticity and strength of the interfacial film, where the sample without salt exhibited the highest dilatational and shear storage modulus in all the frequencies considered.

EPITOPES IDENTIFICATION OF BROADLY NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST Corynebacterium diphtheriae EXOTOXIN

Background. Better and high-potency vaccines against diphtheria are urgently needed to provide broader protection against diverse strains and subtypes. Identification of novel broadly neutralizing epitopes targeted by protective antibodies could aid in such efforts. Aim. In this study we focused on the search of binding sites identification of anti diphtheria toxin monoclonal antibodies and their neutralizing activity to block binding of recombinant exotoxin derivates with host receptors. Methods. Vero cells were cultured in the complete RPMI-1640 medium under standard conditions and used for flow cytometry assay. Recombinant antigens and products of tryptic hydrolysis of CRM197 and SbB were characterized by Ni2+-NTA affinity chromatography and SDS-PAGE under reducing conditions with following ECL Western-Blot using several hybridomas clones of anti-diphtheria toxin monoclonal antibodies. Results. ECL western blot film results for clone 9.1-E11 showed the specific binding both to whole CRM197 molecule, and to almost all fragments of CRM197 formed as a result of limited proteolysis. In particular, a band corresponding to SbB in molecular weight can be identified. Thus, epitope region of the CRM197 molecule specific to 9.1-E1 mAbs is located within the structure of SbB. At the same time 16.4-E9 clone antibodies had high specificity to R-domain of SbB. In addition, both hybridoma clones antibodies have neutralizing activity against the DT binding subunit, which is a key factor in blocking between cell receptor and it ligand, C.diphtheriae exotoxin. Conclusions. The results obtained indicate that obtained antibodies are prospective for improving new diagnostic tools and therapeutic agents, which are used for treatment and understanding of the molecular mechanisms of diphtheria pathogenesis.

Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αs1 K34–αs2 Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins.Graphical abstract

Marker of schizophrenia with enduring negative symptoms

IntroductionThe relevance of this study is determined by the need to search for biological markers of schizophrenia. The detection and validation of such molecules can become the basis for the creation of additional paraclinical diagnostic methods or contribute to the creation of targets for individual pharmacotherapy, which is an important task of modern fundamental medicine.ObjectivesComparative proteomic analysis of serum in schizophrenic patients with positive and negative symptoms.MethodsThe study includes 10 healthy donors and 27 patients with schizophrenia. Samples preparation included: serum purification from major proteins via affinity chromatography, 1D-PAGE proteins separation, in-gel tryptic hydrolysis, LC-MS/MS mass-spectrometry (Orbitrap Q-exactive HF mass spectrometer, Agilent Technologies). Identification of proteins was carried out using Mascot software Ver. 2.1 («Matrix Science», USA). Proteins for quantitative analysis were selected in view of the DISGENET database. Quantitative LC-MS-SRM analysis of selected protein was performed on QQQ TSQ Vantage (Thermo Scientific) with labeled peptide standards.ResultsReceptor-interacting serine/threonine-protein kinase 1 was selected for quantitative assessment. Significant differences were revealed in the RIPK1 concentrations in the serum of schizophrenic patients with negative and positive symptoms (p=0.02). The serum concentration of RIPK1 in patients with negative symptoms is tenfold in patients with positive symptoms.ConclusionsReceptor-interacting serine/threonine-protein kinase 1 can be considered a biomarker of negative symptoms of schizophrenia based on a significant increase in serum concentration. Mass spectrometric analysis was carried out of the “Human Proteome” Core Facility of the Institute of Biomedical Chemistry Moscow. Support by Grant of RSF № 18-15-00053P.DisclosureNo significant relationships.

The Anxiolytic-like Properties of a Tryptic Hydrolysate of Bovine αs1 Casein Containing α-Casozepine Rely on GABAA Receptor Benzodiazepine Binding Sites but Not the Vagus Nerve.

