Milk: A Natural Guardian for the Gut Barrier.

The gut barrier plays an important role in health maintenance by preventing the invasion of dietary pathogens and toxins. Disruption of the gut barrier can cause severe intestinal inflammation. As a natural source, milk is enriched with many active constituents that contribute to numerous beneficial functions, including immune regulation. These components collectively serve as a shield for the gut barrier, protecting against various threats such as biological, chemical, mechanical, and immunological threats. This comprehensive review delves into the active ingredients in milk, encompassing casein, α-lactalbumin, ß-lactoglobulin, lactoferrin, the milk fat globular membrane, lactose, transforming growth factor, and glycopeptides. The primary focus is to elucidate their impact on the integrity and function of the gut barrier. Furthermore, the implications of different processing methods of dairy products on the gut barrier protection are discussed. In conclusion, this study aimed to underscore the vital role of milk and dairy products in sustaining gut barrier health, potentially contributing to broader perspectives in nutritional sciences and public health.

Effects of protein genetic variants on their phosphorylation levels, milk composition, milk proteome, and milk coagulation ability in Chinese Holstein bovine milk.

Milk samples were collected from 3625 Chinese Holstein cows to assess the effects of κ-casein (κ-CN) and ß-lactoglobulin (ß-LG) genetic variants on its milk coagulation properties. The results show that Chinese Holstein cows have a higher frequency of the κ-CN AA and AB variants, and ß-LG of the AB and AA variants. Of these, κ-CN B variants, the ß-LG AA and BB variants were more frequent in milk showing good coagulation. The effects of the genetic variants on milk composition, milk proteome, and protein phosphorylation sites were studied. The results showed that higher concentrations of protein and dry matter were found in κ-CN BE variant. Moreover, large variations in milk proteome among different κ-CN and ß-LG variants were observed. Highly phosphorylated for κ-CN, especially Ser97, was observed in cows with the κ-CN BE variant, but no effect of ß-LG variants on phosphorylation site was found. Of the various factors examined, variation of κ-CN phosphorylation sites Ser97 may be the most important in affecting casein structure and milk coagulation ability. Some milk protein contents were found to be negative factors for milk coagulation. In summary, this study showed that κ-CN genetic variants contained different milk compositions and phosphorylation site Ser97 influenced milk coagulation.

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.

New Perspective on the Interaction Behavior Between Riboflavin and ß Lactoglobulin-ß Casein Complex by Biophysical Techniques.

Riboflavin (RF) is a vitamin that only exists in plants and microorganisms and must be procured externally by humans. On the other hand, there are two major allergic factors in cow's milk, including ß-lactoglobulin (ßLG) and ß-casein (ßCN), while their allergic properties can be eliminated by binding to micronutrients. In this regard, we examined the binding process of RF to ßLG and ßCN in the binary and ternary systems by different spectroscopies such as zeta potential, electric conductivity, and molecular modeling. According to the result of the fluorescence spectrum regarding the interaction of RF with ßLG and ßCN in binary and ternary systems, an increase in RF concentration declined the fluorescence intensity of three systems and also caused the quenching of proteins. Static quenching plays a pivotal role in the formation of stable interactions. The obtained thermodynamic parameters by Van't Hoff equation ascertained the predominance of hydrogen bonds and van der Waals interaction in all the systems. Considering how the negative value of ΔH0 resulted in the negative value of ΔG0, the systems were assumed to be enthalpy driven. The outcomes of circular dichroism (CD) disclosed that the attachment of RF to the targets of systems increased their a-helix content, which particularly included the binding of RF to ßLG that led to the conversion of ß-sheet to α-helix content. As indicated by the results of zeta potential, the low concentration of RF contained the dominance of hydrophobic forces in the interactions, whereas the enlargement of this concentration prevailed electrostatic forces. Moreover, conductometry measurements showed an extension in the rate of ionizable groups due to the addition of RF to the systems, which may increase the probability of an interaction between RF, ßCN, and ßLG in binary and ternary systems. In consistency with the outcomes of molecular dynamics simulation, the data of molecular docking approved the capability of RF in forming strong and stable interactions with ßCN and ßLG.

