Search Results (223)

During gastrointestinal digestion, dietary proteins are hydrolyzed into peptides and free amino acids that modulate enteroendocrine function and satiety-related hormone secretion along the gut–brain axis, thereby contributing to obesity prevention. We investigated whey protein concentrate (WPC) as a source of bioactive peptides and evaluated the effects of its digests on cholecystokinin (CCK) secretion in STC-1 enteroendocrine cells by integrating the standardized INFOGEST in vitro digestion protocol, peptidomics (LC–MS/MS), and in silico bioactivity prediction. In STC-1 cells, the <3 kDa intestinal peptide fraction exhibited the strongest CCK stimulation, positioning these low-molecular-weight peptides as promising bioactive components for satiety modulation and metabolic health applications. Peptidomic analysis of this fraction identified short sequences derived primarily from β-lactoglobulin (β-La) and α-lactalbumin (α-La), enriched in hydrophobic and aromatic residues, including neuropeptide-like sequences containing the Glu–Asn–Ser–Ala–Glu–Pro–Glu (ENSAEPE) motif of β-La f(108–114). In silico bioactivity profiling with MultiPep predicted antihypertensive, angiotensin-converting enzyme (ACE)–inhibitory, antidiabetic, dipeptidyl peptidase-IV (DPP-IV)–inhibitory, antioxidant, antibacterial, and neuropeptide-like activities. Overall, digestion of WPC released low-molecular-weight peptides and amino acids that enhanced CCK secretion in vitro; these findings support their potential use in nutritional strategies to enhance satiety, modulate appetite and energy intake, and improving cardiometabolic health. Full article

Cheese whey, the major by-product of the dairy industry, poses an environmental challenge due to its high organic load but simultaneously represents a nutrient-dense matrix suitable for biotechnological valorization. This review synthesizes recent advances positioning whey as (i) a fermentation substrate for lactic acid bacteria, yeasts/fungi, and microalgae, enabling the production of functional biomass, organic acids, bioethanol, exopolysaccharides, enzymes, and wastewater bioremediation; (ii) a platform for postbiotic generation, supporting cell-free preparations with functional activities; and (iii) a food-grade encapsulating material, particularly through whey proteins (β-lactoglobulin, α-lactalbumin), which can form emulsions, gels, and films that protect biotics and bioactive compounds during processing, storage, and gastrointestinal transit. We analyze key operational variables (whey type and pretreatment, supplementation strategies, batch and continuous cultivation modes), encapsulation routes (spray drying, freeze-drying, and hybrid protein–polysaccharide systems), and performance trade-offs relevant to industrial scale-up. Finally, we outline future directions, including precision fermentation, mixed-culture processes with in situ lactase activity, microfluidics-enabled encapsulation, and life-cycle assessment, to integrate product yields with environmental performance. Collectively, these strategies reframe whey from a high-impact waste into a circular bioeconomy resource for the food, nutraceutical, and environmental sectors. Full article

The prediction of polypeptide chain fragmentation during digestion (proteolysis) of protein substrates by trypsin was carried out for globular β-lactoglobulin (β-LG) and micellar β-casein (β-CN). Despite significant differences in the protein structures of these substrates, the concentrations of peptide fragments are calculated as functions of time or degree of hydrolysis using the same equations derived from the general proteolysis model. This model considers the opening of protein substrates in the course of proteolysis, the so-called demasking process, and the subsequent hydrolysis of specific peptide bonds at different rates determined by the amino acid sequence of hydrolyzed sites. The use of this model for in silico prediction of proteolysis is discussed. An algorithm for calculating demasking rate constants based on the experimental distribution of peptide fragments is presented. The calculated concentration dependence on the degree of hydrolysis of peptide bonds was compared with the experimental data for the intermediate and final peptide fragments of β-LG and β-CN. The predicted and experimental concentration curves for the final products were compared based on their curvatures. For both substrates, the predicted redistribution of peptide fragments in the course of proteolysis was found to be consistent with the experimental one. Full article

