Peptide Composition Research Articles

Prediction of inhibitory peptides against E.coli with desired MIC value

In the past, several methods have been developed for predicting antibacterial and antimicrobial peptides, but only limited attempts have been made to predict their minimum inhibitory concentration (MIC) values. In this study, we developed predictive models for MIC values of antibacterial peptides against Escherichia coli (E. coli), comprised of 3143 peptides for training and 786 peptides for validation, with experimentally determined MIC values. We found that the Composition Enhanced Transition and Distribution (CeTD) attributes significantly correlate with MIC values. Initially, we attempted to estimate MIC using BLAST similarity searches but found them inadequate. Subsequently, we employed machine learning regression models that integrated various features, including peptide composition, binary profiles and embeddings from large language models. Feature selection techniques, particularly mRMR, were utilized to refine our model inputs. Our Random Forest regressor built using default parameters achieved a correlation coefficient (R) of 0.78, R2 of 0.59, and RMSE of 0.53 on the validation set. Our best model outperformed existing methods when benchmarked on an independent dataset of 498 anti-E. coli peptides. Additionally, we screened anti-E. coli proteins in the proteomes of three probiotic bacterial strains and created a web-based platform, “EIPpred”, enabling users to design peptides with desired MIC values (https://webs.iiitd.edu.in/raghava/eippred).

Electrospinning of Self-Assembling Oligopeptides into Nanofiber Mats: The Impact of Peptide Composition and End Groups.

Low-molecular-weight oligopeptides can be electrospun into nanofiber mats. However, the mechanism underlying their electrospinnability is not well-understood. In this study, we used solid-phase peptide synthesis to produce the oligopeptide FFKK, to which the aromatic end-capping groups naphthalene, pyrene, and tetraphenylporphyrin were attached. Nuclear magnetic resonance, circular dichroism, and electrospray ionization mass spectrometry were used to characterize the oligopeptide structures. We investigated the effect of end-caps and oligopeptide concentration on their self-assembly as well as on their electrospinnability in fluorinated solvents. All oligopeptides with aromatic end-caps were amenable to electrospinning. Attenuated total reflectance Fourier transform infrared spectroscopy and microrheology results support the hypothesis that at sufficiently high concentrations, the self-assembled structures interact strongly, which facilitates electrospinning. Moreover, the results from this fundamental study can be extended to nonpeptidic small molecules possessing strong intermolecular interactions.

A comparative analysis of sequence composition in different lots of a phage display peptide library during amplification

BackgroundTo develop efficient selection strategies and improve the discovery of promising ligands, it is highly desirable to analyze the sequence composition of naïve phage display libraries and monitor the evolution of their peptide content during successive rounds of amplification. In the current study, we performed a comparative analysis of the compositional features in different lots of the same naïve phage display library and monitored alterations in their peptide compositions during three rounds of amplification.MethodsWe conducted three rounds of duplicate serial amplification of two different lots of the Ph.D.™-12 phage display library. DNA from the samples was subjected to Next-Generation Sequencing (NGS) using an Illumina platform. The NGS datasets underwent a variety of bioinformatic analyses using Python and MATLAB scripts.ResultsWe observed substantial heterogeneity in the sequence composition of the two lots indicated by differences in the enhanced percentage of wildtype clones, reduced diversity (number of unique sequences), and increased enrichment factors (EFs) during amplification as well as by observing no common sequence between lots and decreased number of common sequences between the naïve library and the consecutive rounds of amplification for each lot. We also found potential propagation-related target-unrelated peptides (TUPs) with the highest EFs in the two lots, which were displayed by the fastest-propagating phage clones. Furthermore, motif analysis of the most enriched subpopulation of amplified libraries led to the identification of some motifs hypothesized to contribute to the increased amplification rates of the respective phage clones.ConclusionOur results highlight tremendous heterogeneity in the peptide composition of different lots of the same type of naïve phage display library, and the divergent evolution of their compositional features during amplification rounds at the amino acid, peptide, and motif levels. Our findings can be instrumental for phage display researchers by bringing fundamental insights into the vast extent of non-uniformity between phage display libraries and by providing a clear picture of how these discrepancies can lead to different evolutionary fates for the peptide composition of phage pools, which can have profound impacts on the outcome of phage display selections through biopanning.

