Comparison of the functional properties of RuBisCO protein isolate extracted from sugar beet leaves with commercial whey protein and soy protein isolates.

BACKGROUND: RuBisCO was extracted from sugar beet leaves using soft and food-compatible technologies. Proximate composition, solubility, emulsifying, foaming and gelling properties of the protein isolate were determined. All these properties were systematically benchmarked against commercial whey and soy protein isolates used in food applications. RESULTS: RuBisCO protein isolate (RPI) contained 930 g kg-1 of crude protein. Protein solubility was higher than 80% at pH values lower than 4.0 or higher than 5.5. Foaming capacity of RPI was better at pH 4.0 than at pH 7.0. Interestingly, 10 g kg-1 protein foams were more stable (pH 7.0 and 4.0) than foams obtained with whey or soy protein. Moreover, 10 g kg-1 RPI emulsions at pH 4.0 or 7.0 exhibited good stability, being similar to whey protein isolate. Remarkable gelling properties were observed at pH 7.0, where 50 g kg-1 protein solutions of RPI formed self-supporting gels while more concentrated solutions were needed for whey or soy protein. CONCLUSION: RuBisCO showed comparable or superior functional properties to those of currently used whey and soy protein isolates. These results highlight the high potential of sugar beet leaf protein isolate as a nutritious and functional food ingredient to face global food security and protein supply. © 2018 Society of Chemical Industry.

Inhibition and Promotion of Heat-Induced Gelation of Whey Proteins in the Presence of Calcium by Addition of Sodium Caseinate.

Heat-induced aggregation and gelation of aqueous solutions of whey protein isolate (WPI) in the presence of sodium caseinate (SC) and CaCl2 was studied at pH 6.6. The effect of adding SC (0-100 g/L) on the structure of the aggregates and the gels was investigated by light scattering and confocal laser scanning microscopy at different CaCl2 concentration ([CaCl2] = 0-30 mM). The gelation process was studied by oscillatory shear rheology. At the whey protein concentrations studied here (34 and 60 g/L), no gels were formed in the absence of CaCl2 and SC. However, WPI solutions gelled above a critical CaCl2 concentration that increased with increasing SC concentration. In the absence of CaCl2, WPI gels were formed only above a critical SC concentration. The critical SC concentration needed to induce WPI gelation decreased weakly when CaCl2 was added. In an intermediate range of CaCl2 concentrations, gels were formed both at low and high SC concentrations, but not at intermediate SC concentrations. Finally, at high CaCl2 concentrations gels were formed at all SC concentrations. The gelation rate and the gel structure of the gels formed at low and high casein concentrations were very different. The effect of SC on the thermal gelation of WPI was interpreted by competition for Ca2+, a chaperon effect, and microphase separation.

Complex equilibria, speciation, and heteroprotein coacervation of lactoferrin and ß-lactoglobulin.

There has been a resurgence of interest in complex coacervation, a form of liquid-liquid phase separation (LLPS) in systems of oppositely charged macroions, but very few reports describe the somewhat anomalous coacervation between acidic and basic proteins, which occurs under very narrow ranges of conditions. We sought to identify the roles of equilibrium interprotein complexes during the coacervation of ß-lactoglobulin dimer (BLG2) with lactoferrin (LF) and found that this LLPS arises specifically from LF(BLG2)2. We followed the progress of complexation and coacervation as a function of r, the LF/BLG molar ratio, using turbidity to monitor the degree of coacervation and proton release and dynamic light scattering (DLS) to assess the stoichiometry and abundance of complexes. Isothermal titration calorimetry (ITC) showed that initial complex formation is endothermic, but a large exotherm related to coacervate formation obscured other regions. On the basis of turbidimetry, proton release, and DLS, we propose a speciation diagram that presents the abundance of various complexes as a function of r. Although multiple species could be simultaneously present, distinct regions could be identified corresponding to equilibria among particular protein pairs.

Structure of bovine ß-lactoglobulin-lactoferrin coacervates.

