Protein-Induced DNA Dumbbell Amplification (PINDA) and its applications to food hazards detection.

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of ß-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

Protonation State-Induced Unfolding of Protein Secondary Structure in the Gas Phase.

The combination of infrared spectroscopy (IR) and ion mobility mass spectrometry (IM-MS) has revealed that protein secondary structures are retained upon transformation from aqueous solution to the gas phase under gentle conditions. Yet the details about where and how these structural elements are embedded in the gas phase remain elusive. In this study, we employ long time scale molecular dynamics (MD) simulations to examine the extent to which proteins retain their solution structures and the impact of protonation state on the stability of secondary structures in the gas phase. Our investigation focuses on two well-studied proteins, myoglobin and ß-lactoglobulin, representing typical helical and ß-sheet proteins, respectively. Our simulations accurately reproduce the experimental collision cross section (CCS) data measured by IM-MS. Based on accurately reproducing previous experimental collision cross section data and dominant secondary structural species obtained from IM-MS and IR, we confirm that both proteins largely retain their native secondary structural components upon passing from aqueous solution to the gas phase. However, we observe significant reductions in secondary structure contents (19.2 ± 1.2% for myoglobin and 7.3 ± 0.6% for ß-lactoglobulin) in specific regions predominantly composed of ionizable residues. Further mechanistic analysis suggests that alterations in protonation states of these residues after phase transition induce changes in their local interaction networks and backbone dihedral angles, which potentially promote the unfolding of secondary structures in the gas phase. We anticipate that similar protonation state induced unfolding may be observed in other proteins possessing distinct secondary structures. Further studies on a broader array of proteins will be essential to refine our understanding of protein structural behavior during the transition to the gas phase.

Regioselective analysis of heat-induced conformational changes of ß-lactoglobulin by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics.

ß-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native ß-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25-75 °C) and complete transitions (80-90 °C). Regioselective conformational changes of BLG-A in the native state (25 °C), sequential (70 °C) and complete transition (90 °C) were determined by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 (both N-terminal chain), K60 (ß-sheet C), K75 (ß-sheet D), K77 (DE loop), K83 (ß-sheet E), K100 and K101 (FG loop). The residues K14 (ß-sheet A1), K47 (ß-sheet B), K69, K70 (both ß-sheet D), and K91 (ß-sheet F) were not involved in conformational changes.

Association of polymorphic variants of prolactin (PRL) and beta-lactoglobulin (BLG) genes with resistance/susceptibility to mastitis in holstein cows.

The work aims to analyze the associations of polymorphic variants of the PRL and BLG genes with resistance and susceptibility to mastitis in Holstein cows. The experimental study consisted of the selection of biomaterial samples from 250 heads of Holstein cows aged 3 years divided into two groups (healthy and with a confirmed diagnosis of mastitis). The determination of animal genotypes was carried out using polymerase chain reaction and restriction fragment length polymorphism. The study of the nature of the association of polymorphic variants of the PRL and BLG gene with resistance/increased risk of mastitis established a significant deviation from the theoretically expected distribution of bBLG-HaeIII genotypes in the group of animals suffering from mastitis (the value of χ2 was 0.24). The bBLG-HaeIIIBB genotype can act as a marker of an increased risk of developing mastitis in Holstein cows; its frequency in the group of sick animals exceeds the frequency in the control group by more than 2 times (44.0 compared to 17.0%, respectively). The bBLG-HaeIIIAB genotype is significantly associated with mastitis resistance in Holstein cows; its frequency is 2 times lower than in the control group (28.0 compared to 54.0%).

Exploration of dynamic interaction between ß-lactoglobulin and casein micelles during UHT milk process.

The protein aggregation induced by UHT treatment shortens the shelf life of UHT milk. However, the mechanism of ß-Lg induced casein micelle aggregation remains unclear. Herein, the dynamic interaction between ß-Lg and casein micelles during UHT processing was investigated by experimental techniques and molecular dynamics simulations. Results showed that ß-Lg decreased the stability of casein micelles, increased their size and zeta potential. Raman and FTIR spectra analysis suggested that hydrogen and disulfide bonds facilitated their interaction. Cryo-TEM showed that the formation of the casein micelle/ß-Lg complex involved rigid binding, flexible linking, and severe cross-linking aggregation during UHT processing. SAXS and MST demonstrated ß-Lg bound to κ-casein on micelle surfaces with a dissociation constant (Kd) of 3.84 ± 1.14 µm. Molecular docking and dynamic simulations identified the interacting amino acid residues and clarified that electrostatic and van der Waals forces drove the interaction. UHT treatment increased hydrogen bonds and decreased total binding energy. The non-covalent binding promoted the formation of disulfide bonds between ß-Lg and casein micelles under heat treatment. Ultimately, it was concluded that non-covalent interaction and disulfide bonding resulted in casein micelle/ß-Lg aggregates. These findings provided scientific insights into protein aggregation in UHT milk.

