Whey Protein Β-lactoglobulin Research Articles

Electrostatic interactions mediated defibrillation of β-lactoglobulin fibrils using Keggin Polyoxometalates

The whey protein β-lactoglobulin (βLG) forms fibrils similar to the amyloid fibrils in the neurodegenerative diseases due to its higher predisposition of β-sheets. This study shed light on the understanding different inorganic Keggin polyoxometalates (POMs) interaction with the protein βLG fibrils. POMs such as Phosphomolybdic acid (PMA), silicomolybdic acid (SMA), tungstosilicic acid (TSA), and phosphotungstic acid (PTA) were used due to their inherent higher anionic charges. The interaction studies were monitored with fluorescence spectra and Thioflavin T assay for both the βLG monomers and the fibrils initially to elucidate the binding ability of the POMs. The binding of POMs and βLG is also demonstrated by molecular docking studies. Zeta potential studies showed the electrostatic mediated higher interactions of the POMs with the protein fibrils. Isothermal titration calorimetry (ITC) studies showed that the molybdenum containing POMs have higher affinity to the protein fibrils than the tungsten. This study could help understanding formation of food grade protein fibrils which have profound importance in food industries.

Exploring Functionality Gain for (Recombinant) β-Lactoglobulin Through Production and Processing Interventions

AbstractInterventions in the upstream production and further processing of recombinant food proteins affect its properties when used for food application. Often the efficiency of particular interventions is evaluated based on molecular purity and yield rather than final functional properties. Yet, the formulation of foods, including the amount of protein required, can be affected when the functional properties have changed. In this explorative study, we exemplify how far we can extend the functionality range of the major whey protein β-lactoglobulin (BLG), in terms of foaming and (heat-set) gelling, through various interventions. Slight changes in the amino acid sequence of BLG affected its functional properties significantly. Foams were up to ten times more stable, when selecting different natural isoforms of BLG (isoform A instead of B) or when inducing targeted cysteine mutations. The isoform B yielded stronger thermally induced gels (+ 40%) compared to isoform A. During downstream processing of recombinantly secreted BLG, limited purification of up to ~ 67 wt% enabled reasonable foaming properties and superior gelation, while a lower purity of ~ 22 wt% resulted in poor performance in both cases. Post-processing allowed conversion of native whey protein into soluble amyloid-like aggregates. These aggregates resulted in better foam stability (i.e., approximately four times longer than non-aggregated protein), but did not improve gelation. The presented study demonstrates that one should consider not only protein yield and purity, but also functional properties when developing recombinant proteins for food application. In turn, these functional properties are a result of the complete upstream and downstream chain.

Binding of Per- and Polyfluoroalkyl Substances to β-Lactoglobulin from Bovine Milk.

Per- and polyfluoroalkyl substances (PFAS) are known for their high environmental persistence and potential toxicity. The presence of PFAS has been reported in many dairy products. However, the mechanisms underlying the accumulation of PFAS in these products remain unclear. Here, we used native mass spectrometry and molecular dynamics simulations to probe the interactions between 19 PFAS of environmental concern and two isoforms of the major bovine whey protein β-lactoglobulin (β-LG). We observed that six of these PFAS bound to both protein isoforms with low- to mid-micromolar dissociation constants. Based on quantitative, competitive binding experiments with endogenous ligands, PFAS can bind orthosterically and preferentially to β-LG's hydrophobic ligand-binding calyx. β-Cyclodextrin can also suppress binding of PFAS to β-LG owing to the ability of β-cyclodextrin to directly sequester PFAS from solution. This research sheds light on PFAS-β-LG binding, suggesting that such interactions could impact lipid-fatty acid transport in bovine mammary glands at high PFAS concentrations. Furthermore, our results highlight the potential use of β-cyclodextrin in mitigating PFAS binding, providing insights toward the development of strategies to reduce PFAS accumulation in dairy products and other biological systems.

Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein β-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native β-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with β-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

Assessment of the Efficiency of Technological Processes to Modify Whey Protein Antigenicity.