Lactoferrin (LF) and ß-lactoglobulin (BLG) are among the protein pairs that exhibit heteroprotein coacervation, a unique and relatively unexamined type of liquid-liquid phase separation (LLPS). In prior work we found that LF and BLG undergo coacervation at highly constrained conditions of pH, ionic strength and protein stoichiometry. The molar stoichiometry in coacervate and supernatant is LF : BLG2 1 : 2 (where BLG2 represents the 38 kDa BLG dimer), suggesting that this is the primary unit of the coacervate. The precise balance of repulsive and attractive forces among these units, thought to stabilize the coacervate, is achieved only at limited conditions of pH and I. Our purpose here is to define the process by which such structural units form, and to elucidate the forces among them that lead to the long-range order found in equilibrium coacervates. We use confocal laser scanning microscopy (CLSM), small angle neutron scattering (SANS), and rheology to (1) define the uniformity of interprotein spacing within the coacervate phase, (2) verify structural unit dimensions and spacing, and (3) rationalize bulk fluid properties in terms of inter-unit forces. Electrostatic modeling is used in concert with SANS to develop a molecular model for the primary unit of the coacervate that accounts for bulk viscoelastic properties. Modeling suggests that the charge anisotropies of the two proteins stabilize the dipole-like LF(BLG2)2 primary unit, while assembly of these dipoles into higher order equilibrium structures governs the macroscopic properties of the coacervate.

Modulating Weak Protein-Protein Cross-Interactions by the Addition of Free Amino Acids at Millimolar Concentrations.

In this paper, we quantify weak protein-protein interactions in solution using cross-interaction chromatography (CIC) and surface plasmon resonance (SPR) and demonstrate that they can be modulated by the addition of millimolar concentrations of free amino acids. With CIC, we determined the second osmotic virial cross-interaction coefficient (B23) as a proxy for the interaction strength between two different proteins. We perform SPR experiments to establish the binding affinity between the same proteins. With CIC, we show that the amino acids proline, glutamine, and arginine render the protein cross-interactions more repulsive or equivalently less attractive. Specifically, we measured B23 between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that B23 increases when amino acids are added to the solution even at millimolar concentrations, corresponding to protein/ligand stoichiometric ratios as low as 1:1. With SPR, we show that the binding affinity between proteins can change by 1 order of magnitude when 10 mM glutamine is added. In the case of Lys and one whey protein isolate (WPI), it changes from the mM to the M range, thus by 3 orders of magnitude. Interestingly, this efficient modulation of the protein cross-interactions does not alter the protein's secondary structure. The capacity of amino acids to modulate protein cross-interactions at mM concentrations is remarkable and may have an impact across fields in particular for specific applications in the food or pharmaceutical industries.

Heteroprotein complex coacervation: bovine ß-lactoglobulin and lactoferrin.

Lactoferrin (LF) and ß-lactoglobulin (BLG), strongly basic and weakly acidic bovine milk proteins, form optically clear coacervates under highly limited conditions of pH, ionic strength I, total protein concentration C(P), and BLG:LF stoichiometry. At 1:1 weight ratio, the coacervate composition has the same stoichiometry as its supernatant, which along with DLS measurements is consistent with an average structure LF(BLG2)2. In contrast to coacervation involving polyelectrolytes here, coacervates only form at I < 20 mM. The range of pH at which coacervation occurs is similarly narrow, ca. 5.7-6.2. On the other hand, suppression of coacervation is observed at high C(P), similar to the behavior of some polyelectrolyte-colloid systems. It is proposed that the structural homogeneity of complexes versus coacervates with polyelectrolytes greatly reduces the entropy of coacervation (both chain configuration and counterion loss) so that a very precise balance of repulsive and attractive forces is required for phase separation of the coacervate equilibrium state. The liquid-liquid phase transition can however be obscured by the kinetics of BLG aggregation which can compete with coacervation by depletion of BLG.

Tuning the structure of protein particles and gels with calcium or sodium ions.

The effect of the addition of NaCl or CaCl2 on the structure of protein particles and gels was investigated in detail for aqueous solutions of the globular milk protein ß-lactoglobulin at 40g/L and pH 7.0. When heated in the presence of NaCl or at very low CaCl2 concentrations, the proteins form small strand-like particles, but if more than about two Ca(2+) ions per protein are present, larger spherical particles (microgels) are formed, which increase in size with increasing CaCl2 concentration. The effect of the heating temperature was investigated between 62 and 85 °C. At lower heating temperatures, more Ca(2+) ions per protein are needed to drive the formation of microgels. Particle size measurements done with dynamic light scattering suggest that the aggregation occurs via a nucleation and growth process. The nuclei grow either by fusion or by addition of denatured proteins. If more than three Ca(2+) ions per protein are added, particulate gels are formed by random association of the microgels. Similar particulate gels are also formed at high NaCl concentrations (>200 mM), but by a different mechanism. In this case, the randomly aggregated small strands formed at the early stage of the heating process formed dense spherical domains at a later stage of the heating process by microphase separation that randomly associated to form a particulate gel.