Fingerprint Profile Analysis of Eupolyphaga steleophaga Polypeptide Based on UHPLC-MS and Its Application

Background and Objectives: As a medicinal and food homologous substance, Eupolyphaga steleophaga is renowned for its potential health benefits, including anti-tumor effects, immune system support, and anti-inflammatory properties. Eupolyphaga steleophaga polypeptides have demonstrated significant biological activity, including the regulation of coagulation and lipid metabolism. However, the peptide composition of Eupolyphaga steleophaga requires further clarification to facilitate quality control improvements and a deeper investigation into its pharmacological effects. Therefore, this study aimed to simulate the digestive absorption process of Eupolyphaga steleophaga following oral administration and identify its enzymatic components to enhance quality control. Methods: The digestive absorption process was simulated using artificial gastric fluid and pepsin. A fingerprinting method based on ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS)(Acquire UPLC-Synapt G2-Si HDMS, Waters Corporation, Milford, MA, USA) was developed to identify 63 enzymatic components. The enzymolysis polypeptide fingerprint detection method was used to analyze 10 batches of Eupolyphaga steleophaga sourced from Harbin No. 4 Traditional Chinese Medicine Factory. Chromatographic collection was performed using an ACQUITY UPLC BHE C18 column. Gradient elution was carried out using a mixture of 0.1% formic acid with acetonitrile and 0.1% formic acid with water, with an average flow rate of 0.3 mL/min, a column temperature of 40 °C, and an injection volume of 2 μL. The mass spectrometry (MS) conditions were set as follows: the ion source was operated in positive electrospray ionization (ESI+) mode, with a capillary voltage of 2.8 kV and a sampling cone voltage of 40 V. The ion-source temperature was maintained at 110 °C, while the desolvation temperature was set to 400 °C. The cone gas flow rate was 50 L/h, and the desolvation gas flow rate was 800 L/h. The range for the collection of mass-to-charge ratios (m/z) was between 50 and 1200. Results: The UHPLC-MS method demonstrated high accuracy, repeatability, and stability, successfully identifying 63 enzymatic components of Eupolyphaga steleophaga. Furthermore, polypeptide markers for 63 selected components were identified in all 10 batches of Eupolyphaga steleophaga medicinal materials. This approach was validated by including numerical values such as retention times and peak areas, confirming its reliability for quality control enhancement. Conclusions: This novel UHPLC-MS approach serves as a powerful tool for advancing quality control strategies in veterinary medicine, particularly for animal-derived medicines. It lays a solid foundation for subsequent pharmacological studies of Eupolyphaga steleophaga polypeptides, offering a more reliable means to explore their biological activities and therapeutic potential.

Peptidomic Differences Between Natural Musk and Secretions from Forest Musk Gland Cells Cultured In Vitro

Background The Chinese forest musk deer, a solitary ungulate renowned for its musk secretion, is under extinction threat despite its extensive use in traditional Chinese medicine and perfumery. The demand for musk significantly outstrips its natural supply. Purpose This study aimed to explore the differences in peptide profiles between cultured musk gland cells and natural musk to develop sustainable alternatives for musk production and aid in the conservation of musk deer. Materials and Methods Musk samples from Chinese forest musk deer and cultured cells were collected and analyzed using the EASY-nLC 1000 system. Peptide extraction, nano LC–MS/MS analysis, and advanced peptidomics techniques were employed to identify and compare peptide compositions. Results The study found significant differences in secreted and natural musk peptide compositions. Cultured cells could partially replicate the complex peptide environment of natural musk, with some unique peptides identified in natural samples not present in cultured secretions. Conclusion While cultured musk gland cells show potential in mimicking natural musk synthesis, notable differences in peptide profiles suggest the importance of the gland’s microenvironment and the deer’s physiological conditions. This research establishes a foundation for optimizing culture techniques to enhance the therapeutic qualities of synthesized musk, contributing to the sustainable production of musk and the conservation of the species.