Whey is a by-product that represents a cheap source of protein with a high nutritional value, often used to improve food quality. When used as a raw material to produce hypoallergenic infant formulas (HIF), a processing step able to decrease the allergenic potential is required to guarantee their safe use for this purpose. In the present paper, thermal treatments, high hydrostatic pressure (HHP), and enzymatic hydrolysis (EH) were assessed to decrease the antigenicity of whey protein solutions (WPC). For monitoring purposes, a competitive ELISA method, able to detect the major and most allergenic whey protein β-lactoglobulin (BLG), was developed as a first step to evaluate the efficiency of the processes. Results showed that EH together with HHP was the most effective combination to reduce WPC antigenicity. The evaluation method proved useful to monitor the processes and to be employed in the quality control of the final product, to guarantee the efficiency, and in protein antigenicity reduction.

Fibrillation of β-lactoglobulin at pH 2.0: Impact of cysteine substitution and disulfide bond reduction

Fibrillar aggregation is a promising route to enhance protein functionality for food, while prefibrillar oligomers in vivo are suspected to be pathogenic. Here, the role of intramolecular disulfide bonds in fibrillation (80 °C, pH 2) of the major whey protein β-lactoglobulin (BLG) was studied. Different cysteine-modified variants were used for this investigation, removing one or both disulfide bonds in BLG.Denaturation occurred upon heating at pH 2.0 up to 1 h, but residual structure elements prevented aggregation of intact bovine BLG. Heating for longer caused acidic hydrolysis, which resulted in the release of peptides with enhanced aggregation tendency. Partial destabilisation by removal of C66–C160 (by recombinant substitution) or both disulfide bonds (by chemical cleavage) was found to affect this hydrolysis rate, but only cleavage of both disulfide bonds accelerate the aggregation kinetics. No major impact on the fibrillar morphology was observed. In contrast, recombinant removal of both disulfide bonds led to complete structural disruption and instantaneous aggregation of intact BLG at pH 2 prior to heating. These small aggregates (10–50 nm) led to the formation of worm-like aggregates within 1 h of heating, which slowed down the acidic hydrolysis and inhibited further aggregation into fibrils.We concluded that fibrillation can be accelerated by structural destabilisation through disulfide bond cleavage, while complete destabilisation can actually hinder it. These insights can be used for the production of functional protein aggregates, and possibly for the avoidance of pathogenic fibrillation of proteins.

A Comparative Study between A Protein Based Amorphous Formulation and Other Dissolution Rate Enhancing Approaches: A Case Study with Rifaximin.

Amorphous solid dispersions (ASDs) based on proteins as co-formers have previously shown promising potential to improve the solubility and bioavailability of poorly water-soluble drugs. In particular, whey proteins have shown to be promising co-formers and amorphous stabilizers in ASD formulations, including at high drug loading. In this study, the feasibility of the whey protein β-lactoglobulin (BLG) as a co-former in ASDs was compared to the more traditional ASD co-formers based on synthetic polymers (hydroxypropyl methylcellulose acetate succinate and Eudragit® L) as well as to a nanocrystalline formulation. The poorly water-soluble drug rifaximin (RFX) was chosen as the model drug. All drug/co-former formulations were prepared as fully amorphous ASDs by spray drying at 50% (w/w) drug loading. The BLG-based ASD had the highest glass transition temperature and showed a faster dissolution rate and higher drug solubility in three release media with different pH values (1.2, 4.5, and 6.5) compared to the polymer-based ASDs and the nanocrystalline RFX. In conclusion, BLG is a promising co-former and amorphous stabilizer of RFX in ASD formulations, superior to the selected polymer-based ASD systems or the nanocrystalline formulation.

Insight into the binding behavior, structure, and thermal stability properties of β-lactoglobulin/Amoxicillin complex in a neutral environment

Antibiotics are extensively used in agriculture and husbandry, thus threatening human health due to their direct exposure to dairy products. Therefore, it is essential to investigate whether milk proteins are unintended carriers of antibiotics. In this research, therefore, we studied the impact of Amoxicillin on the conformation and structural stability of whey protein β-lactoglobulin by using spectroscopic techniques combined with molecular docking and simulation approaches. Further, the thermal stability of the resultant complex was explored. Amoxicillin was observed to effectively quench β-lactoglobulin's intrinsic fluorescence, suggesting that Amoxicillin was successfully bound to the β-lactoglobulin and a stable complex between them was established. The thermodynamic parameters demonstrated that van der Waals forces and hydrogen bonds dominated the spontaneous bonding process. According to circular dichroism (CD) and Fourier transform infrared (FTIR) analyses, the conformational structure of the β-lactoglobulin was altered upon the complexation with Amoxicillin. Further, this interaction significantly enhanced the thermal stability of β-lactoglobulin compared to sole β-lactoglobulin solutions. Molecular docking theoretically showed the preferred binding locus of Amoxicillin within the β-lactoglobulin structure. Molecular dynamics (MD) simulation results further validated the stability of the β-lactoglobulin-Amoxicillin complex. The findings obtained here are helpful for elucidating the potential of β-lactoglobulin to bind and transport harmful substances into the body, thus opening up new avenues for food security.