Low-flow liquid chromatography has become the primary tool for advanced chromatographic analysis and is an indispensable technique for the sensitive detection of biomolecules. In this study, we developed a new 4-tritylphenyl methacrylate-based monolithic nano-column with an internal diameter of 20 µm for bioanalytical separations in nano-liquid chromatography (nano-LC). The composition of the monolith was optimized with regard to the monomer and porogenic solvent. The column was characterized using Fourier Transformed Infrared Spectroscopy (FT-IR) spectroscopy, scanning electron microscopy (SEM) and chromatographic analyses. Chromatographic characterization was performed using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), which facilitate hydrophobic and π–π interactions. Run-to-run and column-to-column reproducibility values were found to be <2.51% and 2.4–3.2%, respectively. The final monolith was then used to separate six standard proteins, including β-lactoglobulin A, carbonic anhydrase, ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C) and myoglobin (Myo), as well as three dipeptides: Alanine-tyrosine (Ala-Tyr), Glycine-phenylalanine (Gly-Phe) and L-carnosine. The nano-column was then applied to profiling peptides and proteins in the MCF-7 cell line, enabling high-resolution peptide analysis. Full article

Donkey milk is increasingly recognized as a functional food due to its unique nutritional profile and richness in bioactive compounds. This longitudinal observational study investigated changes in both chemical composition (total solids, protein, fat, lactose, and ash) and immune-active proteins (lactoferrin, α-lactalbumin, β-lactoglobulin, and lysozyme) across lactation. A total of 153 donkey milk samples were collected from five farms from very early (1–3 days in milk) to late lactation (30–210 days in milk). Chemical composition was determined using mid-infrared spectroscopy, while the concentrations of the immune-active proteins were determined by ELISA Quantitative Sandwich. Chemical analysis showed high values in colostrum, including total solids (10.13%), protein (3.1%), and ash (0.73%), which declined progressively during lactation to 8.45%, 1.14%, and 0.64%, respectively. Fat varied modestly, between 0.55 and 0.25%, while lactose remained stable at 5.75–6.41%. In parallel, bioactive proteins measured between 31 and 210 days exhibited distinct trajectories. Lactoferrin increased from 0.07 to 0.14 mg/mL, α-lactalbumin peaked mid-lactation at 2.58 mg/mL (compared with 1.91 mg/mL early and 2.25 mg/mL late), β-lactoglobulin declined from 0.84 to 0.55 mg/mL, and lysozyme decreased from 0.95 mg/mL early to 0.64 mg/mL late. Across lactation, we observed dilution of total solids and protein, relatively stable lactose and fat, and distinct trajectories of lactoferrin, α-lactalbumin, β-lactoglobulin, and lysozyme, indicating that donkey milk modulates rather than loses its protective protein profile. These results refine reference values for donkey milk and support its nutraceutical relevance for human nutrition and health. Full article

A common strategy for a protein’s functionality modification is the covalent binding of phenolic compounds (PCs) under alkaline conditions. Whether intentionally applied or arising during food processing and storage, such reactions are highly relevant, as alkaline pH promotes oxidation, covalent adduct formation, and polymerization, thereby altering both PC and protein properties. However, the interplay of these reactions and the impact of PC structure remain insufficiently understood. This study aimed at characterizing covalent binding products of structurally related PCs with tryptic peptides of the model protein β-lactoglobulin (β-Lg) at pH 9. Emphasis was given on substitution patterns and steric effects influencing polymerization and peptide adduct building. Hydroxycinnamic acid and flavonoid derivatives differing in hydroxyl substitution and carrying polar (glycosidic) groups were selected. Incubation products were characterized by HPLC–DAD and high-resolution mass spectrometry. Results showed that both mono- and dihydroxy PC undergo oxidation under alkaline conditions, but with distinct reactivity. Monohydroxy PCs form only limited peptide adducts due to resonance stabilization and steric hindrance. In contrast, dihydroxy PCs displayed a higher reactivity, producing more polymerization products and covalent adducts. Their enhanced reactivity is linked to the ability of quinone formation with reduced electrostatic repulsion, while additional polar substituents promote interactions with polar amino acids. At the same time, these substituents impose steric constraints on PC polymerization, modulating oligomer size and thereby influencing peptide binding. Overall, the findings highlight structural determinants of PC reactivity and provide mechanistic insight into the balance between polymerization and covalent peptide modification under alkaline conditions. Full article