Glycosylation-modified antigens as a tolerance-inducing vaccine platform prevent anaphylaxis in a pre-clinical model of food allergy.

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We develop a tolerance-inducing vaccine technology that utilizes glycosylation-modified antigens to induce antigen-specific non-responsiveness. The glycosylation-modified antigens are administered intravenously (i.v.) or subcutaneously (s.c.) and traffic to the liver or lymph nodes, respectively, leading to preferential internalization by antigen-presenting cells, educating the immune system to respond in an innocuous way. In a mouse model of cow's milk allergy, treatment with glycosylation-modified ß-lactoglobulin (BLG) is effective in preventing the onset of allergy. In addition, s.c. administration of glycosylation-modified BLG shows superior safety and potential in treating existing allergies in combination with anti-CD20 co-therapy. This platform provides an antigen-specific immunomodulatory strategy to prevent and treat food allergies.

Modeling of Proteolysis of ß-Lactoglobulin and ß-Casein by Trypsin with Consideration of Secondary Masking of Intermediate Polypeptides.

The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for ß-lactoglobulin (ß-LG) and ß-casein (ß-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening. This led to the limitations in the red shift of fluorescence and the degree of hydrolysis with a long time of hydrolysis of ß-LG and ß-CN at a constant substrate concentration and reduced trypsin concentrations. The proposed proteolysis model included demasking of initially masked bonds in the protein globule or micelle, secondary masking of intermediate polypeptides, and their subsequent slow demasking. The hydrolysis of peptide bonds was modeled taking into account different hydrolysis rate constants for different peptide bonds. It was demonstrated that demasking competes with secondary masking, which is less noticeable at high trypsin concentrations. Modeling of proteolysis taking into account two demasking processes and secondary masking made it possible to simulate kinetic curves consistent with the experimental data.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Genetic variants associated with two major bovine milk fatty acids offer opportunities to breed for altered milk fat composition.

BACKGROUND: Although bovine milk is regarded as healthy and nutritious, its high content of saturated fatty acids (FA) may be harmful to cardiovascular health. Palmitic acid (C16:0) is the predominant saturated FA in milk with adverse health effects that could be countered by substituting it with higher levels of unsaturated FA, such as oleic acid (C18:1cis-9). In this work, we performed genome-wide association analyses for milk fatty acids predicted from FTIR spectroscopy data using 1811 Norwegian Red cattle genotyped and imputed to a high-density 777k single nucleotide polymorphism (SNP)-array. In a follow-up analysis, we used imputed whole-genome sequence data to detect genetic variants that are involved in FTIR-predicted levels of C16:0 and C18:1cis-9 and explore the transcript profile and protein level of candidate genes. RESULTS: Genome-wise significant associations were detected for C16:0 on Bos taurus (BTA) autosomes 11, 16 and 27, and for C18:1cis-9 on BTA5, 13 and 19. Closer examination of a significant locus on BTA11 identified the PAEP gene, which encodes the milk protein ß-lactoglobulin, as a particularly attractive positional candidate gene. At this locus, we discovered a tightly linked cluster of genetic variants in coding and regulatory sequences that have opposing effects on the levels of C16:0 and C18:1cis-9. The favourable haplotype, linked to reduced levels of C16:0 and increased levels of C18:1cis-9 was also associated with a marked reduction in PAEP expression and ß-lactoglobulin protein levels. ß-lactoglobulin is the most abundant whey protein in milk and lower levels are associated with important dairy production parameters such as improved cheese yield. CONCLUSIONS: The genetic variants detected in this study may be used in breeding to produce milk with an improved FA health-profile and enhanced cheese-making properties.

Alleviating Aspirin-Induced Gastric Injury by Binding Aspirin to ß-Lactoglobulin.