Conformational analysis and water dynamics: a molecular dynamics study on the survival of a β-lactoglobulin peptide in the archaeological record

One of the greatest successes of the application of Palaeoproteomics to Archaeology is its use by a number of authors to track evidence of dairying practice, both in terms of its origin and the selection of animal species. To this end, the whey protein β-lactoglobulin entrapped in pottery and dental calculus is widely studied because it is so frequently recovered but why is it differentially preserved? Hydrolysis plays a big part in the breakdown of proteins. Therefore, it is essential to explore the role of water in degradation to uncover some of the patterns linked to protein survival. One approach to understand the hydrolytic process is to examine the molecular behaviour of this protein and in particular of the peptide most commonly recovered: T125PEVDXEALEK135. In this study, we use Molecular Dynamics, with the Amber14SB forcefield and the SPC/E water model in Gromacs 2020, to first explore the dynamics of this peptide in bulk water. Despite the difficulties in describing reactive processes with classical methods, we were able to identify geometric arrangements between water and protein residues which are similar to the ones described in the literature for protein hydrolysis. These arrangements helped to identify potential sites for hydrolysis along the bovine β-lactoglobulin T125PEVDDEALEK135 amino acid chain.

Tryptophan self-assembly yields cytotoxic nanofibers containing amyloid-mimicking and cross-seeding competent conformers.

Dietary consumption of Trp via protein-based foods is essential for the maintenance of crucial metabolic processes including the synthesis of proteins and several vital metabolites such as serotonin, melatonin, acetyl CoA, and NADP. However, the abnormal build-up of Trp is known to cause familial hypertryptophanemia and several brain-related medical complications. The molecular mechanism of the onset of such Trp-driven health issues is largely unknown. Here, we show that Trp, under the physiologically mimicked conditions of temperature and buffer, undergoes a concentration driven self-assembly process, yielding amyloid-mimicking nanofibers. Viable H-bonds, π-π interactions and hydrophobic contacts between optimally coordinated Trp molecules become important factors for the formation of a Trp nanoassembly that displays a hydrophobic exterior and a hydrophilic interior. Importantly, Trp nanofibers were found to possess high affinity for native proteins, and they act as cross-seeding competent conformers capable of nucleating amyloid formation in globular proteins including whey protein β-lactoglobulin and type II diabetes linked insulin hormone. Moreover, these amyloid mimicking Trp nanostructures showed toxic effects on neuroblastoma cells. Since the key symptoms in hypertryptophanemia such as behavioural defects and brain-damaging oxidative stress are also observed in amyloid related disorders, our findings on amyloid-like Trp-nanofibers may help in the mechanistic understanding of Trp-related complications and these findings are equally important for innovation in applied nanomaterials design and strategies.

Comparison of non-covalent binding interactions between three whey proteins and chlorogenic acid: Spectroscopic analysis and molecular docking

Non-covalent binding interactions between three whey proteins β-lactoglobulin (β-Lg), α-lactalbumin (α-La) and bovine serum albumin (BSA) with chlorogenic acid (CA) were investigated using spectroscopic analysis and molecular docking. Fluorescence study showed that CA quenched the fluorescence of three whey proteins through static mode. The binding number was equal to 1 for three proteins and binding affinity in declined order was: α-La > β-Lg > BSA. Thermodynamic parameters revealed contribution of hydrophobic force in three systems. Fluorescence resonance energy transfer (FRET) measurements indicated that energy transfer occurs between three proteins and CA in probability of α-La > BSA > β-Lg. Variation in surface charge indicated the involvement of electrostatic interaction. Surface hydrophobicity (H0) were declined with decrease degree of α-La > β-Lg > BSA. α-La and β-Lg were unfolded with more flexible structure while BSA skeleton was more compact after interacting with CA. Modeling study revealed that the most likely binding sites for the three proteins were outer surface, cleft and subdomain I for β-Lg, α-La and BSA respectively. Docking results also suggested the contribution of hydrophobic interaction and hydrogen bond (β-Lg, α-La) for formation of molecular nano complexes between whey proteins and CA.