Adaptability to the environment of protease by secondary structure changes and application to enzyme-selective hydrolysis.

The reactions involving enzymes are significantly influenced by various environmental factors. Clarity of how the activity and structure of proteases impact their function is crucial for more efficient application of enzymes as a tool. The impact of temperature, pH, and ionic strength on changes in protease activity, secondary structure, and protein conformation during enzymatic hydrolysis were investigated in this study. The enzymatic activity and secondary structure of acid-base protease were found to undergo significant modifications under different physical conditions, as demonstrated by UV spectrophotometry and FTIR spectroscopy analysis. Specifically, variations in α-helix and ß-fold content were observed to correlate with changes in enzyme activity. Molecular simulation analysis revealed that physical conditions have varying effects on the protease, particularly influencing enzyme activity and secondary structure. Evaluation of the proteases indicated alterations in both enzyme activity and structure. This treatment selectively hydrolyzed ß-lactoglobulin and reduced sensitization. These findings offer novel perspectives on the functionalities and regulatory mechanisms of proteases, as well as potential industrial applications.

Carboxymethyl chitosan coating infused with linalool-loaded molten globular ß-Lactoglobulin nanoparticles for extended preservation of fresh-cut apples.

This investigation employed molten globule state ß-lactoglobulin nanoparticles (MG-BLGNPs) for encapsulating linalool (LN) combined with carboxymethyl chitosan (CMC) coating to enhance the shelf-life of fresh-cut apples. The effect of different MG structures on the encapsulation efficiency of BLGNPs and the properties of coating was studied. Structural characterization and molecular simulation showed structural differences between heat-induced MG state (70-BLGNPs, heated at 70 °C for 1 h) and sodium dodecyl sulfate-co-heat-induced MG state (SDS/70-BLGNPs, treated with 0.192 mg/mL SDS for 10 min, then heated at 70 °C for 1 h), with the latter being more unfolded. LN self-assembles into MG-BLGNPs, among the generated particles, SDS/70-BLG@LN exhibits stronger binding effect and higher LN loading capacity. Integration of MG-BLG@LN into CMC enhanced coating's mechanical properties and adhesion to fresh-cut apples. The SDS/70-BLG@LN/CMC coating showed superior preservation on fresh-cut apples during storage, reducing enzymatic browning, membrane lipid oxidation, and microbial growth while maintaining hardness and overall quality.

Reducing the Allergenicity of ß-Lactoglobulin by Covalent Modification with Different Contents of Epigallocatechin Gallate (EGCG): In Vitro and In Vivo Studies.

ß-Lactoglobulin (ßLG) is a major allergen in bovine milk protein. This study was designed to investigate changes in ßLG structure, digestibility, and allergenicity induced by covalent binding modification with different contents of (-)-epigallocatechin 3-gallate (EGCG). The reaction of EGCG conjugation with ßLG reached saturation at a molar ratio of 1:60 ßLG:EGCG. Conjugation with EGCG altered the ßLG structure, decreased IgE-binding capacity, and increased digestibility in a dose-dependent manner. In vivo studies showed that covalent conjugation with EGCG can reduce ßLG-induced allergic symptoms with reducing levels of IgE, histamine, and mast cell protease-1 (mMCP-1) and the percentage of sensitized mast cells. Allergenicity was reduced more effectively in saturated ßLG-EGCG conjugates compared to semisaturated conjugates. Observed changes in IFN-γ, IL-4, IL-5, IL-10, and TGF-ß levels suggested that ßLG-EGCG conjugates were able to promote Th1/Th2 immune balance. These findings further our understanding of the relationship between the degree of polyphenol conjugation and the allergenicity of food allergens.

Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

Key structural factors that determine the in vitro enzymatic digestibility of amylose-complexes.

The effects of complexing conditions on the formation of amylose-lipid-protein complexes and relationships between structure and digestion of amylose-lipid and amylose-lipid-protein complexes were poorly understood. The objective of this study was to investigate the effects of complexing time (0, 0.5, 2, 4 and 6 h) and temperature (60, 70, 80, 90 and 100 °C) on the structure and in vitro amylolysis of amylose-lauric acid (AM-LA) and amylose-lauric acid-ß-lactoglobulin (AM-LA-ßLG) complexes, and to understand the relationships between structure and in vitro digestiblity of these complexes. Longer complexing time and higher complexing temperature promoted the formation of greater amounts of the more stable type II crystallites than type I crystallites in both AM-LA and AM-LA-ßLG complexes, which in turn decreased the rate and extent of the complexes digestion to a greater extent. Correlation analyses between parameters for structure and digestion kinetics showed that both the quantity of AM-LA and AM-LA-ßLG complexes and the quality of their arrangement into V-type crystallites influenced their rate and extent of digestion. This study demonstrates that AM-LA and AM-LA-ßLG complexes can be prepared with designed structural and functional properties tailored for various applications.