Fermented mare’s milk, or koumiss, has been consumed for centuries across Central Asia for its nutritional and therapeutic value. Mare’s milk differs from bovine milk by its near 1:1 casein-to-whey ratio, high lysozyme and lactoferrin, abundant immunoglobulins, and low β-lactoglobulin, which enhance digestibility, reduce allergenicity, and increase antimicrobial activity. During fermentation, lactic acid bacteria and yeasts transform this substrate into a reservoir of bioactive proteins, peptides, and metabolites. Multi-omics profiling has identified more than 2300 peptides and over 350 metabolites, including sequences with angiotensin-converting enzyme (ACE)-inhibitory, antioxidant, antimicrobial, and immunomodulatory activities. Preclinical and limited clinical data indicate potential benefits for lipid metabolism, cardiovascular function, and gut health. Mechanistically, these effects appear to arise from synergistic actions of native proteins, fermentation-derived peptides, and probiotic consortia. Technological advances such as rational starter culture design, controlled proteolysis, and microencapsulation offer strategies to enhance bioactive yield and stability. However, standardized fermentation protocols and clinical validation remain necessary to position koumiss as a scientifically supported functional food. Full article

Bioactive peptides (BAPs) from dairy products have garnered increasing attention as natural agents with health-promoting properties, including antihypertensive, antioxidant, antimicrobial, immunomodulatory, opioid, and antidiabetic activities. This systematic review synthesizes research published between 2014 and 2024, retrieved from Scopus and PubMed, and selected according to PRISMA guidelines. A total of 192 studies met the inclusion criteria, collectively reporting over 3200 distinct peptides, with antihypertensive sequences, predominantly angiotensin-converting enzyme (ACE) inhibitors, constituting the largest category (n = 1237). β-casein was the principal precursor across bioactivities, followed by αs1-casein, β-lactoglobulin, and α-lactalbumin. Peptides were primarily produced via enzymatic hydrolysis, microbial fermentation, and gastrointestinal digestion, with peptide profiles influenced by the type of milk, microbial strains, and processing conditions. While cow’s milk remained the dominant source, investigations into goat, sheep, camel, buffalo, and donkey milk revealed species-specific biopeptides. Recent advances in proteomics have enhanced peptide identification and bioactivity prediction, enabling the discovery of novel sequences. These findings underscore the significant potential of dairy-derived BAPs as functional food components and nutraceutical ingredients, while highlighting the need for further in vivo validation, bioavailability studies, and broader exploration of underrepresented milk sources. Full article

β-lactoglobulin (β-Lg), the major whey protein containing nine lysine residues, serves as an ideal model for studying protein glycation and thermal processing safety in dairy products. This study systematically compared three different high-temperature treatment methods, namely superheated steam (SS), hot air (HA), and oil bath (OB), to investigate their effects on the spatial conformation and glycation product formation of proteins in the β-Lg-glucose system. The results show that compared with OB and HA, SS has a lower degree of glycation, lower consumption of free amino groups, and less unfolding of the protein’s three-dimensional structure. It leads to a lower proportion of α-helix transformation into β-sheet and random coil in the protein. SS resulted in the least browning and produced less 5-hydroxymethylfurfural, pentosidine, fluorescent advanced glycation end products, and melanogenin, yet produced the highest amount of Carboxymethyllysine. Mass spectrometry analysis shows that lysine residues were the primary glycation sites. Therefore, this work provides molecular-level insights into how different heating techniques modulate protein glycation and structural stability, supporting the potential of superheated steam as a gentler alternative to control glycation for β-Lg in food thermal processing. Full article