Purpose: Gastric injury is a major issue for long-term administration of aspirin. In this work, we tried to explore the possibility of using BLG to alleviate aspirin-induced gastric injury, because of excellent abilities of BLG in loading drug molecules. Methods: Various spectroscopic techniques and molecular docking methods were applied to investigate the interaction mechanism between BLG and aspirin. Animal experiments were performed to figure out the effects of taking aspirin-BLG on the stomach. Results: Our results demonstrate that aspirin could bind with BLG to form stable aspirin-BLG complex (the binding constant Kb = 2.051 × 103 M-1). The formation process is endothermic (∆H>0) and the main acting force is hydrophobic force. Our data also show that the aspirin-BLG complex is formed with a higher affinity in simulated gastric fluid and could remain stable for several hours, which might arise from its special binding mode under acidic condition and the resistance of BLG to gastric digestion. Furthermore, animal models (rats with aspirin-induced gastric damage) were built. The results of animal experiments reveal that the oral administration of aspirin-BLG could cause less damage to gastric tissue, and it also hardly triggers obvious inflammatory responses. Conclusion: This study would contribute to an in-depth understanding of the interaction mechanism between BLG and aspirin. It is reasonable to believe that using BLG to bind with aspirin would be a potential way to alleviate the aspirin-induced gastric injury.

The ß-casein (CSN2) A2 allelic variant alters milk protein profile and slightly worsens coagulation properties in Holstein cows.

Milk proteins genetic variants have long attracted interest as they are associated with important issues relating to milk composition and technological properties. An important debate has recently opened at an international level on the role of ß-casein (ß-CN) A1 and A2 polymorphisms, toward human health. For this reason, a lot of efforts has been put into the promotion of A2 milk by companies producing and selling A1-free milk, leading the farmers and breeders to switch toward A2 milk production without paying attention on the potential effect of the processability of milk into cheese. The aim of the present work was to evaluate the effects of ß-CN, specifically the A1 and A2 allelic variants, on the detailed milk protein profile and cheese-making traits in individual milk samples of 1,133 Holstein Friesian cows. The protein fractions were measured with reversed-phase (RP)-HPLC (expressed in g/L and % N), and the cheese-making traits, namely milk coagulation properties, cheese yield, and curd nutrient recoveries assessed at the individual level, with a nano-scale cheese-making procedure. The ß-CN (CSN2), κ-CN (CSN3), and ß-lactoglobulin (LGB) genetic variants were first identified through RP-HPLC and then confirmed through genotyping. Estimates of the effects of protein genotypes were obtained using a mixed inheritance model that considered, besides the standard nuisance variables (i.e., days in milk, parity, and herd-date), the milk protein genes located on chromosome 6 (CSN2, CSN3) and on chromosome 11 (LGB), and the polygenic background of the animals. Milk protein genes (CSN2, CSN3, and LGB) explained an important part of the additive genetic variance in the traits evaluated. The ß-CN A1A1 was associated with a significantly lower production of whey proteins, particularly of ß-lactoglobulin (-8.2 and -6.8% for g/L and % N, respectively) and α-lactalbumin (-4.7 and -4.4% for g/L and % N, respectively), and a higher production of ß-CN (6.8 and 6.1% for g/L and % N, respectively) with respect to the A2A2 genotype. Regarding milk cheese-making ability, the A2A2 genotype showed the worst performance compared with the other genotypes, particularly with respect to the BA1, with a higher rennet coagulation time (7.1 and 28.6% compared with A1A1 and BA1, respectively) and a lower curd firmness at 30 min. Changes in milk protein composition through an increase in the frequency of the A2 allele in the production process could lead to a worsening of the coagulation and curd firming traits.

Approach to Study pH-Dependent Protein Association Using Constant-pH Molecular Dynamics: Application to the Dimerization of ß-Lactoglobulin.