A New Method for Growth Factor Enrichment from Dairy Products by Electrodialysis with Filtration Membranes: The Major Impact of Raw Product Pretreatment.

This study is focused on fractionation of insulin-like growth factor I (IGF-I) and transforming growth factor-ß2 (TGF-ß2) using a new electro-based membrane process calledelectrodialysis with filtration membranes (EDFM). Before EDFM, different pretreatments were tested, and four pH conditions (4.25, 3.85, 3.45, and 3.05) were used during EDFM. It was demonstrated that a 1:1 dilution of defatted colostrum with deionized water to decrease mineral content followed by the preconcentration of GFs by UF is necessary and allow for these compounds to migrate to the recovery compartment during EDFM. MS analyses confirmed the migration, in low quantity, of only α-lactalbumin (α-la) and ß-lactoglobulin (ß-lg) from serocolostrum to the recovery compartment during EDFM. Consequently, the ratio of GFs to total protein in recovery compartment compared to that of feed serocolostrum solution was 60× higher at pH value 3.05, the optimal pH favoring the migration of IGF-I and TGF-ß2. Finally, these optimal conditions were tested on acid whey to also demonstrate the feasibility of the proposed process on one of the main by-products of the cheese industry; the ratio of GFs to total protein was 2.7× higher in recovery compartment than in feed acid whey solution, and only α-la migrated. The technology of GF enrichment for different dairy solutions by combining ultrafiltration and electrodialysis technologies was proposed for the first time.

Exploration of Novel Plasmin Inhibitor from ß-Lactoglobulin for Enhancing the Storage Stability of UHT Milk.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. ß-Lactoglobulin (ß-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in ß-Lg. Herein, plasmin inhibitory peptides were explored from ß-Lg using experimental and computational techniques. The results revealed that increased denaturation of ß-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the ß-Lg/plasmin complex. Denatured ß-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to ß-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from ß-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Purification of α-lactalbumin and ß-lactoglobulin from cow milk.

Whey, a valuable byproduct of dairy processing, contains essential proteins like ß-lactoglobulin (ßLG) and α-lactalbumin (αLA), making it a focus of research for its nutritional benefits. Various techniques, including chromatography and membrane filtration, are employed for protein extraction, often requiring multiple purification steps. One approach that has gained prominence for the purification and concentration of proteins, including those present in whey, is the use of polyethylene glycol (PEG) in aqueous two-phase systems. Our study simplifies this process by using PEG alone for whey protein purification. This approach yielded impressive results, achieving 92 % purity for ßLG and 90 % for αLA. These findings underscore the effectiveness of PEG-based purification in isolating whey proteins with high purity.

Adsorption of ß-Lactoglobulin on Thiol-Functionalized Mesoporous Silica.

SBA-15 mesoporous materials were synthesized with different pore sizes (5 and 10 nm) and thiol-functionalized groups and then characterized to describe their ability to differentially adsorb ß-lactoglobulin (BLG), a globular protein with an ellipsoid shape measuring 6.9 nm in length and 3.6 nm in width. All adsorption experiments showed that the adsorption capacities of mesoporous materials for BLG were dependent on the duration of contact between the two materials (mesoporous material and BLG) and the initial BLG concentration. It was also shown that the pore sizes and thiol groups of SBA-15-based adsorbents are important factors for the BLG adsorption capacities. Among the tested adsorbents, thiol-functionalized SBA-15 with a 10 nm pore size (SBA-15-SH-10) showed the highest adsorption capacity (0.560 g·g-1) under optimal experimental conditions. Kinetics studies demonstrated that the adsorption occurs predominantly inside the pores, with interactions occurring on heterogeneous surfaces. In addition, the thermodynamic parameters indicate a spontaneous and exothermic behavior of the BLG adsorption process onto the thiol-functionalized SBA-15 mesoporous adsorbent. Finally, the characterization of the SBA-15-SH-10 adsorbent at 308 K showed the occurrence of an oxidation reaction of the thiol groups to sulfonate groups during the adsorption process as confirmed by Raman spectroscopy. The spectra recorded after adsorption of the protein showed that this adsorption did not affect the secondary structure of the protein.

Effects of high pressure processing on structural changes, aggregation, and binding mechanisms of ß-Lactoglobulin with typical polyphenols.