Biodegradable, ionic thermoelectric composites via self-assembly of dipeptides and deep eutectic solvents

The growing demand for biodegradable conductive composites is driven by the need to mitigate electronic waste and advance bioelectronics for healthcare applications. Self-assembled peptide composites, particularly diphenylalanine (FF) derivatives, represent a promising class of materials for such electronics due to their inherent biodegradability and ease of hybridization with functional materials. However, the integration of ionic species with these peptides is often limited by the disruption of non-covalent interactions between FF derivatives. In this study, we developed biodegradable, ionic thermoelectric composites by co-assembling Fmoc-FF with deep eutectic solvents (DESs) composed of choline chloride (ChCl) and ethylene glycol (EG). Spectroscopic analyses revealed that Fmoc-FF formed eutectogels through π-π interactions between Fmoc groups, resulting in a highly porous colloidal network. The Fmoc-FF eutectogels exhibited an ionic conductivity of up to 47.5 mS·cm−1 and a Seebeck coefficient of 7.39 mV·K−1, making them suitable for heat harvesting. Additionally, they were entirely degraded within 48 h under proteolytic conditions, confirming their biodegradability. The eutectogels also displayed self-healing and shear-thinning behaviors, highlighting compatibility with additive manufacturing techniques for device integration.Graphical

Analysis of Body Composition and Levels of Antimicrobial Peptides in Patients with Basal Cell Carcinoma: A Preliminary Study.

Background: Excessive body fatness is the basis of many diseases, especially civilization-related ones. The aim of this study is to analyze the body composition and serum levels of selected antimicrobial peptides (AMPs) in patients with basal cell carcinoma (BCC), in comparison to healthy controls (HCs), and investigate whether any specific parameter significantly increases the risk of BCC development. Methods: The body composition and measurements of serum levels of cathelicidin and human-beta-defensin-2 were analyzed in a group of 100 subjects (50 patients with BCC and 50 HCs). Results: There were statistically significant differences between the visceral fat rating (BCC 11.7 vs. control 10.1), cathelicidin (BCC 1022.6 vs. control 428.4), defensin-2 (BCC 1.2 vs. control 0.4), age (BCC 68.7 vs. control 62.4), and the visceral fat/muscle ratio (BCC 0.24 vs. control 0.21). Conclusions: It seems that excessive fat, especially visceral fat, may pose a risk of developing skin cancer. Therefore, it should be taken into account when caring for patients and they should be made aware that losing body weight may be important not only in reducing the risk of hypertension or diabetes but also cancer diseases. There are numerous well-known risk factors for developing skin cancer, but few are modifiable. Among these modifiable factors is the patient's weight and body composition, so improvaing lifestyle is crucial in the prevention of skin cancers.

Dosage and organic acid residue of myelin oligodendrocyte glycoprotein35–55 peptide influences immunopathology and development of Bacillus Calmette-Guérin induced experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) serves as a model for studying multiple sclerosis, with immunization strategies utilizing myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, emulsified in adjuvant enriched with Mycobacterium tuberculosis (Mtb). This study examined the effects of Bacillus Calmette-Guérin (BCG) as an adjuvant, alongside the impact of MOG35-55 peptide doses and their residual counter ions on EAE development. We found that BCG can be effectively used to induce EAE with similar incidence and severity as heat-killed H37Ra, contingent upon the appropriate MOG35-55 peptide dose. Different immunization doses of MOG35-55 peptide significantly affect EAE development, with higher doses leading to a paradoxical reduction in disease activity, probably due to peripheral tolerance mechanisms. Furthermore, doses of MOG35-55 peptides with acetate showed a more pronounced effect on disease development compared to those containing trifluoroacetic acid (TFA), suggesting the potential influence of residual counter ions on EAE activity. We highlighted the feasibility of applying BCG to the establishment of EAE for the first time. Our findings emphasized the importance of MOG35-55 peptide dosage and composition in modulating EAE development, offering insights into the mechanisms of autoimmunity and tolerance. This could have implications for autoimmune disease research and the design of therapeutic strategies.