Background: β-lactoglobulin (BLG) is a protein found within whey protein (WP) that is rich in essential amino acids, most notably, leucine (LEU). LEU is considered the most potent EAA in the postprandial stimulation of muscle protein synthesis (MPS), such that suboptimal protein/essential amino acid (EAA) doses containing higher LEU content elicit muscle anabolism comparable to larger protein doses. Our objective was to test the effects of naturally LEU-rich BLG (~10 g protein) versus isonitrogenous whey protein isolate (WPI, ~10 g) on MPS. Methods: Ten healthy young men (26 ± 2 y; 179 ± 2 cm; 81 ± 3 kg) received BLG (1.57 g LEU) or WPI (1.02 g LEU) in a randomised double-blind cross-over fashion. A primed constant intravenous infusion of [1,2 ¹³C₂] LEU was used to determine MPS (isotope ratio mass spectrometry) at baseline and in response to feeding (FED) and feeding-plus-exercise (FED-EX; 6 × 8 unilateral leg extensions; 75% 1-RM). Plasma insulin and EAA’s were quantified. Results: Plasma EAA, branched-chain amino acid (BCAA), and LEU concentrations increased rapidly following both protein supplements but exhibited a significantly greater EAA/BCAA/leucinemia following BLG (p < 0.05 for all). MPS increased significantly in both FED (~52%) and FED-EX (~58%) states, with no significant differences between supplements. Conclusions: Both BLG and WPI effectively stimulated MPS doses in young healthy males, with BLG offering an advantage in EAA/BCAA/LEU bioavailability. It follows that future research should explore the potential of BLG in populations exhibiting anabolic resistance and exercise anabolism deficiency, such as older adults as well as frail and clinical populations, to assess its utility in preserving muscle mass under conditions of suboptimal protein intake. Full article

The prevalence of food allergies has been steadily increasing in recent years. β-lactoglobulin (β-LG), the main allergenic protein of milk and dairy allergies, is more commonly observed in infants and children. In this study, a β-LG-specific aptamer was selected using the combinatorial chemistry process known as systematic evolution of ligands by exponential enrichment (SELEX), and a quartz crystal microbalance with dissipation monitoring (QCM-D)-based aptasensor was developed using a novel surface functionalization technique, which mimics an artificial cell membrane on the QCM-D sensor surface, creating a physiologically relevant environment for the binding of the target to the sensor. Through SELEX combined with next-generation sequencing (NGS), the aptamer Apt 356 was identified. Its binding to β-LG was confirmed via dot blot analysis. The selected Apt 356 was then used for the development of a QCM-D-based sensor. To fabricate the sensor, the quartz surface was functionalized with a supported lipid bilayer (SLB). The β-LG-specific aptamer was immobilized onto this SLB. The results demonstrated that the QCM-D system allows real-time observation and evaluation of the binding of β-LG. While there have been some studies on aptasensors for the β-LG protein, to the best of our knowledge, this is the first QCM-D-based aptasensor developed specifically for β-LG protein detection. Full article

Innovative changes to our current food system are needed, and one solution is cultivated meat, which uses modern engineering, materials science, and biotechnology to produce animal protein. This article highlights the advantages of incorporating whey protein isolate (WPI) and β-lactoglobulin (β-LG) into hydrogel networks to aid cell growth on cultivated meat scaffolds. The protein and polysaccharide (i.e., alginate) components of the scaffolds are food-grade and generally regarded as safe ingredients, enabling the transition to more food-safe, edible, and nutritious scaffolds. The impact of WPI and varying properties on cell performance was evaluated; alginate concentration and the addition of proteins into the hydrogels significantly altered their stiffness and strength. The results of this study demonstrate the innocuous nature of novel scaffolds and reveal enhanced cell proliferation on WPI and β-LG-modified groups compared to standard biomaterial controls. This work serves as a stepping stone for more comprehensive analyses of WPI, β-LG, and alginate scaffolds for use in cultivated meat research and production. Full article

