Peptidomic profile of human milk as influenced by fortification with different protein sources: An in vitro dynamic digestion simulation.

Fortification of human milk (HM) is often necessary to meet the nutritional requirements of preterm infants. The present experiment aimed to establish whether the supplementation of HM with either an experimental donkey milk-derived fortifier containing whole donkey milk proteins, or with a commercial bovine milk-derived fortifier containing hydrolyzed bovine whey proteins, affects peptide release differently during digestion. The experiment was conducted using an in vitro dynamic system designed to simulate the preterm infant's digestion followed by digesta analysis by means of LC-MS-MS. The different fortifiers did not appear to influence the cumulative intensity of HM peptides. Fortification had a differential impact on the release of either donkey or bovine bioactive peptides. Donkey milk peptides showed antioxidant/ACE inhibitory activities, while bovine peptides showed opioid, dipeptil- and propyl endo- peptidase inhibitory and antimicrobial activity. A slight delay in peptide release from human lactoferrin and α-lactalbumin was observed when HM was supplemented with donkey milk-derived fortifier.

Discrimination of overheated pasteurized milk using mass spectrometry-based proteomics.

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides-INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK-were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

Peptidome profiling of human, bovine, and donkey colostrum through label-free quantitative analysis reveals proteolysis of milk proteins.

Proteins and peptides play vital roles in different biological processes in vivo. As a dynamic hydrolysis system, milk is rich in proteins and proteases and provides a constant supply of endogenous bioactive peptides to newborn mammals. Previous studies have primarily focused on researching bioactive peptides by adding exogenous enzymes to milk samples. However, such an approach overlooks the significance of endogenous peptides and parent proteins that naturally exist in milk. Herein, we analyzed and compared parent proteins and their releasing peptides in human colostrum (HC), bovine colostrum (BC), and donkey colostrum (DC). The predominant proteins and hydrolyzed peptides in the three types of milk were identified. Among them, peptides were found to possess common bioactivities, including ACE inhibitory, antioxidant, antibacterial and immunomodulatory properties in HC, BC, and DC. Furthermore, the biological functions of these parent proteins were clarified using bioinformatics. These insights offer a novel perspective on natural bioactive peptides and the potential utilization of specific parent proteins and peptides to develop infant formulae derived from diverse milk sources.

Milk serum peptidomics revealed the age gelation of direct UHT milk.

Age gelation is undesirable for direct UHT (dUHT) milk, which is closely related to protein hydrolysis. However, little information is available for the role of serum peptides during the age gelation. In this study, the composition and protein morphology of serum phase were characterized by RP-HPLC, ICP-MS and TEM. The results showed significant increases in soluble proteins, free amino acids, calcium, and phosphorus from casein micelles, indicating protein hydrolysis and peptide release into the serum phase. 23,466 peptides derived from caseins and other proteins were identified in serum phase by peptidomics. The serum peptide profiles of age gelation milk changed dramatically. Peptide fingerprinting revealed that plasmin and cathepsin contributed to the protein hydrolysis during age gelation, with a significant increase in their activity observed. 23 characteristic peptides were ultimately selected as potential indicators for age gelation. These findings provide new insights into the age gelation of UHT milk.

Lipid matrix-specific pretreatment method for enhancing the extractability and allergenicity maintenance of bovine milk allergens in ELISA detection.

The presence of various components in the food matrix makes allergen detection difficult and inaccurate, and pretreatment is an innovative breakthrough point. Food matrices were categorised based on their composition. Subsequently, a pretreatment method was established using a combination of ultrasound-assisted n-hexane degreasing and weakly alkaline extraction systems to enhance the detection accuracy of bovine milk allergens. Results showed that more allergens were obtained with less structural destruction, as demonstrated using immunological quantification and spectral analysis. Concurrently, allergenicity preservation was confirmed through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, a KU812 cell degranulation model, and western blotting. The method exhibited good accuracy (bias, 8.47%), repeatability (RSDr, 1.52%), and stability (RSDR, 5.65%). In foods with high lipid content, such as chocolate, the allergen content was 2.29-fold higher than that of commercial kits. Laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM) analyses revealed a significant decrease in fat content after post-pretreatment using our method. In addition, colloidal stability surpassed that achieved using commercial kits, as indicated through the PSA and zeta potential results. The results demonstrated the superiority of the extractability and allergenicity maintenance of lipid matrix-specific pretreatment methods for improving the accuracy of ELISA based allergen detection in real food.