Protein-protein association is often mediated by electrostatic interactions and modulated by pH. However, experimental and computational studies have often overlooked the effect of association on the protonation state of the protein. In this work, we present a methodological approach based on constant-pH molecular dynamics (MD), which aims to provide a detailed description of a pH-dependent protein-protein association, and apply it to the dimerization of ß-lactoglobulin (BLG). A selection of analyses is performed using the data generated by constant-pH MD simulations of monomeric and dimeric forms of bovine BLG, in the pH range 3-8. First, we estimate free energies of dimerization using a computationally inexpensive approach based on the Wyman-Tanford linkage theory, calculated in a new way through the use of thermodynamically based splines. The individual free energy contribution of each titratable site is also calculated, allowing for identification of relevant residues. Second, the correlations between the proton occupancies of pairs of sites are calculated (using the Pearson coefficient), and extensive networks of correlated sites are observed at acidic pH values, sometimes involving distant pairs. In general, strongly correlated sites are also slow proton exchangers and contribute significantly to the pH-dependency of the dimerization free energy. Third, we use ionic density as a fingerprint of protein charge distribution and observe electrostatic complementarity between the monomer faces that form the dimer interface, more markedly at the isoionic point (where maximum dimerization occurs) than at other pH values, which might contribute to guide the association. Finally, the pH-dependent dimerization modes are inspected using PCA, among other analyses, and two states are identified: a relaxed state at pH 4-8 (with the typical alignment of the crystallographic structure) and a compact state at pH 3-4 (with a tighter association and rotated alignment). This work shows that an approach based on constant-pH MD simulations can produce rich detailed pictures of pH-dependent protein associations, as illustrated for BLG dimerization.

UHT treatment and storage of liquid infant formula affects protein digestion and release of bioactive peptides.

Ready-to-feed liquid infant formulas (IF) were subjected to direct (D) or indirect (ID) ultra-high-temperature (UHT) treatment and then stored at 40 °C under aseptic conditions for 60-120 days simulating global transportation which accelerates the Maillard reaction. Low pasteurized and unstored IF (LP) was included as a control for the UHT treatments. Simulated infant in vitro digestion was conducted. SDS-PAGE indicated that protein aggregate formation correlated with thermal treatment, being greatest after 60 days of storage. Limited protein digestion was observed after pepsin treatment for 2 h. Beta-lactoglobulin (ß-Lg), alpha-lactalbumin (α-La) and protein aggregates remained undigested after 2 h of pepsin digestion in LP and D, but less ß-Lg and α-La remained in ID. The digestion of ß-Lg and α-La was enhanced in D and ID stored for 60 days, but aggregates remained undigested. After pepsin and pancreatin digestion, large amounts of ß-Lg remained undigested in the LP, but digestion increased after UHT treatment (ID > D) and increased further after storage for 60 and 120 days, indicating that heat treatment and storage facilitate the digestion of unaggregated proteins. No aggregates remained after pancreatin digestion of LP, D, ID and D stored for 60 days, but were present in ID stored for 60 days. Aggregates were mainly disulphide-linked, but dityrosine linkages were detected in D and ID stored for 120 days. LC-MS/MS indicated limited proteolysis arising from endogenous milk proteases prior to in vitro digestion, being highest in D. Peptide numbers increased following pepsin and further during pancreatin digestion (ß-casein > ß-Lg > ß-La), and released ß-Lg peptides, typically 5-8 amino acids in length, contained several bioactivities, e.g., dipeptidyl-peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibition.

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.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - part B: Neutral pH.

Interactions between the dimeric form of ß-lactoglobulin and vanillic acid were investigated at pH 7.2, using a variety of spectroscopic techniques and molecular dynamics (MD) simulations. FTIR and CD studies showed alterations in the secondary structure of the protein upon its interaction with the ligand. Fluorescence measurements indicated that the dimeric complex with the phenolic acid produced a large dissociation constant (KD) compared to the monomeric counterpart at acidic pH (part A of this series). Stoichiometry of 1:1 was identified for the ß-lactoglobulin-vanillic acid complex by Job plot analysis at neutral pH suggesting two ligand molecules can participate in binding with the dimer. Molecular docking and MD simulations suggested that the top-ranked binding sites of the ligand were located at the entrance of each ß-barrel structure of the dimer. These simulations also allowed identification of the contribution of water molecules, in the form of protein-water-ligand bridging interactions, to the complexes.

Interactions of ß-Lactoglobulin with Bovine Submaxillary Mucin vs. Porcine Gastric Mucin: The Role of Hydrophobic and Hydrophilic Residues as Studied by Fluorescence Spectroscopy.