(1) Background: A tryptic hydrolysate of bovine αs1-casein (CH) exerts anxiolytic-like properties in many species, including humans. This is mainly related to the presence of α-casozepine (α-CZP), which yields these properties in rodents. This study evaluates, in a rat model, the roles of the vagus nerve and the benzodiazepine binding site of GABAA receptors in the mode of action of CH. (2) Methods: The conditioned defensive burying test was used to evaluate anxiety. (3) Results: Participation of the vagus nerve in the mode of action of CH was excluded, as the global anxiety score in vagotomised rats was not significantly different from that of non-vagotomised animals. The blocking of the binding sites of benzodiazepines with flumazenil antagonised CH anxiolytic-like properties. (4) Conclusions: The vagus nerve does not play a role in the anxiolytic-like properties of CH. On the other hand, this anxiolytic-like activity relies on the benzodiazepine binding site of the GABAA receptors. This result is consistent with previous in vitro studies and, more specifically with the discovery of α-CZP, the peptide responsible for the anxiolytic-like properties of CH.

Reference LC-MS/MS method to detect fresh cheeses adulteration with whey

This study evaluated the potential of a reference method to detect fresh cheeses adulteration with whey by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Qualitative results were expressed as presence or absence of the marker peptide TPEVDDEALEK, obtained by tryptic hydrolysis of β-lactoglobulin. Sample preparation was based on defatting with cold acetone and protein solubilization in ammonium bicarbonate and urea buffer (pH = 8.0). Reversed phase liquid chromatography used a C18 column for separation of the analyte, whose retention time was 4.12 min. Mass spectrometry was carried out with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode for the precursor ion (m/z 624) and the quantitation (m/z 573) and confirmation transitions (m/z 820; m/z 920) of the peptide. Method validation was carried out in quantitative terms, to set the baseline concentration of the marker peptide in 69 authentic samples, and in qualitative terms, to set the action level that distinguish authentic from adulterated cheeses (350 mg kg−1). Sensitivity was enough to detect cheeses with 10% adulteration and the detection limit was set to 21 mg kg−1. The simple extraction procedure allowed high-throughput analysis of 33 real samples. Results were compared to SDS-PAGE electrophoresis, whose limitations for accurate quantitation were easily overcome by LC-MS/MS. The developed method ensured precision, accuracy, sensitivity, and specificity needed for the unequivocal detection of non-compliant cheeses made with cow or buffalo milk, without dealing with the highly toxic chemical species required for SDS-PAGE. This method can be extended in the future to detect similar adulterations in fresh cheeses prepared with milk from other animal species, as well as in other dairy products.

A method to identify and quantify the complete peptide composition in protein hydrolysates

Automated approaches from proteomics are used to characterise peptides for food applications and in protein digests. Peptide annotations and confidence in these annotations are then based on the fragment spectra. Low reproducibility in repeat analyses has been reported even for annotations with high confidence. When analysing protein hydrolysates (in food) it is important to determine criteria that yield highly reproducible annotations. This study provides a structured approach to determine these criteria. Tryptic hydrolysates of α-lactalbumin, β-lactoglobulin and β-casein were analysed manually and automatically, using an UPLC-PDA-MS method for untargeted identification and absolute label-free quantification of peptides. A lock mass with two components was introduced resulting in an average mass error of 1 ppm. Processing filters were set to ensure reliable annotations based on MS/MS fragmentation, while maintaining maximum amount of information. Peptides in the individual hydrolysates with an MS intensity above the limit of annotation represented 99% of total MS intensity and were 100% consistently annotated between four replicates. Amino acid and peptide sequence coverages for the individual protein hydrolysates were 99–100% and 89–95%, respectively. Mixing the hydrolysates resulted in a loss of 11% of the peptide annotations above the LOA and lower reproducibility (97%) for the remaining annotations, as well as more co-eluting peptides. Calculated concentrations of co-eluting peptides in mixed hydrolysates varied 37 ± 21% from the value for single hydrolysates. The proposed approach allows complete description of peptide composition with highly repeatable annotations and quantification of peptides even in mixed hydrolysates.

High-Performance Thin-Layer Chromatography-Immunostaining as a Technique for the Characterization of Whey Protein Enrichment in Edam Cheese.