A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

Revealing the diversity of endogenous peptides and parent proteins in human colostrum and mature milk through peptidomics analysis.

Endogenous peptides and their parent proteins are important nutritional components with diverse biological functions. The objective of this study was to analyze and compare endogenous peptides and parent proteins found in human colostrum (HC) and human mature milk (HM) using a 4D label-free technique. In total, 5162 and 940 endogenous peptides derived from 258 parent proteins were identified in human milk by database (DB) search and de novo, respectively. Among these peptides, 2446 differentially expressed endogenous peptides with various bioactivities were identified. The Gene Ontology analysis unveiled the cellular components, biological processes, and molecular functions associated with these parent proteins. Metabolic pathway analysis suggested that neutrophil extracellular trap formation had the greatest significance with 24 parent proteins. These findings will offer a fresh perspective on the development of infant formula powder, highlighting the potential for incorporating these changes to enhance its nutritional composition and benefits.

Milk Antiviral Proteins and Derived Peptides against Zoonoses.

Milk is renowned for its nutritional richness but also serves as a remarkable reservoir of bioactive compounds, particularly milk proteins and their derived peptides. Recent studies have showcased several robust antiviral activities of these proteins, evidencing promising potential within zoonotic viral diseases. While several publications focus on milk's bioactivities, antiviral peptides remain largely neglected in reviews. This knowledge is critical for identifying novel research directions and analyzing potential nutraceuticals within the One Health context. Our review aims to gather the existing scientific information on milk-derived antiviral proteins and peptides against several zoonotic viral diseases, and their possible mechanisms. Overall, in-depth research has increasingly revealed them as a promising and novel strategy against viruses, principally for those constituting a plausible pandemic threat. The underlying mechanisms of the bioactivity of milk's proteins include inhibiting viral entry and attachment to the host cells, blocking replication, or even viral inactivation via peptide-membrane interactions. Their marked versatility and effectiveness stand out compared to other antiviral peptides and can support future research and development in the post-COVID-19 era. Overall, our review helps to emphasize the importance of potentially effective milk-derived peptides, and their significance for veterinary and human medicines, along with the pharmaceutical, nutraceutical, and dairy industry.

Whey proteins as multifunctional food materials: Recent advancements in hydrolysis, separation, and peptidomimetic approaches.

Whey protein derived bioactives, including α-lactalbumin, ß-lactoglobulin, bovine serum albumin, lactoferrin, transferrin, and proteose-peptones, have exhibited wide ranges of functional, biological and therapeutic properties varying from anticancer, antihypertensive, and antimicrobial effects. In addition, their functional properties involve gelling, emulsifying, and foaming abilities. For these reasons, this review article is framed to understand the relationship existed in between those compound levels and structures with their main functional, biological, and therapeutic properties exhibited either in vitro or in vivo. The impacts of hydrolysis mechanism and separation techniques in enhancing those properties are likewise discussed. Furthermore, special emphasize is given to multifunctional effects of whey derived bioactives and their future trends in ameliorating further food, pharmaceutical, and nutraceutical products. The underlying mechanism effects of those properties are still remained unclear in terms of activity levels, efficacy, and targeted effectiveness. For these reasons, some important models linking to functional properties, thermal properties and cell circumstances are established. Moreover, the coexistence of radical trapping groups, chelating groups, sulfhydryl groups, inhibitory groups, and peptide bonds seemed to be the key elements in triggering those functions and properties. Practical Application: Whey proteins are the byproducts of cheese processing and usually the exploitation of these food waste products has increasingly getting acceptance in many countries, especially European countries. Whey proteins share comparable nutritive values to milk products, particularly on their richness on important proteins that can serve immune protection, structural, and energetic roles. The nutritive profile of whey proteins shows diverse type of bioactive molecules like α-lactalbumin, ß-lactoglobulin, lactoferrin, transferrin, immunoglobulin, and proteose peptones with wide biological importance to the living system, such as in maintaining immunological, neuronal, and signaling roles. The diversification of proteins of whey products prompted scientists to exploit the real mechanisms behind of their biological and therapeutic effects, especially in declining the risk of cancer, tumor, and further complications like diabetes type 2 and hypertension risk effects. For these reasons, profiling these types of proteins using different proteomic and peptidomic approaches helps in determining their biological and therapeutic targets along with their release into gastrointestinal tract conditions and their bioavailabilities into portal circulation, tissue, and organs. The wide applicability of those protein fractions and their derivative bioactive products showed significant impacts in the field of emulsion and double emulsion stabilization by playing roles as emulsifying, surfactant, stabilizing, and foaming agents. Their amphoteric properties helped them to act as excellent encapsulating agents, particularly as vehicle for delivering important vitamins and bioactive compounds. The presence of ferric elements increased their transportation to several metal-ions in the same time increased their scavenging effects to metal-transition and peroxidation of lipids. Their richness with almost essential and nonessential amino acids makes them as selective microbial starters, in addition their richness in sulfhydryl amino acids allowed them to act a cross-linker in conjugating further biomolecules. For instance, conjugating gold-nanoparticles and fluorescent materials in targeting diseases like cancer and tumors in vivo is considered the cutting-edges strategies for these versatile molecules due to their active diffusion across-cell membrane and the presence of specific transporters to these therapeutic molecules.