Effect of Different Nutritional Supplements on Glucose Response of Complete Meals in Two Crossover Studies.

Postprandial hyperglycemia is an important risk factor in the development and progression of type-2 diabetes and cardiometabolic diseases. Therefore, maintaining a low postprandial glucose response is key in preventing these diseases. Carbohydrate-rich meals are the main drivers of excessive glycemic excursions during the day. The consumption of whey protein premeals or mulberry leaf extract was reported to reduce postprandial glycemia through different mechanisms of action. The efficacy of these interventions was shown to be affected by the timing of the consumption or product characteristics. Two randomised crossover studies were performed, aiming to identify the optimal conditions to improve the efficacy of these nutritional supplements in reducing a glycemic response. The acute postprandial glycemic response was monitored with a continuous glucose monitoring device. The first study revealed that a preparation featuring 10 g of whey protein microgel reduced the postprandial glucose response by up to 30% (p = 0.001) and was more efficient than the whey protein isolates, independently of whether the preparation was ingested 30 or 10 min before a complete 320 kcal breakfast. The second study revealed that a preparation featuring 250 mg mulberry leaf extract was more efficient if it was taken together with a complete 510 kcal meal (−34%, p < 0.001) rather than ingested 5 min before (−26%, p = 0.002). These findings demonstrate that the efficacy of whey proteins premeal and mulberry leaf extracts can be optimised to provide potential nutritional solutions to lower the risk of type-2 diabetes or its complications.

On the crucial importance of the pH for the formation and self-stabilization of protein microgels and strands.

Stable suspensions of protein microgels are formed by heating salt-free ß-lactoglobulin solutions at concentrations up to about C = 50 g·L(-1) if the pH is set within a narrow range between 5.75 and 6.1. The internal protein concentration of these spherical particles is about 150 g·L(-1) and the average hydrodynamic radius decreases with increasing pH from 200 to 75 nm. The formation of the microgels leads to an increase of the pH, which is a necessary condition to obtain stable suspensions. The spontaneous increase of the pH during microgel formation leads to an increase of their surface charge density and inhibits secondary aggregation. This self-stabilization mechanism is not sufficient if the initial pH is below 5.75 in which case secondary aggregation leads to precipitation. Microgels are no longer formed above a critical initial pH, but instead short, curved protein strands are obtained with a hydrodynamic radius of about 15-20 nm.

The impact of thermal processing on the simulated infant gastrointestinal digestion, bactericidal and anti-inflammatory activity of bovine lactoferrin - An in vitro study.

Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95 °C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.

Protein choices targeting thermogenesis and metabolism.

BACKGROUND: Dietary proteins stimulate thermogenesis and satiety more than does carbohydrate or fat; however, less is known about the differences between protein sources. OBJECTIVE: The objective was to determine the differential effects of 3 proteins on energy metabolism, satiety, and glucose control. DESIGN: Energy metabolism, satiety, and glucose control were measured in 23 lean, healthy subjects on separate occasions, before and 5.5 h after consumption of 4 isocaloric test meals in a randomized, double-blind, crossover design. Three meals consisting of 50% protein (whey, casein, or soy), 40% carbohydrate, and 10% fat and a fourth meal consisting of 95.5% carbohydrate were compared with a glucose meal that provided the same glucose load as the protein meals. RESULTS: The thermic effect was greater after the whey (14.4 ± 0.5%) than after the casein (12.0 ± 0.6%; P = 0.002) and soy (11.6 ± 0.5%; P = 0.0001) meals and was greater after the whey, casein, and soy meals than after the high-carbohydrate meal (6.6 ± 0.5%; P < 0.0001). Cumulative fat oxidation tended to be greater after the whey meal (16.2 ± 1.1 g) than after the soy meal (13.7 ± 1.0 g; P = 0.097) and was greater after the whey and soy meals than after the high-carbohydrate meal (10.9 ± 0.9 g; P < 0.05). The glycemic response to glucose was attenuated 32% by the proteins (P < 0.001) at the expense of a greater insulin response after whey than after glucose (154%; P = 0.02), casein (143%; P = 0.07), and soy (151%; P = 0.03). Subjective appetite sensations indicated that casein and soy were more satiating than whey (P < 0.01), but whey was more "liked" compared with casein and soy (P = 0.025 and P = 0.09, respectively). CONCLUSION: The results suggest that different protein sources could be used to modulate metabolism and subsequently energy balance.