Underutilized Mexican crop, runner bean (Phaseolus coccineus): A comprehensive review of nutritional and functional implications

Introduction. The scarlet runner bean (Phaseolus coccineus L.), also known as the runner or ayocote bean, is one of five Phaseolus species domesticated in Mesoamerica. It holds considerable economic importance, ranking third after P. vulgaris and P. lunatus. Despite its relevance, research on its bioactive compounds and potential health benefits still needs to be conducted. Objective: This review explores the nutritional composition, anti-nutritional factors, and bioactive compound levels of P. coccineus, emphasizing their implications for consumer health. Methodology: A comprehensive literature review, following PRISMA 2020 guidelines, was conducted across electronic databases, identifying 163 relevant studies. After applying inclusion and exclusion criteria, 13 studies were selected for analysis. Results: These studies highlighted P. coccineus as a potential source of nutraceutical properties and innovative food products aimed at enhancing nutritional quality. The bioactive compounds, including antioxidants and peptides, suggest benefits in preventing chronic diseases and promoting food security. Conclusions: However, further research is required to fully understand the composition of phenolic compounds, bioactive peptides, and anti-nutritional factors in P. coccineus, promoting its consumption as a functional food and novel dietary ingredient. Expanding its use in novel food formulations could enhance its dietary value and positively impact public health.

Application of AprX from Pseudomonas paralactis for the improvement of the emulsifying properties of milk, plant and insect protein and estimation of their hydrolysate’s bitter potential

AbstractProtein properties can be modified by selective enzymatic hydrolysis. In this study, the alkaline metalloendopeptidase AprX (Serralysin; EC 3.4.24.40) from Pseudomonas paralactis was used for the tailored hydrolysis of different food proteins resulting in the production of protein hydrolysates with improved emulsifying properties. Sodium caseinate, wheat gluten and buffalo worm protein were used for AprX hydrolysis at 40 °C and pH 8 to cover a spectrum of different protein sources. A maximum degree of hydrolysis (DH) of 13.1 ± 0.2%, 14.2 ± 0.1% and 20.7 ± 0.1% was reached for sodium caseinate, wheat gluten and the worm protein, respectively. The corresponding hydrolysate properties were analyzed regarding their particle size, peptide composition, solubility, viscosity, surface hydrophobicity and interfacial tension. The emulsifying properties were investigated by the oil-droplet size, ζ-potential and stability of emulsions prepared from the hydrolysates. Using partially hydrolyzed sodium caseinate (DH = 10.6%) as an emulsifier lead to an eightfold increase of the emulsion stability (t1/2 = 180 ± 0 min) compared to unhydrolyzed sodium caseinate. The emulsion stability using wheat gluten hydrolysates (DH = 11.9%) was increased 30-fold (t1/2 = 45 ± 5 min). Simultaneously, the solubility of gluten was increased by 60%. Buffalo worm hydrolysates (DH = 14.6%) had a twofold (t1/2 = 85 ± 5 min) increased emulsion stability. In conclusion, AprX can be used to improve the solubility and emulsifying properties of food proteins at a relatively high DH.

Recent Advances in the Application of Foodborne Substances in Hyperuricemia.

Hyperuricemia (HUA) is a purine metabolism disorder characterized by the excessive production or inadequate excretion of uric acid. Current pharmacological strategies targeting uric acid reduction have potential adverse effects. Following the concept of "homology of medicine and food", food ingredients are increasingly being explored to prevent HUA and gout, with xanthine oxidase (XOD) emerging as a crucial therapeutic target in managing HUA. Recent scientific investigations have determined that uric acid-lowering substances come from various food sources, such as seafood, dairy products, and agricultural products. These bioactive substances have attracted wide attention because of their effective antihyperuricemia and XOD inhibitory ability. In this study, the pathogenesis, many side effects of uric acid-lowering drugs, and some components of uric acid-lowering drugs are mainly described, with emphasis on the source, composition, preparation technology, and mechanism of uric acid-lowering peptides.

Thiol-linked hyaluronic acid-mediated encapsulation of RCR-stabilized gold nanoclusters for hyaluronidase sensing and cellular imaging

Encapsulating peptide-stabilized gold nanoclusters (AuNCs) with thiolated hyaluronic acid (HA-SH) and selectively adding cysteine to the peptide sequence increased their photoluminescence. We found that peptide compositions with cysteine in the middle emitted the most. RCR-stabilized AuNCs can be purified using size-exclusion chromatography to characterize their optical characteristics, chemical composition, and possible structure. Our findings show that RCR-stabilized AuNCs have a unique chemical structure, microsecond photoluminescence lifetime, good quantum yield, and near-infrared emission peak. Due to Au-S bonding and electrostatic interactions, RCR-stabilized AuNCs were encapsulated with HA-SH to create nanocomposites. HA-SH-AuNCs had a longer emission peak, greater particle size, and better photostability than RCR-stabilized AuNCs. HAase break down HA in HA-SH-AuNCs, changing their structure and size. Thus, centrifugation makes it easier to separate HA-SH-AuNCs from HAase-digested ones. Similar to earlier sensors, HA-SH-AuNCs have great sensitivity and selectivity for HAase, with a linear range of 0.5–6.0 U/mL and a detection limit of 0.39 U/mL. They were useful for urine HAase determination, with spike recovery of 103 % to 107 %. HA-SH-AuNCs further served as a platform for targeted imaging of CD44 receptor-expressing cancer cells, demonstrating bioimaging and clinical diagnostic potential.