The aim of this study was to investigate binding interactions between ß-lactoglobulin (BLG) and two different mucins, bovine submaxillary mucins (BSM) and porcine gastric mucin (PGM), using intrinsic and extrinsic fluorescence spectroscopies. Intrinsic fluorescence spectra showed an enhanced decrease of fluorescence intensity of BLG at all pH conditions when BLG was mixed with PGM rather than with BSM. We propose that, unlike BSM, the tertiary structure of PGM changes and the hydrophobic regions are exposed at pH 3 due to protonation of negatively charged residues. Results suggest that PGM also facilitated the structural unfolding of BLG and its binding with PGM by a hydrophobic interaction, especially at acidic pH, which was further supported by extrinsic fluorescence spectroscopy. Hydrophobic interaction is suggested as the dominant interaction mechanism between BLG and PGM at pH 3, whereas electrostatic interaction is the dominant one between BLG and BSM.

Covalent and non-covalent interactions of cyanidin-3-O-glucoside with milk proteins revealed modifications in protein conformational structures, digestibility, and allergenic characteristics.

Currently, there is a need to explore the effects of different types of protein-anthocyanin complexations, as well as the possible changes in the nutrition and allergenicity of the formed complexes. Here, we systematically investigated the covalent and non-covalent interactions between cyanidin-3-O-glucoside (C3G) and two major milk proteins, α-casein (α-CN) and ß-lactoglobulin (ß-LG). Fluorescence quenching data showed that, under non-covalent conditions, C3G quenched the fluorescence of the two proteins via a static process, with the interaction forces being revealed; for covalent products, decreased fluorescence intensities were observed with red shifts in the λmax. Multiple spectroscopic analyses implied that C3G-addition induced protein structural unfolding through transitions between the random coil and ordered secondary components. With a two-stage simulated gastrointestinal (GI) digestion model, it was seen that covalent complexes, not their non-covalent counterparts, showed reduced protein digestibility, ascribed to structural changes resulting in the unavailability of enzyme cleaving sites. The GI digests displayed prominent 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging abilities (3.8-11.1 mM Trolox equivalents per mL digest), in contrast to the markedly reduced 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacities. Additionally, covalent protein-C3G complexes, but not their non-covalent counterparts, showed lower IgE-binding levels in comparison to the native control. This study provides new understanding for the development of anthocyanin-milk protein systems as functional ingredients with health-beneficial properties.

Effects of anthocyanins on ß-lactoglobulin glycoxidation: a study of mechanisms and structure-activity relationship.

We elucidated the underlying mechanisms of the anti-glycoxidation effects of five structurally different anthocyanins on glycated-ß-lactoglobulin (ß-Lg). The results indicated that anthocyanins structurally inhibited the formation of advanced glycation end-products, where petunidin-3-rutinoside-(p-coumaryl)-5-glucoside (Pt-Gl) exerted higher effects than those of others (p < 0.05). Through the three main steps of glycoxidation, anthocyanins trapped intermediate dicarbonyls and blocked some of the glycation sites of ß-Lg. UPLC-ESI-Q-TOF-MS characterized that these anthocyanins structurally formed mono- and di-GO/MGO adducts, and Pt-Gl formed adducts with both dicarbonyls. More importantly, Pt-Gl interacted with some of the glycation sites of ß-Lg such as Lys100, Lys101, and Arg124. Structurally, it was found that high-molecular weight anthocyanins with coumaric acid acylation seem to be better than others, which was followed by di- and mono-glycoside anthocyanins. Overall, GO/MGO-trapping and ß-Lg-anthocyanin binding are revealed as the key mechanisms of the anti-glycoxidation effects of anthocyanins on ß-Lg, which could be used as effective glycation inhibitors in protein-rich matrices.

Formation of ß-Lactoglobulin Self-Assemblies via Liquid-Liquid Phase Separation for Applications beyond the Biological Functions.