The binding capacity of ß-Lactoglobulin (BLG) is crucial for delivering polyphenols, influenced by structural changes. High pressure processing (HPP) has the potential to modify BLG's structure and aggregation, but its specific impact on BLG-polyphenol interactions is uncertain. This study used circular dichroism spectroscopy and molecular dynamics simulations to reveal HPP-induced structural changes in BLG, supported by particle size analysis indicating aggregation. Seven structurally diverse polyphenols (quercetin-QR, hesperetin-HSP, dihydromyricetin-DHM, gallic acid-GA, (-)-epicatechin-EC, resveratrol-RES, and secoisolariciresinol diglucoside-SDG) were investigated to comprehensively analyze their binding patterns using fluorescence spectroscopy and molecular docking. HPP reduced BLG's ordered structure and increased its aggregation. Binding affinities peaked at 400 MPa for DHM, QR, HSP, GA, and RES, while SDG and EC exhibited maximum affinities at atmospheric pressure and 600 MPa, respectively. Elevated pressures enhanced BLG-polyphenol interactions, particularly at residues 44GLU and 160CYS, with van der Waals forces dominating the binding free energy.

Effect of ultrasound-pretreated starch on the formation, structure and digestibility of starch ternary complexes from lauric acid and ß-lactoglobulin.

Starch, lipids, and proteins are key macronutrients in starchy foods. Their interactions during processing can form starch-lipid-protein ternary complexes, significantly affecting food quality. Ultrasonic treatment, as a common processing method, is expected to regulate the quality of starchy foods by influencing the formation of ternary complexes. This study aimed to understand the effect of ultrasonic pretreatment on the formation of starch-lipid-protein ternary complexes using various types of starches. Wheat starch (WS), maize starch (MS), and potato starch (PS) were gelatinized and treated with ultrasound at various power densities (0-40 W/L) to form complexes with lauric acid (LA) and ß-lactoglobulin (ßLG), respectively. Ultrasound increased the amylose content of gelatinized WS, MS, and PS and shifted their chain length distribution towards the short chains. Results from Fourier transform infrared spectroscopy, laser confocal micro-Raman, X-ray diffraction, and differential scanning calorimetry showed that the largest amount of WS-LA-ßLG complexes was formed at the ultrasonic power density of 10 W/L, and MS-LA-ßLG and PS-LA-ßLG complexes at 20 W/L. Additionally, ultrasound enhanced the content of resistant starch (RS) in the starch-LA-ßLG complexes. The RS content increased from 14.12 % to 18.31 % for WS-LA-ßLG, and from 19.18 % and 20.69 % to 27.60 % and 28.63 % for MS-LA-ßLG and PS-LA-ßLG complexes, respectively. This study presents an approach for facilitating the formation of ternary complexes, contributing to the development of low-GI functional foods.

How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target.

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to ß-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

ß-Lactoglobulin-based amorphous solid dispersions: A graphical review on the state-of-the-art.

Proteins have recently caught attention as potential excipients for amorphous solid dispersions (ASDs) to improve oral bioavailability of poorly water-soluble drugs. Notably, the studies have highlighted whey protein isolates, particularly ß-lactoglobulin (BLG), as promising candidates in amorphous stabilization, dissolution and solubility enhancement, achieving drug loadings of 50 wt% and higher. Consequently, investigations into the mechanisms underlying the solid-state stabilization of amorphous drugs and the enhancement of drug solubility in solution have been conducted. This graphical review provides a comprehensive overview of recent findings concerning BLG-based ASDs. Firstly, the dissolution performance of BLG-based ASDs is compared to more traditional polymer-based ASDs. Secondly, the drug loading onto BLG and the resulting amorphous stabilization mechanisms is summarized. Thirdly, interactions between BLG and drug molecules in solution are described as the mechanisms governing the improvement of drug solubility. Lastly, we outline the impact of the spray drying process on the secondary structure of BLG, and the resulting differences in amorphous stabilization and drug dissolution performance between α-helix-rich and ß-sheet-rich BLG-based ASDs.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

The mechanism of resveratrol stabilization and degradation by synergistic interactions between constituent proteins of whey protein.

Whey protein isolate (WPI) is mainly composed of ß-lactoglobulin (ß-LG), α-lactalbumin (α-LA) and bovine serum albumin (BSA). The aim of this study was to compare and analyze the influence of WPI and its three main constituent proteins, as well as proportionally reconstituted WPI (R-WPI) on resveratrol. It was found that the storage stability of resveratrol was protected by WPI, not affected by R-WPI, but reduced by individual whey proteins at 45°C for 30 days. The rank of accelerated degradation of resveratrol by individual whey proteins was BSA > α-LA > ß-LG. The antioxidant activity, localization of resveratrol and oxidation of carrier proteins were determined by ABTS, H2O2 assay, synchronous fluorescence, carbonyl and circular dichroism. The non-covalent interactions and disulfide bonds between constituent proteins improved the antioxidant activity of the R-WPI-resveratrol complex, the oxidation stability of the carrier and the solvent shielding effect on resveratrol, which synergistically inhibited the degradation of resveratrol in R-WPI system. The results gave insight into elucidating the interaction mechanism of resveratrol with protein carriers.