Peptidomic Analysis of Potential Bioactive Peptides in Mare Milk Under Different Heat Treatment Conditions.

Active peptides in mare milk have unique biological activities, but how the bioactive protein in mare's milk changes under the influence of temperature has not been fully studied. Therefore, in this study, the differential expression of bioactive peptides potentially present in horse milk under different heat treatment conditions was investigated for the first time using peptidomic and bioinformatic techniques. We collected a total of 15 samples. In this study, we divided the samples into five groups, specifically, 65 °C, 30 min; 72 °C, 15 min; 83 °C; 10 min; 95 °C, 5 min; and an untreated group as a control, which involved four different temperature treatments, in order to understand changes in bioactive peptides and to identify the sequence of bioactive peptides. In the experiment, a total of 2341 active peptides were identified. The amino acid composition of the potential active peptides remained stable across different temperatures, but their abundance varied with temperature. In all, 23 peptides from 20 different proteins were identified, with the highest number of active peptides identified at 72 °C. Through database searches, we found that a majority of these peptides were within β-lactoglobulin and immunoglobulin domain proteins, which are known for their potential biological activities. These findings provide a theoretical foundation for the development of peptides with different bioactivities as potential functional foods.

Metabolomic Analysis Reveals the Linkage between Sleep-Enhancing Effects and Metabolite Biomarkers and Pathways of Different Casein Hydrolysates in Chronic Unpredictable Mild Stressed Mice.

Casein hydrolysates have been proven to exert varying sleep-enhancing and anxiolytic effects due to their distinct release of potential peptides. However, their underlying sleep-enhancing mechanisms at the metabolic level remain unclear. This study aims to investigate the potential sleep-enhancing mechanism of casein hydrolysates through an integrated approach of untargeted and targeted metabolomics in CUMS-induced anxiety mice for the first time. The results showed seven potential biomarkers were identified and screened using orthogonal partial least-squares discriminant analysis, random forest model, and pathway analysis, including ornithine, l-proline, l-prolinamide, inhibitory neurotransmitters gamma-aminobutyric acid, 5-HIAA, fumaric acid, and 4-oxoglutaramate. Moreover, casein hydrolysates exerted sleep-enhancing effects through multiple metabolic pathways, mainly including the GABAergic system, tryptophan metabolism, and cAMP response signaling pathway, which was validated by targeted metabolomics and vital protein expressions. It was interesting that casein hydrolysates with diverse representative peptide compositions exhibited varying activity, which could be attributed to distinct alterations in metabolites via different pathways.

Hydroxyprolyl-Glycine in 24 H Urine Shows Higher Correlation with Meat Consumption than Prolyl-Hydroxyproline, a Major Collagen Peptide in Urine and Blood.

Background. Urinary collagen peptides, the breakdown products of endogenous collagen, have been used as biomarkers for various diseases. These non-invasive biomarkers are easily measured via mass spectrometry, aiding in diagnostics and therapy effectiveness. Objectives. The objective of this study was to investigate the effects of consuming collagen-containing meat on collagen peptide composition in human blood and urine. Methods. Ten collagen peptides in 24 h urine were quantified. Results. Prolyl-hydroxyproline (Pro-Hyp) was the most abundant peptide. Except for hydroxyprolyl-glycine (Hyp-Gly), levels of other minor collagen peptides showed high correlation coefficients with Pro-Hyp (r = 0.42 vs. r > 0.8). Notably, 24 h urinary Hyp-Gly showed a correlation coefficient of r = 0.72 with meat consumption, significantly higher than the coefficient for Pro-Hyp (r = 0.37). Additionally, the levels of Pro-Hyp and Hyp-Gly in the blood of seven young women participants increased similarly after consuming fish meat, while before ingestion, only negligible amounts of Hyp-Gly were present. To examine which peptides are generated by the degradation of endogenous collagen, mouse skin was cultured. The amount of Pro-Hyp released from the skin was approximately 1000-fold higher than that of Hyp-Gly. Following consumption of collagen-containing meat, both Pro-Hyp and Hyp-Gly are released in blood and excreted into urine, although Pro-Hyp is primarily generated from endogenous collagen even under physiological conditions. Conclusions. Therefore, in 24 h urine samples, the non-negligible fraction of Pro-Hyp is contributed by endogenous collagen, making 24 h urine Hyp-Gly level a potential biomarker for evaluating meat consumption on the day.