Interfacial film formation and film stability of high hydrostatic pressure-treated β-lactoglobulin

The mechanical stability of interfacial protein films is limited through the intermolecular interaction of the adsorbed proteins. Since the intermolecular interactions vary in dependence of the protein structure, the investigation of intentionally induced structural modifications (i.e., through high hydrostatic pressure) of interfacial active proteins is required to mechanistically understand their structure-function-relationship. The aim of this study was to link the structural analysis of pressure-treated whey protein β-lactoglobulin at the oil/water-interface with the formation of the interfacial protein film and the resulting film stability against mechanical stress. For this purpose, we treated β-lactoglobulin in water at pH 7 with hydrostatic pressures of 600 MPa for 10 min at 20 °C and analyzed the protein structure in oil/water-emulsion with Fourier-transform-infrared-spectroscopy, extrinsic fluorescence, and ζ-potential. We characterized the molecular density and interfacial film properties via Langmuir trough analysis, electron-paramagnetic-resonance-spectroscopy, interfacial shear and dilatational rheology. The structural flexibility of pressure-treated β-lactoglobulin favored its unfolding and a fast interfacial protein film formation that resulted in pronounced intermolecular interactions, including van der Waals interactions, hydrophobic associations and disulfide bonds. The high interfacial molecule density combined with many and strong intermolecular interactions stabilized the pressure-treated protein film against shear deformation and small dilatational deformation. However, against high dilatational deformation, the pressure-treated β-lactoglobulin film showed a lower stability due to a slow migration towards unoccupied interfacial area. Hence, our research contributes to the control of the mechanical protein film stability, and thus the colloidal stability of emulsions in dependence of the protein structure at the oil/water-interface.

Investigating the Binding Interaction between β-Lactoglobulin and Vitamin B12: A Spectroscopic and Computational Approach

Binding interaction of the major whey protein β-Lactoglobulin (βLG) with vitamin B12 is crucial for understanding the potency of βLG as a transporter for vitamin B12. Hence, the binding interaction of the βLG with vitamin B12 was studied using spectroscopic (UV-Visible absorption spectroscopy, steady-state & time-resolved fluorescence spectroscopy, synchronous fluorescence spectroscopy (SFS), circular dichroism (CD), and fluorescence correlation spectroscopy (FCS)) and computational (molecular docking and molecular dynamics (MD) simulation) tools. The quenching of the intrinsic fluorescence of βLG by vitamin B12 confirms the binding of vitamin B12 with βLG. The nature of the quenching is found to be static due to ground state complex formation. The calculated thermodynamic parameters suggest the binding process is hydrophobic. Synchronous fluorescence spectroscopy revealed that vitamin B12 affects the tryptophan residue microenvironment of the protein. Vitamin B12 does not alter the secondary and tertiary structure of βLG, as concluded from the CD and FCS experiments. From the molecular docking results, vitamin B12 is found to bind at the β- barrel site of βLG. The MD simulation results suggest that vitamin B12 bound βLG complex is stable. The secondary structural element analysis of MD simulation results show no alteration in protein secondary structure after binding with vitamin B12, which is matched with the experimental findings. This biophysical binding interaction study of βLG with vitamin B12 might have promising applications in the pharmaceutical and food industries.

β-Lactoglobulin-gold nanoparticles interface and its interaction with some anticancer drugs – an approach for targeted drug delivery

