Magnetic resonance imaging (MRI) and relaxation spectrum analysis as methods to investigate swelling in whey protein gels

Effective means for controlled delivery of nutrients and nutraceuticals are needed. Whey protein-based gels, as a model system and as a potential delivery system, exhibit pH-dependent swelling when placed in aqueous solutions. Understanding the physics that govern gel swelling is thus important when designing gel-based delivery platforms. The extent of swelling over time was monitored gravimetrically. In addition to gravimetric measurements, magnetic resonance imaging (MRI) a real-time noninvasive imaging technique that quantified changes in geometry and water content of these gels was utilized. Heat-set whey protein gels were prepared at pH 7 and swelling was monitored in aqueous solutions with pH values of 2.5, 7, and 10. Changes in dimension over time, as characterized by the number of voxels in an image, were correlated to gravimetric measurements. Excellent correlations between mass uptake and volume change (R(2)= 0.99) were obtained for the gels in aqueous solutions at pH 7 and 10, but not for gels in the aqueous solution at pH 2.5. To provide insight into the mechanisms for water uptake, nuclear magnetic resonance (NMR) relaxation times were measured in independent experiments. The relaxation spectrum for the spin-spin relaxation time (T(2)) showed the presence of 3 proton pools for pH 7 and 10 trials and 4 proton pools for pH 2.5 trials. Results demonstrate that MRI and NMR relaxation measurements provided information about swelling in whey protein gels that can constitute a new means for investigating and developing effective delivery systems for foods.     

离子强度和温度对乳清蛋白凝胶的影响

本实验主要研究凝胶温度和CaCl2 浓度对乳清蛋白冷凝胶的影响。结果表明：较低的凝胶温度和增加CaCl2浓度能够致使乳清蛋白形成清亮的凝胶;在0、10、20℃凝胶温度条件下,增加CaCl2 浓度使得凝胶硬度有所增加;乳清蛋白凝胶的持水性在凝胶温度为0、10℃,CaCl2 浓度为20、40mmol/L 时受到影响;除了0℃ 和20mmol/LCaCl2 条件下,低温能够使乳清蛋白形成较高的凝胶硬度和持水性。凝胶温度和CaCl2 浓度是影响乳清蛋白冷凝胶的关键因素。     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Monitoring the effects of divalent ions (Mn and Ca) in heat-set whey protein gels

Exploring the effects of cations in whey protein-based gels (WPG) is of importance when these gels are used for controlled release applications in food systems. The objective of this study was to evaluate both water uptake and cation release from heat-set WPGs. Magnetic Resonance Imaging and NMR relaxometry were employed to study the uptake and release. A non-paramagnetic (Ca⁺²) and a paramagnetic cation (Mn⁺²) were incorporated into the WPG as model divalent cations. Cylindrical pieces of WPGs with manganese were immersed in water (pH 2.40, 7.00, 10.40) or in EDTA solution whereas WPGs with calcium were immersed in water at pH 2.40. Water uptake by the gels was influenced by both ionic environment and pH. The release of Mn⁺² from WPG was enhanced by the presence of EDTA. Relaxation spectra of Mn⁺²-loaded gels were significantly influenced by pH of the suspending fluid and by the presence of EDTA. Results of relaxometry experiments, obtained with Ca⁺²-loaded gels immersed in water at pH 2.40, indicated a strong correlation (R² > 0.99) between relative areas of the proton pools and the amount of calcium released to the medium. Results support the use of MRI and NMR to monitor cation release and water uptake in WPG, non-destructively.     

Formulation,process conditions,and biological evaluation of dairy mixed gels containing fava bean and milk proteins: Effect on protein retention in growing young rats