Computational exploration of naturally derived peptides inhibitory mechanisms against ACE enzyme, from interactions to structural-dynamics

Naturally derived peptides have gained significant attention because of their potential to reduce blood pressure. These peptides can be derived from various sources, including snake venom, marine organisms, cow's milk, seahorses, and plants. Investigating the underlying mechanisms of these peptides in lowering blood pressure is crucial for replacing synthetic drugs currently in use. In this regard, we conducted in silico studies, such as molecular docking, classical molecular dynamics (MD) simulations, and QM/MM simulation methods, on two naturally derived peptides (PAGPRGPA and WALKGYK) and Angiotensin II (ANGII) as the enzyme substrates for binding to sACE. The results of 500 ns MD calculations showed that the WALKGYK peptide occupies the ACE binding site, similar to the ANGII and BPPb peptides, two distinguished C-domain-specific inhibitors. Furthermore, QM/MM calculations demonstrated that no peptide bond cleavage was mediated by Zn2+ at the catalytic site of the peptides. However, during the 500 ns MD simulation, the backbone oxygens of LYS4 and GLY5 of the WALKGYK peptides were tightly coordinated to the Zn2+ ion. Free energy calculations also confirmed the higher affinity of the WALKGYK peptides for binding to sACE. In addition, structural analysis correlation showed a different pattern in PAGPRGPA compared to WALKGYK and ANGII. Despite the similarity of the peptide PAGPRGPA to typical ACE peptide inhibitors with hydrophobic ends, the electrostatic composition of the WALKGYK peptide showed a higher tendency towards ACE inhibition. Therefore, peptide residue compositions, such as WALKGYK, can be considered for designing new inhibitors with fewer side effects for sACE C-terminal inhibitors.

Effect of protease reaction conditions on volatile compounds generated in Maillard reaction products from soy protein hydrolysates

Maillard reaction products (MRPs) produced by heating protease-catalyzed soy protein hydrolysates (SPHs) with cysteine and ribose can generate meat-like flavors. However, the impact of protease reaction conditions on the volatile compound composition of MRPs has not been thoroughly investigated. In this study, seven SPHs were prepared using two proteases, flavourzyme and trypsin, over reaction times of 10, 120, and 1440 min. The volatile compound compositions, including sulfur-containing compounds, aldehydes, pyrazines, and furans, of the seven SPHs and the corresponding seven MRPs varied according to the protease reaction conditions and the Maillard reaction. Differences in pH, free amino acid composition, and peptide composition were responsible for these variations. Notably, soy-derived peptides containing unique cysteine sequences, such as PGCPST, DETICT, ECQIQK, and HCQR, were significantly reduced during the Maillard reaction, suggesting that these sequences may serve as precursors to volatile compounds.

Биологически активные гидролизаты белков молока и их комплексы включения с циклодектринами