The protein-nanoparticle interface plays a crucial role in drug binding and stability, in turn enhancing efficacy in targeted drug delivery. In the present study, whey protein β-lactoglobulin (BLG) is conjugated with gold nanoparticles (AuNP) and its interaction with curcumin (CUR) and gemcitabine (GEM) has been explored. Further, AuNP-BLG conjugate interactions with anticancer drugs were characterized using dynamic light scattering (DLS), zeta potential, UV-visible, Raman spectroscopy, fluorescence, circular dichroism along with molecular dynamics simulation. The cytotoxicity studies were performed using breast cancer cell lines (MCF-7). ∼8 µM of BLG resides on AuNP (∼29 nm) surface revealed by DLS. Raman scattering of AuNP-BLG conjugate showed orientation of the central calyx of BLG towards solvent. BLG fluorescence confirmed the interaction between AuNP-BLG conjugate with drugs and indicated strong binding and affinity (for CUR KD = 3.71 x 108 M −1, n = 1.83, and for GEM KD = 3.78 x 103 M −1, n = 0.94), enhanced in the presence of AuNP. CD and Raman analysis exhibited selective hydrophilic and hydrophobic conformations induced by drug binding. Computational studies on BLG-drug complexes revealed that the residues Pro38, Leu39 and Met107 are largely associated with CUR binding, while GEM interaction is via hydrophilic contacts which significantly matches with spectroscopic investigation. IC50 values were calculated for all components of this loading system on MCF-7. The possible mechanisms of interaction between AuNP-BLG with anticancer drugs has been explored at the molecular level. We believe that these conjugates could be considered in the targeted drug delivery studies for cancer research. Communicated by Ramaswamy H. Sarma

Administration of Extensive Hydrolysates From Caseins and Lactobacillus rhamnosus GG Probiotic Does Not Prevent Cow's Milk Proteins Allergy in a Mouse Model.

BackgroundEarly nutrition may influence the development of food allergies later in life. In the absence of breastfeeding, hydrolysates from cow’s milk proteins (CMP) were indicated as a prevention strategy in at risk infants, but their proof of effectiveness in clinical and pre-clinical studies is still insufficient. Thanks to a validated mouse model, we then assessed specific and nonspecific preventive effects of administration of extensive hydrolysates from caseins (eHC) on the development of food allergy to CMP. The additional nonspecific effect of the probiotic Lactobacillus GG (LGG), commonly used in infant formula, was also assessed.MethodsGroups of young BALB/cByJ female mice were pretreated by repeated gavage either with PBS (control mice), or with PBS solution containing non-hydrolyzed milk protein isolate (MPI), eHC or eHC+LGG (eq. of 10 mg of protein/gavage). All mice were then experimentally sensitized to CMP by gavage with whole CM mixed with the Th2 mucosal adjuvant Cholera toxin. All mice were further chronically exposed to cow’s milk. A group of mice was kept naïve. Sensitization to both caseins and to the non-related whey protein β-lactoglobulin (BLG) was evaluated by measuring specific antibodies in plasma and specific ex vivo Th2/Th1/Th17 cytokine secretion. Elicitation of the allergic reaction was assessed by measuring mMCP1 in plasma obtained after oral food challenge (OFC) with CMP. Th/Treg cell frequencies in gut-associated lymphoid tissue and spleen were analyzed by flow cytometry at the end of the protocol. Robust statistical procedure combining non-supervised and supervised multivariate analyses and univariate analyses, was conducted to reveal any effect of the pretreatments.ResultsPBS pretreated mice were efficiently sensitized and demonstrated elicitation of allergic reaction after OFC, whereas mice pretreated with MPI were durably protected from allergy to CMP. eHC+/-LGG pretreatments had no protective effect on sensitization to casein (specific) or BLG (non-specific), nor on CMP-induced allergic reactions. Surprisingly, eHC+LGG mice demonstrated significantly enhanced humoral and cellular immune responses after sensitization with CMP. Only some subtle changes were evidenced by flow cytometry.ConclusionNeither specific nor nonspecific preventive effects of administration of casein-derived peptides on the development of CMP food allergy were evidenced in our experimental setup. Further studies should be conducted to delineate the mechanisms involved in the immunostimulatory potential of LGG and to clarify its significance in clinical use.

High temperature induced structural changes of apo-lactoferrin and interactions with β-lactoglobulin and α-lactalbumin for potential encapsulation strategies