Developing advanced antimicrobial agents is critically imperative to address antibiotic-resistant infection crises. MXenes have emerged as a potential nanomedicine for antibacterial applications, but they suffer from suboptimal photothermal conversion efficiency and inherent cytotoxicity. Herein, we report the synthesis of MXene (Ti₃C₂)-based nanohybrids and hybrid membranes through firstly interfacial conjugation of self-assembled β-lactoglobulin nanofibers (β-LGNFs)-inspired copper sulfide nanoparticles (CuS NPs) onto MXene nanosheets, and subsequent vacuum filtration of the created β-LGNF-CuS/MXene nanohybrids. The constructed β-LGNF-CuS/MXene nanohybrids exhibit excellent photothermal conversion performances and satisfactory biocompatibility and minimal cytotoxicity toward mammalian cells, ascribing to the introduction of highly biocompatible β-LGNFs into the hybrid system. In addition, the fabricated β-LGNF-CuS/MXene hybrid membranes demonstrate high efficiency in antibacterial application through the synergistic photothermal and material-related antibacterial effects of both MXene and CuS NPs. Therefore, the ideas and findings shown in this study are useful for inspiring researchers to design and fabricate functional and biocompatible 2D material-based hybrid membranes for antimicrobial applications. Full article

The functional properties of proteins are closely related to their structure and conformation. The effects of glycosylation and pH on the structural and conformational changes in whey protein isolate (WPI) were investigated using multispectral technology. More and higher-molecular-weight molecules of WPI–dextran conjugates (WDCs) with increased degrees of glycosylation (DGs) in SDS-PAGE occurred at the expense of band intensities of α-lactalbumin, β-lactoglobulin, and bovine serum albumin. The higher wavenumber shift in FTIR peaks of WPI after glycosylation in the Amide I, II, and III regions and the decrease in its intensity occurred. The maximum absorption wavelength (λ_max) of UV-Vis spectra of WPI before and after glycosylation in the range of 260–290 nm showed no significant difference in a pH range of 2.0–10.0. Moreover, the UV-Vis absorption intensities of WDCs at λ_max around 278 nm were highly and positively correlated with their DGs. The λ_max and intensities of total intrinsic fluorescence spectra of Tyr and Trp residues in WDCs with an increase in DGs had an obvious redshift and decrease, respectively. Although the intensities of synchronous fluorescence spectra of individual Tyr or Trp residues in WDCs with an increase in DGs also gradually decreased, the λ_max of the former and latter had a blueshift and redshift, respectively. UV-Vis absorption and fluorescence spectroscopies indicated that the changes in the λ_max and intensity of WPI were closely related to the protonation states of carbonyl groups and free amino groups and the degree of glycosylation. This work may be beneficial for understanding the structural and conformational changes in proteins by measuring the microenvironment around Tyr and/or Trp residues in proteins using UV-Vis absorption and synchronous fluorescence spectroscopies, providing a promising technique for quantitatively monitoring the degree of glycosylation (DG) in a rapid and practical way without any chemical reagents using UV-Vis absorption spectroscopy. Full article

The whey protein (WP) fraction represents 18–20% of the total milk nitrogen content. It was originally considered a dairy industry waste, but upon its chemical characterization, it was found to be a precious source of bioactive components, growing in popularity as nutritional and functional food ingredients. This has generated a remarkable increase in interest in applications in the different sectors of nutrition, food industry, and pharmaceutics. WPs comprise immunoglobulins and proteins rich in branched and essential amino acids, and peptides endowed with several biological activities (antimicrobial, antihypertensive, antithrombotic, anticancer, antioxidant, opioid, immunomodulatory, and gut microbiota regulation) and technological properties (gelling, water binding, emulsification, and foaming ability). Currently, various process technologies and biotechnological methods are available to recover WPs and convert them into BioActive Peptides (BAPs) for commercial use. Additionally, in silico approaches could have a significant impact on the development of novel foods and/or ingredients and therapeutic agents. This review provides an overview of current and emerging methods for the production, selection, and application of whey peptides, offering insights into bioactivity profiling and potential therapeutic targets. Recent updates in legislation related to commercialized WPs-based products are also presented. Full article