Bioactive peptides in hydrolysates of bovine and camel milk proteins: A review of studies on peptides that reduce blood pressure, improve glucose homeostasis, and inhibit pathogen adhesion.

The prevalence of diet-related chronic conditions including hypertension and cardiovascular disease, and diabetes mellitus has increased worldwide. Research regarding the use of food-derived bioactive peptides as an alternative strategy to mitigate chronic diseases is on the rise. Milk is recognized as one of the main dietary protein sources for health beneficial bioactive compounds. Hundreds of in vitro studies have suggested that milk-derived bioactive peptides offer multiple biological and physiological benefits, and some but not all were confirmed in vivo with animal models for hypertension, hyperglycemia, and pathogen adhesion. However, only a limited number of health benefits have been confirmed by randomized clinical trials. This review provides an overview of the current clinical studies that target hypertension, postprandial hyperglycemic, and adhesion of enteric pathogen with bioactive peptides derived from bovine and camel milk, with a focus on the factors affecting the efficacy of orally ingested products.

The role of milk fat globule size in modulating the composition of postbiotics produced by Bacillus subtilis and their effect on mammary epithelial cells.

Milk lipids are secreted into the milk collecting ducts as milk fat globule (MFG) where they are exposed to microflora of the udder. We hypothesized that MFG size modulates the metabolic fingerprint of B. subtilis. Accordingly, small and large (2.3 and 7.0 µm, respectively) MFG were isolated from cow milk and used as a substrate for B. subtilis. Small MFG enhanced growth, whereas large MFG enhanced biofilm formation. Bacteria incubated with small MFG had increased concentration of metabolites related to energy production whereas metabolome of the bacteria incubated with large MFG had reduced concentrations of metabolites important for biofilm formation. Postbiotics from bacteria grown on large MFG exacerbated the proinflammatory response of MEC to LPS, and changed the expression of key enzymes involved in lipid and protein synthesis. Our results suggest that MFG size modulate growth trajectories and metabolome of B. subtilis, and consequently the stress response of host cells.

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples.

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S-S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S-S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S-S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity.

Differential effects of milk proteins on amino acid digestibility, post-prandial nitrogen utilization and intestinal peptide profiles in rats.

OBJECTIVE: The aim of this study was to analyze the protein digestibility and postprandial metabolism in rats of milk protein matrices obtained by different industrial processes. MATERIAL AND METHODS: The study was conducted on Wistar rats that consumed a meal containing different 15N-labeled milk proteins. Four milk matrices were tested: native micellar caseins (C1), caseins low in calcium (C2 low Ca2+), a matrix containing a ratio 63:37 of caseins and whey proteins (CW2) and whey proteins alone (W). Blood and urine were collected during the postprandial period and rats were euthanized 6 h after meal intake to collect digestive contents and organs. RESULTS: Orocaecal digestibility values of amino acids ranged between 96.0 ± 0.2% and 96.6 ± 0.4% for C1-, C2 low Ca2+- and W-matrices, while this value was significantly lower for CW2 matrix (92.4 ± 0.5%). More dietary nitrogen was sequestered in the splanchnic area (intestinal mucosa and liver) as well as in plasma proteins after ingestion of W matrix, especially compared to the C1- and C2 low Ca2+-matrices. Peptidomic analysis showed that more milk protein-derived peptides were identified in the caecum of rats after the ingestion of the matrices containing caseins compared to W matrix. CONCLUSION: We found that demineralization of micellar caseins did not modify its digestibility and postprandial metabolism. The low digestibility of the modified casein-to-whey ratio matrix may be ascribed to a lower accessibility of the protein to digestive enzymes due to changes in the protein structure, while the higher nitrogen splanchnic retention after ingestion of whey was probably due to the fast assimilation of its protein content. Finally, our results showed that industrial processes that modify the structure and/or composition of milk proteins influence protein digestion and utilization.