Proteins are like miracle machines, playing important roles in living organisms. They perform vital biofunctions by further combining together and/or with other biomacromolecules to form assemblies or condensates such as membraneless organelles. Therefore, studying the self-assembly of biomacromolecules is of fundamental importance. In addition to their biological activities, protein assemblies also exhibit extra properties that enable them to achieve applications beyond their original functions. Herein, this study showed that in the presence of monosaccharides, ethylene glycols, and amino acids, ß-lactoglobulin (ß-LG) can form assemblies with specific structures, which were highly reproducible. The mechanism of the assembly process was studied through multi-scale observations and theoretical analysis, and it was found that the assembling all started from the formation of solute-rich liquid droplets via liquid-liquid phase separation (LLPS). These droplets then combined together to form condensates with elaborate structures, and the condensates finally evolved to form assemblies with various morphologies. Such a mechanism of the assembly is valuable for studying the assembly processes that frequently occur in living organisms. Detailed studies concerning the properties and applications of the obtained ß-LG assemblies showed that the assemblies exhibited significantly better performances than the protein itself in terms of autofluorescence, antioxidant activity, and metal ion absorption, which indicates broad applications of these assemblies in bioimaging, biodetection, biodiagnosis, health maintenance, and pollution treatment. This study revealed that biomacromolecules, especially proteins, can be assembled via LLPS, and some unexpected application potentials could be found beyond their original biological functions.

Co-encapsulation of (-)-epigallocatechin-3-gallate and piceatannol/oxyresveratrol in ß-lactoglobulin: effect of ligand-protein binding on the antioxidant activity, stability, solubility and cytotoxicity.

The co-encapsulation of multiple bioactive components in a carrier may produce synergistic effects and improve health benefits. In this study, the interactions of ß-lactoglobulin (ß-LG) with epigallocatechin-3-gallate (EGCG) and/or piceatannol (PIC)/oxyresveratrol (OXY) were investigated by multispectroscopic techniques, isothermal titration calorimetry, and molecular docking. The static quenching mechanism of ß-LG by EGCG, PIC and OXY was confirmed by fluorescence spectroscopy and UV-vis absorption difference spectroscopy. The binding sites of these three polyphenols in ß-LG were identified by site marking fluorescence experiments and molecular docking. The thermodynamic parameters of the ß-LG + EGCG/PIC/OXY binary complex and ß-LG + EGCG + PIC/OXY ternary complex were obtained from fluorescence data and used to analyze the main driving force for complex formation. The exothermic binding process was further confirmed by isothermal titration calorimetry. The α-helical content, particle size and morphology of free and ligand-bound ß-LG were determined by circular dichroism spectroscopy, dynamic light scattering and transmission electron microscopy, respectively. The effect of EGCG, PIC and OXY on the conformation of ß-LG was studied by Fourier transform infrared spectroscopy. In addition, the maximum synergistic antioxidant activity between EGCG and PIC/OXY was obtained by response surface analysis. The effects of ß-LG in the binary and ternary systems on the antioxidant activity, stability, solubility and cytotoxicity of the polyphenols were also studied. Finally, the different cytotoxicities of the complexes and nanoparticles of the binary and ternary systems were compared. The results of this study are expected to provide a theoretical basis for the development of ß-LG-based carriers co-encapsulating a variety of bioactive components.

The bile salt content of human bile impacts on simulated intestinal proteolysis of ß-lactoglobulin.

The gastrointestinal hydrolysis of food proteins has been portrayed in scientific literature to predominantly depend on the activity and specificity of proteolytic enzymes. Human bile has not been considered to facilitate proteolysis in the small intestine, but rather to assist in intestinal lipolysis. However, human bile can potentially influence proteins that are largely resistant to gastric digestion, and which are mainly hydrolysed after they have been transferred to the small intestine. We used purified and food-grade bovine milk ß-lactoglobulin (ßLg) to assess the impact of bile salts (BS) on the in vitro gastrointestinal digestion of this protein. Quantitative analysis showed that the proteolysis rate increased significantly with increasing BS concentration. The effect was consistent regardless of whether individual BS or real human bile samples, varying in BS concentrations, were used. The total BS content of bile was more important than its BS composition in facilitating the proteolysis of ßlg. We also show that the impact of human bile observed during the digestion of purified ßLg and ßLg-rich whey protein isolate can be closely replicated by the use of individual BS mixed with phosphatidylcholine. This could validate simple BS/phosphatidylcholine mixtures as human-relevant substitutes of difficult-to-obtain human bile for in vitro proteolysis studies.