Food formulation and process conditions can indirectly influence AA digestibility and bioavailability. Here we investigated the effects of formulation and process conditions used in the manufacture of novel blended dairy gels (called “mixed gels” here) containing fava bean (Vicia faba) globular proteins on both protein composition and metabolism when given to young rats. Three mixed dairy gels containing casein micelles and fava bean proteins were produced either by chemical acidification (A) with glucono-δ-lactone (GDL) or by lactic acid fermentation. Fermented gels containing casein and fava bean proteins were produced without (F) or with (FW) whey proteins. The AA composition of mixed gels was evaluated. The electrophoretic patterns of mixed protein gels analyzed by densitometry evidenced heat denaturation and aggregation via disulfide bonds of fava bean 11S legumin that could aggregate upon heating of the mixtures before gelation. Moreover, fermented gels showed no particular protein proteolysis compared with gel obtained by GDL-induced acidification. Kinetics of acidification were also evaluated. The pH decreased rapidly during gelation of GDL-induced acid gel compared with fermented gel. Freeze-dried F, A, and FW mixed gels were then fed to 30 young (1 mo old) male Wistar rats for 21 d (n = 10/diet). Fermented mixed gels significantly increased protein efficiency ratio (+58%) and lean mass (+26%), particularly muscle mass (+9%), and muscle protein content (+15%) compared with GDL-induced acid gel. Furthermore, F and FW formulas led to significantly higher apparent digestibility and true digestibility (+7%) than A formula. Blending fava bean, casein, and whey proteins in the fermented gel FW resulted in 10% higher leucine content and significantly higher protein retention in young rats (+7% and +28%) than the F and A mixed gels, respectively. Based on protein gain in young rats, the fermented fava bean, casein, and whey mixed proteins gel was the most promising candidate for further development of mixed protein gels with enhanced nutritional benefits.     

Aerated whey protein gels as a controlled release system of creatine investigated in an artificial stomach

A controlled creatine-release system has been developed from whey protein-based gels. Their functionalization was carried out by aeration and sodium ions induced “cold gelation” processes. The effect of protein concentration in the aerated whey protein gels at pH 7.0 and 8.0 was analyzed. Physicochemical properties of the aerated gels were evaluated. It was possible to obtain the ions induced whey protein aerated gel with well distributed creatine and different microstructure as well as rheological properties. Different protein concentrations and pH enabled obtaining gels with different rheological properties, texture, air fraction, diameter of air bubbles, microstructure and surface roughness. An increase in the protein concentration enhanced the hardness of the samples, regardless of their pH. The mechanical strength of gels prepared at pH 8 were higher than those obtained at pH 7, as was manifested by the smaller storage modulus of the latter. The former gel exhibited a microstructure between particulate and fine-stranded. A stronger gel matrix produced smaller air bubbles. Aerated gels produced at pH 7.0 had higher roughness than those obtained at pH 8.0. Optimal conditions for inclusion of air bubbles into the gel matrix were: 9% protein concentration at pH 8.0 and this aerated gel was selected for digestion in the artificial stomach. There is a small conversion of creatine to creatinine in the artificial stomach digestion process (9.6% after 6 h). The diffusion of creatine crystals from the aerated gel matrix was the mechanism responsible for the release process. Aerated whey protein gels can be used as matrices for time extended releasing of creatine in the stomach.     

Formation of Elastic Whey Protein Gels at Low pH by Acid Equilibration

Abstract: Whey protein gels have a weak/brittle texture when formed at pH ≤ 4.5, yet this pH is required to produce a high-protein, shelf-stable product. We investigated if gels could be made under conditions that produced strong/elastic textural properties then adjusted to pH ≤ 4.5 and maintain textural properties. Gels were initially formed at 15% w/w protein (pH 7.5). Equilibration in acid solutions caused gel swelling and lowered pH because of the diffusion of water and H⁺ into the gels. The type and concentration of acid, and presence of other ions, in the equilibrating solutions influenced pH, swelling ratio, and fracture properties of the gels. Swelling of gels decreased fracture stress (because of decreased protein network density) but caused little change to fracture strain, thus maintaining a desirable strong/elastic fracture pattern. We have shown that whey protein isolate gels can be made at pH ≤ 4.5 with a strong/elastic fracture pattern and the magnitude of this pattern can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time. Practical Application: Low-pH shelf-stable whey protein gels having the strong/elastic texture can be made by forming gels at high pH and equilibrating in acid solutions. Acid equilibration causes the gel to swell and lower the gel pH. Moreover, gel properties can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time.     