Structural, Binding and Functional Properties of Milk Protein-Polyphenol Systems: A Review.

Polyphenols (PP) are linked to health benefits (e.g., prevention of cancer, cardiovascular disease and obesity), which are mainly attributed to their antioxidant activity. During digestion, PP are oxidised to a significant degree reducing their bio-functionality. In recent years, the potential of various milk protein systems, including ß-casein micelles, ß-lactoglobulin aggregates, blood serum albumin aggregates, native casein micelles and re-assembled casein micelles, to bind and protect PP have been investigated. These studies have yet to be systematically reviewed. The functional properties of the milk protein-PP systems depend on the type and concentration of both PP and protein, as well as the structure of the resultant complexes, with environmental and processing factors also having an influence. Milk protein systems protect PP from degradation during digestion, resulting in a higher bioaccessibility and bioavailability, which improve the functional properties of PP upon consumption. This review compares different milk protein systems in terms of physicochemical properties, PP binding performance and ability to enhance the bio-functional properties of PP. The goal is to provide a comprehensive overview on the structural, binding, and functional properties of milk protein-polyphenol systems. It is concluded that milk protein complexes function effectively as delivery systems for PP, protecting PP from oxidation during digestion.

In silico identification of novel ACE and DPP-IV inhibitory peptides derived from buffalo milk proteins and evaluation of their inhibitory mechanisms.

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP-UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver-Burk plots showed that KPW (IC50 = 136.28 ± 10.77 µM) and RGP (104.72 ± 8.37 µM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC50 = 873.92 ± 32.89 µM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52 ± 10.37 and 126.57 ± 8.45 µM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.

Comparative assessment of antioxidant and immunomodulatory properties of skimmed milk protein hydrolysates and their incorporation in beverage mix.

BACKGROUND: Milk-derived protein hydrolysates have generated a great deal of interest recently due to their numerous beneficial health effects. However, there are few comparative studies on protein hydrolysates from different dairy species, their production, characterization, and bioactivity. In the present study, skimmed milk from both major and minor dairy species was hydrolyzed with alcalase, and its protein profiles were studied using tricine polyacrylamide gel electrophoresis and reverse phase-high protein liquid chromatography. The antioxidant and in vitro immunostimulatory properties were determined. RESULTS: Iron chelation activity was highest in hydrolysates of whey (25.00 ± 0.32 mmol L-1 ), casein (25.14 ± 0.34 mmol L-1 ), colostrum (24.52 ± 0.28 mmol L-1 ), and skimmed cattle milk (24.21 ± 0.26 mmol L-1 ). α,α-Diphenyl-ß-picrylhydrazyl scavenging and 2,2'-azobis(2-amidino-propane) dihydrochloride activity was lowest in skimmed donkey milk protein hydrolysates (MPHs) (IC50 : 5.37 ± 0.05 mg mL-1 and 151.59 ± 2.1 mg mL-1 ). Production of nitric oxide and phagocytosis activity in RAW 264.7 (murine macrophage cell line) was significantly higher among whey and buffalo skimmed milk protein hydrolysate-treated groups as compared with the untreated group. The incorporation of whey protein hydrolysate and skimmed buffalo milk protein hydrolysate were sensorially acceptable at 10% level in beverage mix. CONCLUSION: This study comparatively evaluates the antioxidative and immunomodulatory properties of different skimmed MPHs and their potential applications as ingredients in pediatric, geriatric, and other health-promoting foods. © 2022 Society of Chemical Industry.