There has been increasing interest in lactoferrin as a bioactive compound from milk, as it functions as an antimicrobial and also in delivering iron. Despite the potential as a food ingredient, the low bioavailability of lactoferrin has limited opportunities for commercialisation. Therefore, protective encapsulation of lactoferrin has been explored as a means of enhancing its bioavailability. In this study, a novel in silico approach was adopted to explore the possibility of employing whey protein components to encapsulate lactoferrin. The most unstable form of the protein, apo-lactoferrin has been studied, particularly interactions with the whey proteins β-lactoglobulin and α-lactalbumin. Temperature conditions that might be encountered during spray drying (95 and 180 °C) have been modelled in order to provide an in-depth understanding of the interactions at the molecular level. The findings indicate that β-lactoglobulin and α-lactalbumin are able to preserve the two antibacterial regions within the apo-lactoferrin structure (lactoferricin and lactoferampin). Intra-molecular residue contact analyses within apo-lactoferrin illustrate the tendency for agglomeration under these temperature conditions, indicating that it is most likely to be readily dispersible during subsequent rehydration. Furthermore, free energy calculations found electrostatic attractions to be the primary mode of interaction between apo-lactoferrin, β-lactoglobulin, and α-lactalbumin. This data is confirmed by a network analysis that demonstrates how interactions amongst apo-lactoferrin, β-lactoglobulin, and α-lactalbumin are predominantly a result of the acidic and basic amino acid residues of the proteins. Overall, this study provides molecular insights in understanding the interactions between lactoferrin with β-lactoglobulin and α-lactalbumin for potential encapsulation strategies via spray drying.

FTIR analysis of β-lactoglobulin at the oil/water-interface

Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein β-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fourier-transform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing β-lactoglobulin content (0.10–1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20–0.31 wt% β-lactoglobulin (1.80–2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular β-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.

Adhesive Interactions Between Lactic Acid Bacteria and β-Lactoglobulin: Specificity and Impact on Bacterial Location in Whey Protein Isolate.

In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior toward the major whey protein β-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using atomic force microscopy (AFM) and a relation with bacterial distribution in whey protein isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to β-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to β-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing β-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.

Effect of Methylglyoxal-Induced Glycation on the Composition and Structure of β-Lactoglobulin and α-Lactalbumin.

Glycation, and particularly reactions between aldehydes and nucleophiles (thiols, amines), can initiate changes in the structure, solubility, composition, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. This can have adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. Low-molecular-mass dialdehydes such as methylglyoxal (MGO) are much more reactive than parent glucose and therefore potentially highly damaging. These are present at significant levels in some foods. This study investigated whether and how MGO exposure, with or without concurrent heat exposure, affected the major whey proteins β-lactoglobulin and α-lactalbumin. MGO diminished the formation of heat-induced, reducible, intermolecular disulfide cross-links for both proteins, with this being associated, at least in part, with alternative thiol consuming reactions of MGO. At long incubation times, nonreducible protein cross-links were formed in a dose-dependent manner, with LC-MS/MS and UPLC analysis showing the presence of methylglyoxal-lysine dimers (MOLD). UPLC analysis revealed MGO-dependent consumption of specific amino acids in the order Cys > Arg > Lys > Trp for both proteins, with α-lactalbumin affected to a greater extent than β-lactoglobulin. SDS-PAGE revealed altered protein mobility consistent with modification of charged residues. MGO exposure also resulted in increased binding of the hydrophobic dye, 8-anilino-1-naphthalene sulfonic acid, consistent with limited protein unfolding. Overall, these data are consistent with rapid reaction of MGO residues at Cys residues (when available) and surface accessible Arg and Lys residues, with formation of adducts and cross-linked materials. These alternative reactions of dialdehydes diminish direct heat-induced (disulfide) cross-link formation and result in limited protein unfolding.

Manufacturing of demineralized whey concentrates with extended shelf life: Impact of the degree of demineralization on functional and microbial quality criteria

For producing demineralized whey concentrates (dry matter (DM) contents: 12–24%) with high whey protein nativity, the suitability of a gentle two-step preservation process (extended shelf life (ESL) process) combining microfiltration and subsequent thermal treatment was investigated. Concentration and demineralization of whey was achieved by using solely nanofiltration (NF) or NF combined with subsequent diafiltration. The influence of the degree of demineralization (number of diafiltration steps: 0; 0.75; 1.5) and DM content on each process step, whey protein denaturation and shelf life of whey concentrates was studied. Applying the ESL process, a strong bacterial reduction of 5.3–7.8 log cycles was achieved depending on the initial bacterial count. For the heat sensitive whey protein β-lactoglobulin, low thermal denaturation of 10–28% was observed, which was independent of DM content and degree of demineralization. However, the shelf life of NF whey concentrates was strongly dependent on their degree of demineralization and ranged between 2 and 16weeks.