Whey protein-enriched cheese can be produced by means of a high-temperature treatment of a part of the cheese milk. In this way, the nutritional quality of the resulting cheeses can be increased while resources are conserved. High-performance thin-layer chromatography-immunostaining (HPTLC-IS) using specific β-lactoglobulin (β-LG) antibodies was applied to study the implementation and stability of β-LG in two different sample sets of whey protein-enriched Edam model cheeses, including industrial-scale ones. Two methods were compared for the extraction of the proteins/peptides from the cheese samples. By applying tryptic hydrolysis directly from a suspended cheese sample instead of a supernatant of a centrifuged suspension, a better yield was obtained for the extraction of β-LG. When applying this method, it was found that selected epitopes in the tryptic β-LG peptides remain stable over the ripening period of the cheese. For four of the tryptic β-LG peptides detected by immunostaining, the amino acid sequence was identified using MALDI-TOF-MS/MS. One of the peptides identified was the semi-tryptic peptide VYVEELKPTP. A linear relationship was found between the content of this peptide in cheese and the proportion of high-heated milk in the cheese milk. β-LG enrichment factors of 1.72 (n = 3, sample set I) and 1.33 ± 0.19 (n = 1, sample set II) were determined for the cheese samples containing 30% high-heated milk compared to the non-enriched samples. The relative β-LG contents in the cheese samples with 30% high-heated milk were calculated to be 4.35% ± 0.39% (sample set I) and 9.11% ± 0.29% (sample set II) using a one-point calibration. It can be concluded that the HPTLC-IS method used is a suitable tool for the analysis of whey protein accumulation in cheese, being therefore potentially directly applicable on an industrial scale. For more accurate quantification of the whey protein content in cheese, an enhanced calibration curve needs to be applied.

Effect of Alpha-S1-Casein Tryptic Hydrolysate and L-Theanine on Poor Sleep Quality: A Double Blind, Randomized Placebo-Controlled Crossover Trial.

This randomized, placebo-controlled, crossover and double-blind study investigates the effects of RLX2™ containing alpha-s1-casein tryptic hydrolysate and L-theanine in working adults affected by poor sleep quality. The supplement or placebo was randomly and blindly assigned to 39 subjects for four weeks and the changes in the subjective sleep assessment via the Pittsburgh Sleep Quality Index (PSQI), heart rate, blood pressure, salivary cortisol by high-performance liquid chromatography method and alpha power of awake electroencephalogram (EEG) were studied. The data were analyzed in two ways, by crossover and crossover summed up. The latter depicted that RLX2™ improved PSQI total score, sleep latency, sleep duration, sleep habitual efficiency, daytime dysfunction, and increased total and frontal alpha power significantly (p < 0.05). The supplement prolonged the total sleeping time by 45 min in the supplement receiving group compared to the placebo group (p < 0.001). However, only sleep duration and sleep habitual efficiency showed a profound effect in both analyses (p < 0.05). In conclusion, being given its beneficial effects without notable adverse events, it would be advantageous to use these nutraceutical ingredients to promote better sleep quality. Further studies with a larger number participants are warranted to support these findings.

Soybean-Derived Peptides Attenuate Hyperlipidemia by Regulating Trans-Intestinal Cholesterol Excretion and Bile Acid Synthesis.

Increased triglyceride, cholesterol, and low-density lipoprotein (LDL) levels cause hyperlipidemia. Despite the availability of statin-based drugs to reduce LDL levels, additional effective treatments for reducing blood lipid concentrations are required. Herein, soybean hydrolysate prepared via peptic and tryptic hydrolysis promoted trans-intestinal cholesterol excretion (TICE) by increasing ATP-binding cassette subfamily G member 5 (ABCG5) and ABCG8 expression. The peptide sequence capable of promoting TICE was determined via HPLC and LC-MS/MS. Based on this, pure artificial peptides were synthesized, and the efficacy of the selected peptides was verified using cellular and hyperlipidemic mouse models. Soybean hydrolysates, including two bioactive peptides (ALEPDHRVESEGGL and SLVNNDDRDSYRLQSGDAL), promoted TICE via the expression of ABCG5 and ABCG8 in enterocytes. They downregulated expression of hepatic cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and CYP8B1 via expression of fibroblast growth factor 19 (FGF19) in a liver X receptor α (LXRa)-dependent pathway. Administration of bioactive peptides to hyperlipidemic mouse models by oral gavage reduced cholesterol levels in serum via upregulation of ABCG5 and ABCG8 expression in the proximal intestine and through fecal cholesterol excretion, upregulated FGF 15/19 expression, and suppressed hepatic bile acid synthesis. Oral administration of soybean-derived bioactive peptides elicited hypolipidemic effects by increasing TICE and decreasing hepatic cholesterol synthesis.