Effects of malondialdehyde-induced protein modification on water functionality and physicochemical state of fish myofibrillar protein gel

Effects of malondialdehyde (MDA)-induced modification on water distribution in fish myofibrillar proteins (MP) gels were investigated using nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). The oxidative modifications of MP gels were evaluated by surface hydrophobicity, gel strength, water holding capacity (WHC), scanning electron microscopy (SEM) and SDS-PAGE. Influence of heating procedure on water distribution and functional properties of modified MP gels was also investigated. Results from NMR and MRI indicated that the water functionality was strongly affected by the modification level, which corresponded to the changes of water holding capacity of MP upon MDA modification. Compared with unmodified MP gels, the T₂ relaxation times of modified sample increased significantly, indicating an alteration of water-protein interaction upon MDA-induced modification. The fraction of P₂₃ declined from 7.66% to 0.15% as the MDA addition increased from 0 to 50 mM. Moreover, the relaxation components T_2b disappeared with the addition of MDA mainly due to enhanced protein flexibility and surface hydrophobicity. Besides, the P₂₃ (free water) of heated MP samples increased by 5.41 times compared with that of unheated MP samples.     

Whey protein concentrate gels with honey and wheat flour

Structural and functional properties of whey protein concentrate (WPC) gels with different honey and wheat flour contents, prepared at pHs 3.75, 4.2 and 7.0, were analysed. Gel structure was observed by scanning electron microscopy. The apparent transition temperatures for protein denaturation and starch gelatinization were determined by differential scanning calorimetry. Gels were characterised through solubility assays in different extraction solutions and polyacrylamide gel electrophoresis of the soluble protein components. The firmness, elasticity, relaxation time, adhesivity and cohesiveness of gels were determined, and the water-holding capacity and superficial colour of gels were also studied. Results suggest that wheat flour could interact with whey proteins, and produces a decrease in the protein solubility of WPC gels, and in the temperature of whey protein denaturation. The effect of wheat flour on the functional properties of WPC gels was different at acidic than at neutral pH: the presence of wheat flour produced an increase in the relaxation time and in the cohesiveness of gels prepared at pH 3.75, whereas at neutral pH a decrease in both properties was observed. Honey and flour content increased the water-holding capacity and browning of WPC gels.     

低酰基结冷胶-乳清蛋白混合凝胶的凝胶特性

研究了离子种类、离子浓度和基体浓度对低酰基结冷胶/乳清蛋白混合凝胶力学性质、保水性质、光学性质和网络孔径的影响。研究发现,乳清蛋白浓度对混合凝胶凝胶特性影响较小。混合凝胶的断裂应力、断裂应变、不透明性和保水性随着低酰基结冷胶浓度的增大而增大,混合凝胶网络孔径则随着低酰基结冷胶浓度的增大而减小。离子种类和离子浓度对混合凝胶凝胶特性影响显著,混合凝胶的断裂应力和孔径随着离子浓度的增加出现了先增大后降低的变化趋势,断裂应变和保水性则随着离子浓度的增大而减小。离子浓度增大,混合凝胶的不透明指数随之升高,当离子浓度超过某一定值后,不透明指数则基本保持恒定。相对于钠离子而言,钙离子形成的凝胶更强且用量更少。钠离子和钙离子在诱导凝胶形成上不存在协同效应。     

Protein and Salt Effects on Ca2+-Induced Cold Gelation of Whey Protein Isolate

Increasing whey protein concentration (from 6 to 10% w/v) decreased gel opacity but increased gel strength and water-holding capacity (WHC). Increasing CaCl₂, concentration (from 5 to 150 mM) increased gel opacity and gel strength at the high protein concentration (i.e., 10%). However, it lowered gel strength at protein concentration > 10%. Young's modulus and distance to fracture values indicated that gels were most rigid at 30 mM CaCl₂, at which point the extent of aggregation (measured by turbidity) was the highest. Increasing CaCl₂ concentration from 5 to 150 mM slightly affected the WHC of Ca²⁺-induced gels. Protein concentration was the major factor in determining fracture properties and WHC.     

Tenderness improvement of reduced-fat and reduced-salt meat gels as affected by high pressure treating time

This study investigated the effect of high-pressure treating time (HPTT) on the tenderness of reduced-fat reduced-salt (RFRS) meat gels. The result of shear force value showed that the RFRS meat gels exhibited excellent tenderness treated with 200 MPa for 3 min. The mechanism of tenderness enhancement was revealed from the perspective of water migration characteristics in meat batters. After HPP, the characteristics of water molecules in meat batters were investigated using low-field nuclear magnetic resonance (LF-NMR) T₂ relaxometry. The datas of T₂ relaxation time and peak area indicated the free water changed into immobilized water significantly. The result of principal components analysis showed that the sample treated with a proper HPPT (3 min) was significantly different from the others. The tenderness of RFRS meat gel products was improved with the application of 200 MPa, especially treated with 3 min. The improvement of tenderness could be ascribed to the increase of immobilized water. The possible increase of the diameter in three-dimensional gel network might lead to more water molecules entrapped.     