Multiscale characterization of individualized beta-lactoglobulin microgels formed upon heat treatment under narrow pH range conditions.

Aqueous dispersions of demineralized beta-lactoglobulin (beta-lg) were held at 85 degrees C for 15 min at a constant protein concentration of 1 wt % in the pH range of 3.0-7.0. This led to denatured protein content ranging from 20% (pH 3.0) to 90% (pH 5.0). The protein aggregates formed were characterized as to their stability to sedimentation (turbidity), morphology, size, surface charge, ANS surface hydrophobicity, and content in accessible thiol groups. Additionally, the changes in secondary structures of the protein upon heating were followed by Fourier transform infrared spectroscopy (FTIR). Stable dispersions (no sedimentation for 10 min) of individualized beta-lg microgels were obtained at specific pH 4.6 and 5.8, corresponding to an aggregation yield of about 80%. The width of the pH region leading to these microgels was 0.3 pH unit below or above the two specific pH values. Microgels were characterized by a spherical shape and remarkably low polydispersity in size (<0.2). Their z-average hydrodynamic diameter determined by dynamic light scattering (DLS) was between 160 and 220 nm, and their zeta-potential was +30 or -40 mV, depending on the initial pH before heating. Microgels obtained at pH 4.6 displayed a lower binding capacity for ANS and a lower content of accessible thiol groups as compared to those obtained at pH 5.8. Both types of microgels might therefore differ in their internal and interfacial structures. Between pH 4.6 and 5.8, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <4.6 or >5.8. Interestingly, DLS experiments showed that before heating, beta-lg was mainly present in an oligomeric state at pH 4.6 and 5.8. This result was confirmed by FTIR measurements indicating the stronger contribution of the 1616-1624 cm(-1) spectral band corresponding to intermolecular beta-sheets in the pH range of 4.0-6.0. Upon heating, FTIR spectroscopy revealed that individualized microgels were obtained under pH conditions where a balance between attractive forces arising from protein unfolding leading to the formation of intermolecular beta-sheets (1616-1624 cm(-1 )band) and the repulsive electrostatic forces due to the initial protein net charge was achieved.

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes.

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.

Immunostimulatory potential of beta-lactoglobulin preparations: effects caused by endotoxin contamination.

BACKGROUND: The immunomodulating potential residing in cow's milk proteins is currently receiving increasing attention because of growing interest in functional foods and the complex problem of cow's milk allergy. One of the major cow's milk allergens, whey protein beta-lactoglobulin, has previously been shown to mediate cellular activation in both human and murine immune cells. OBJECTIVE: We examined the response to different beta-lactoglobulin preparations in naive immune cells. METHODS: Splenocytes and cells from mesenteric lymph nodes derived from BALB/c mice bred and maintained on a milk-free diet were cultured in vitro with different beta-lactoglobulin preparations. Cell proliferation, cytokine production, and increases in intracellular glutathione were used as cellular activation markers. Moreover, the effect of beta-lactoglobulin on cytokine production in murine bone-marrow-derived dendritic cells was examined. RESULTS: We observed that some commercial beta-lactoglobulin preparations induced pronounced proliferation of both spleen cells and cells from mesenteric lymph nodes; production of TNF-alpha, IL-6, IL-1beta, and IL-10; and an increased level of intracellular glutathione in spleen cell cultures. Furthermore, TNF-alpha, IL-6, IL-1beta, and IL-10 production was induced in murine bone-marrow-derived dendritic cells. Purification of beta-lactoglobulin from raw milk using nondenaturating conditions, however, revealed that the beta-lactoglobulin per se did not possess the immunomodulatory activity. Eventually, the immunostimulatory effect was found to be caused by endotoxin contamination. CONCLUSION: These results identify endotoxin as the main immunostimulatory component present in some commercial beta-lactoglobulin preparations. Moreover, the present study makes it evident that immunomodulatory effects attributed to beta-lactoglobulin need to be reassessed.