Enzymatic hydrolysis of dairy proteins increases their nutritional and biological value while reducing their allergenic potential. The subsequent complexation of peptides with cyclodextrins (CDs) reduces the bitterness of the hydrolyzed proteins. The research objective was to obtain hydrolysates of whey proteins and their cyclodextrin inclusion complexes with peptides, as well as to describe the peptide composition of the cleaved dairy proteins, biological activity, and sensory profile of the hydrolysates and inclusion complexes. The research featured enzymatic whey protein hydrolysates with an extensive hydrolysis degree and their inclusion complexes with β- and γ-CDs. Dairy proteins were hydrolyzed with alcalase, and the hydrolysates obtained were subjected to micro- and ultrafiltration (cut-off limit – 10 kDa). The peptide composition of the hydrolyzed proteins was determined by the methods of high-performance liquid chromatography and chromatography-mass spectrometry. The antimutagenic activity was evaluated using the Ames test whereas the antibacterial effect was studied with the impedimetric method. The antioxidant activity was detected with fluorimetry and spectrophotometry. The method of competitive enzyme immunoassay was applied to reveal the antigenic properties. The bitterness of the experimental sample s was determined by a sensory evaluation. The research delivered the optimal modes for whey protein cleavage with alcalase that made it possible to achieve efficient micro- and ultrafiltration. The resulting hypoallergenic peptide fractions and their inclusion complexes with β- and γ-CDs possessed antioxidant, antibacterial, and antimutagenic properties. The whey proteolysis and subsequent filtration with/without tindalization demonstrated a 265/589-fold decrease in the residual antigenicity. The fluorimetric method showed a 1.79/1.90-fold increase in the antioxidant activity of the hydrolysate in complexes with β- and γ-CDs. Binding of β-CDs to peptides enhanced their antimicrobial activity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. The hydrolysate samples with β-CDs showed less bitterness. Whey proteolysis with alcalase under optimized conditions and subsequent fractionation resulted in a product with high consumer qualities. Enzymatic hydrolysates of dairy proteins and their CD inclusion complexes were able to substitute native protein components. Their bioactive properties, good taste, and low allergenic potential mean good prospects for the functional food industry.

Effect of In Vitro Digestion on Bioactive Peptides Related to Immune and Gut Health in Intact Cow's Milk and Hydrolyzed Protein-Based Infant Formulas.

Background/Objectives: Human milk is the optimal source of nutrition and protection against infection for infants. If breastfeeding is not possible, standard and hydrolyzed infant formulas (IF) are an alternative. Extensively hydrolyzed IFs (eHFs) contain bioactive peptides, but their activities have rarely been evaluated. The aim of this study was to characterize and compare the bioactive peptide profiles of different eHFs and standard IFs before and after in vitro digestion. Methods: Two forms, liquid and powder, of intact protein formula (iPF) and eHF were subjected to in vitro gastrointestinal digestion, mimicking a young infant's gut (age 0-4 months) and an older infant's gut (>6 months). Bioactive peptides of in vitro digested and undigested formulas were analysed with Liquid Chromatography-Mass Spectrometry (LC-MS). Results: In all samples, a variety of peptides with potential bioactive properties were found. Immuno-regulatory peptides, followed by antimicrobial and antioxidative peptides were most frequent, as were peptides promoting wound healing, increasing mucin secretion, regulating cholesterol metabolism, and preventing bacterial infection. Peptides typically found in yoghurt and colostrum were identified in some formula samples. Conclusions: The high amounts of bioactive peptides with various properties in eHFs and iPFs indicate a possible contribution to infection protection, healthy gut microbiomes, and immunological development of infants. eHFs showed similar compositions of bioactive peptides to iPFs, with intermittently increased peptide variety and quantity.

Evaluating the impact of Torulaspora delbrueckii and amino acid concentration on the nitrogen metabolism of Oenococcus oeni

This study investigates the impact of Saccharomyces cerevisiae and Torulaspora delbrueckii inoculation strategies and amino acid supplementation on nitrogen metabolism and malolactic fermentation (MLF) performance in Oenococcus oeni. Oenococcus oeni has low demand of nitrogen and typically prefers peptides over amino acids. In this work we studied the nitrogen metabolism (proteins, peptides, amino acids and biogenic amines (BA)) of three O. oeni strains. Higher initial amino acid concentrations generally accelerated MLF, particularly with the PSU-1 strain, which exhibited stuck fermentation in some conditions with low initial amino acid concentration related with coinoculation of yeasts. Nitrogen metabolism in O. oeni showed a preference for peptide-bound amino acids, and gene expression analyses highlighted a general upregulation in response to increased amino acid concentrations. BA production, particularly cadaverine and putrescine, was strain-dependent and associated with the presence of the odc gene. Besides, the production of 2-phenylethylamine was related with alcoholic fermentation (AF) inoculation strategy. Overall, the study demonstrates the complexity of nitrogen metabolism in O. oeni, emphasizing that it is influenced by both the yeast species used in AF and the initial amino acid composition, highlighting the importance of peptide composition, offering a potential tool for optimizing MLF in winemaking.