Moisture migration in idealized bilayer systems: relationships among water-associated properties, structure, and texture

The effects of composition and thermal treatment on the water sorption and diffusional properties of idealized protein gels arranged in bilayer configurations were determined; these water binding/migration properties were related to the mechanical characteristics of the gels. Samples were prepared from whey protein concentrate (WPC), they consisted of water:WPC ratios of 1.5 to 5.67, and were thermally set for 20–60 min. Moisture migration rates from samples interfaced with filters were determined, as were moisture sorption capacities of samples immersed in water. The physical properties of the gels were assessed by uniaxial compression and microscopy. Results showed that gel strength and consequent extent of protein interaction—as affected by thermal treatment—controlled the ability of the gel structure to absorb water. Sorption was exponentially correlated with gel modulus and linearly correlated with a function of protein content, heating time, and immersion time. Rates of diffusion from interfaced gels were dependent solely on water content. It was concluded that the degree of protein interaction, whether influenced by concentration or thermal treatment, affected network extensibility and thus the capacity of the gels to act as receptors of moisture. Results have implications for the functionality of shelf-stable sandwiches and other multicomponent foods.     

Using multi-slice-multi-echo images with NMR relaxometry to assess water and fat distribution in coated chicken nuggets

Characterizing water and fat distributions in fried foods is of major importance in terms of taste and nutrition. The objective of this study was to introduce a new technique to quantify water and fat distributions of chicken nuggets coated with methylcellulose (MC) through the use of magnetic resonance imaging (MRI) and NMR relaxometry. Multi-slice-multi-echo (MSME-Spin Echo based) images were performed on a 1.03 T spectrometer for the whole chicken nuggets to obtain relaxation time distribution of different regions (core and crust) of the nugget. MSME images provided the relaxation spectra of the core and crust separately and showed that relaxation times and relative areas of the proton pools in crust and core regions are correlated (mostly, R² > 0.85) with moisture and fat content. Results demonstrated that MSME images in combination with T₂ relaxometry are appropriate tools to quantify spatial water and fat distribution in various food products.     

Gelation of Calcium-Reduced and Lipid-Reduced Whey Protein Concentrates as Affected by Total and Ionic Mineral Concentrations

Rheological and microstructural properties of five dialyzed whey protein concentrate (WPC) gels were investigated. Maximum WPC gel hardness as determined by shear stress (ST) was observed at 2.7–4.5 mM Ca and 0.6–1.1 mM Ca²⁺ concentrations with a Ca ionization of 20–25%. Gel cohesiveness by shear strain (SN) correlated with total lipid and phospholipid (PLP) concentrations and percent of lipid unsaturation. Microstructural characteristics of the gels, as determined by light microscopy (LM), confirmed their water holding capacity (WHC) and rheological properties.     

超声协同TG酶处理乳清蛋白/马铃薯蛋白水解物复合凝胶的特性

为开发乳清蛋白/马铃薯蛋白水解物复合凝胶，采用质构仪、流变仪、低场核磁和扫描电镜表征超声与TG酶处理对凝胶性质的影响。结果表明：超声协同TG酶处理凝胶的凝胶强度，胶黏性，回复性和咀嚼性低于对照组（P<0.05），但高于单独TG酶处理组（P<0.05）。在超声协同TG酶处理凝胶样品中，超声时间的增加，温度循环终点时凝胶储能模量与损耗模量增加；超声15和30 min协同TG酶处理样品的溶胀力相较于TG酶单独交联的样品提高了11.55%和55.02%，自由水含量相较TG酶单独交联样品分别提高了62.22%和111.11%。SEM微观结构表明，超声处理使凝胶中蛋白分子聚集体变大，蛋白网络致密但均一性不高；超声协同TG酶处理凝胶的聚集体较小，蛋白网络结构较为均一。研究表明超声协同TG酶交联会显著改变乳清蛋白/马铃薯蛋白水解物复合凝胶的性能及结构，为复合蛋白凝胶性质的调控提供了参考。     