Lactation Activity and Mechanism of Milk-Protein Synthesis by Peptides from Oyster Hydrolysates.

Oyster meat has a tender texture and delicate flavor, and the oyster is an aquatic shellfish with high nutritional and economic values. As they are rich in protein, oysters serve as a good source for the preparation of bioactive peptides. However, research on the lactation effect and mechanism of the synthesis of polypeptides from oyster hydrolysates is yet to be observed. This study aimed to analyze the lactation activity of the fraction UEC4-1 and explore its mechanism. The results show that, in an in vivo experiment, UEC4-1 could significantly increase the concentration of PRL in the serum and mammary tissue and the concentration of PRLR in the mammary tissue in rats with postpartum hypogalactia. UEC4-1 promoted the development of mammary tissue structure, resulting in active lactation. UEC4-1 promoted the proliferation of MCF-10A in a dose-dependent manner and could significantly upregulate the gene expression levels of PRL, PRLR, CSN1S1, CSN2, CSN3 and CCND1. UEC4-1 could also significantly increase the expression of mTOR, AKT1, RPS6KB1 and STAT5A in MCF-10A and improve its phosphorylation level. These results show that UEC4-1 had the ability to upregulate the proliferation and PRL synthesis of MCF-10A and promote lactation. The ability of UEC4-1 to regulate the milk-protein synthesis signaling pathway is the mechanism behind this. Oysters had a remarkable effect on lactating mothers' sweating irritability after childbirth and may serve as an everyday diet to promote lactation. Postpartum dysgalactia is a common problem for lactating women. The study of the oyster's lactation-active peptide can provide dietary nutrition guidance for postpartum lactating mothers, and it has the potential to be used for the development of drugs for the treatment of postpartum hypogalactia or oligogalactia.

Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC-MS/MS-Based Proteomic Approach.

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

In vivo absorptomics: Identification of bovine milk-derived peptides in human plasma after milk intake.

A dedicated two-step purification procedure prior to nanoliquid chromatography-electrospray-tandem mass spectrometry analysis enabled the identification of bovine milk-derived peptides absorbed and circulating in the plasma of three healthy volunteers who received 250 mL of pasteurized milk after a 10-days washout. The appearance and clearance of milk peptides in plasma were monitored at various time points. Overall, 758, 273 and 212 unique peptides derived from 15, 15 and 18 bovine milk proteins, respectively, were identified in the plasma of these volunteers, evidencing a substantial inter-individual variability. Peptides encrypting possible bioactive and/or immunogenic molecules originating from caseins, ß-lactoglobulin and minor milk proteins were detected. Peptide representation data revealed the combined action of endoproteases involved in primary hydrolysis during gastroduodenal digestion and exopeptidases that hydrolyse peptides in the small intestine. It remains to be established whether the half-life and concentration ranges of circulating milk-derived peptides may have any impacts on human health.

Application of preliminary high-pressure processing for improving bioactive characteristics and reducing antigenicity of whey protein hydrolysates.

This study investigated the use of Novo Pro-D® (NPD) and Ficin (FC) as alternative proteases for the production of bioactive peptides with reduced allergenicity from whey protein concentrate (WPC). In addition, the use of high hydrostatic pressure processing as pre-treatment of WPC and its impact on the final characteristics of hydrolysates were also evaluated. NPD treatments generated hydrolysates with a 98% reduction of soluble proteins, greater in vitro antioxidant capacity, and less immunoreactivity when compared to FC ones. However, pre-treatment was an essential tool to improve WPC hydrolysis when FC was used, resulting in hydrolysates with less soluble proteins, enhanced antioxidant capacity, and less allergenicity compared with conventional hydrolysis. As for NPD, the pre-treatment of WPC improved the in vitro antioxidant capacity and resulted in a 100% reduction in immunoreactivity to ß-lactoglobulin in a shorter processing time. Importantly, bioactive peptides generated by FC displayed an improved ability to induce in vitro arterial relaxation, compared with those obtained from NPD process. Therefore, this study provides innovative evidence regarding how the proteases used for production of whey hydrolysates can improve its biological effects, and discloses the use of high hydrostatic pressure combined with enzymatic hydrolysis as a promising alternative to produce hydrolysates with improved properties.