漂洗工艺对秘鲁鱿鱼鱼糜凝胶特性的影响

以秘鲁鱿鱼鱼糜为原料,从漂洗液成分、漂洗液酸碱度、漂洗水温、漂洗次数等方面考察漂洗工艺对秘鲁鱿鱼鱼糜凝胶特性的影响.结果表明,随着漂洗液中钙离子浓度的增加,秘鲁鱿鱼鱼糜的凝胶强度呈先升后降的趋势,漂洗液中氯化钙浓度为4.5 mmol/L,乳酸钙浓度为2.25mmol/L时,秘鲁鱿鱼鱼麋的凝胶强度达到最大值,并且含乳酸钙的漂洗液漂洗效果优于含氯化钙的漂洗液;随着漂洗液中柠檬酸钠浓度的增加,秘鲁鱿鱼鱼糜的凝胶强度也呈现先升后降的趋势,柠檬酸钠浓度0.2%时凝胶强度达到最大值.当漂洗液pH接近中性,漂洗液温度为16℃,漂洗次数为3次时,秘鲁鱿鱼鱼糜凝胶的凝胶效果较好.正交试验发现采用1次清水2次柠檬酸钠1次氯化钙漂洗鱼糜凝胶强度达最大值.     

Dynamics of water in agar gels studied using low and high resolution 1H NMR spectroscopy

The aim of this work was to determine whether the decreased motion of water in agar gels (2.5–12.5% agar) resulted not only from the chemical interaction of the water molecules with the agar macromolecules but also from obstruction by the gel networks. Relaxation experiments were conducted using a 500‐MHz nuclear magnetic resonance (NMR) spectrometer. Diffusion experiments were conducted using a 23‐MHz NMR spectrometer with diffusion times ranging between 15 and 200 ms and a 500‐MHz NMR spectrometer with a fixed diffusion time of 10 ms. This study shows that the interaction of water with hydroxyl groups of agar macromolecules resulted in faster relaxation and slower self‐diffusion of water. The time‐dependent self‐diffusion coefficient of water provides clear evidence of the obstructive effects of the agar gel network on diffusion. This work is the first report on restricted diffusion of water in agar gel systems.     

Gelation of mixed systems whey protein concentrate–gluten in acidic conditions

Gels of whey protein concentrate (WPC)–gluten were prepared by heating WPC–gluten dispersions (10% whey protein/0–5–10% gluten protein, w/w; pH 3.75 or 4.2). Gels were characterized through solubility assays in different extraction solutions, measures of water-holding capacity (WHC), firmness, elasticity and relaxation time, and light microscopy. Differential scanning calorimetry (DSC) of WPC–gluten dispersions was also performed. Gluten increases the firmness and elasticity of gels, mainly at pH 4.2. The WHC also increases with gluten content, being higher at pH 3.75 than at pH 4.2. Solubility assays indicate that electrostatic forces, hydrophobic and H bindings would be involved in maintaining the gel structure of WPC gels at pH 3.75 and 4.2, whereas in mixed gels of WPC–gluten, the principal forces responsible for the maintenance of the gel structure at these pHs would be hydrophobic and H bindings, and in gels prepared at pH 4.2 also disulfide bonds, but in a minor extent. The presence of gluten shifts the apparent transition temperature for whey protein denaturation towards lower temperatures. Gels with gluten present a smooth network with gaps and a more elastic appearance, as observed by light microscopy.     

Effect of pectin methyl esterase (PME) and CaCl2 infusion on the cell integrity of fresh-cut and frozen-thawed mangoes: An NMR relaxometry study

This study describes the use of Nuclear Magnetic Resonance (NMR) relaxometry to understand the effect of pectin methyl esterase (PME) + CaCl2 infusion under different conditions on cell integrity of fresh-cut and frozen–thawed mangoes. Infusion experiments were performed at: atmospheric pressure, vacuum conditions of 50 kPa and 10 kPa. For NMR relaxometry experiments T₂ (spin–spin relaxation time), T₁ (spin–lattice-relaxation times) and 2D T₁–T₂ experiments were performed. Results showed that, as the severity of the vacuum treatment increased, the relaxation times changed significantly (p < 0.05). The number of compartments observed in 1-D relaxation spectra of fresh and frozen–thawed mangoes changed with different treatments. The changes in relaxation times were explained due to formation of a gel formed by the interaction of pectin and calcium. 2D T₁–T₂ relaxation maps showed that compartmentalization was retained after vacuum treatment even for frozen–thawed samples. The study showed that NMR relaxometry is a useful tool to analyze the cell integrity of mangoes exposed to different treatments.