Gelation of edible blue-green algae protein isolate (Spirulina platensis Strain Pacifica): thermal transitions, rheological properties, and molecular forces involved

Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and the development of elastic modulus of Spirulina protein gels. This is also attributed to the strong temperature dependence of hydrophobic interactions. The aggregation, denaturation, and gelation properties of Spirulina algal protein isolate are likely to be controlled from protein-protein complexes rather than individual protein molecules.     

Partition and release of 21 aroma compounds during storage of a pectin gel system

The increasing popularity of low-fat products increases the need for a better understanding of how flavor release is affected by partial substitution of fat with hydrocolloids. Partitioning and release of aroma compounds from four pectin gels with different compositions were studied with static headspace and with a model mouth. Air/product partition coefficients determine the potential extent of aroma release, and mass transfer determines the rate at which aroma compounds are released to the vapor phase. This study showed that the gel network had large effects on the partition of aroma compounds between the gel and vapor phase. The specific properties of the aroma compounds were also of importance for the air/gel partition. Storage of the four gels showed that one of the weaker gels was influencing the concentration of aroma compounds in the headspace, probably caused by formation of a denser network over time.     

Impact of destroying the structure of model gels on volatile release

The release of a strawberry aroma from different composite gels taken as models of fruit preparations and from a sucrose solution was investigated. The composition of the model systems differed with regard to the gelling agent, either pectin or carrageenan, and to the rigidity of the gel. With the use of atmospheric pressure chemical ionization-mass spectrometry, the release profiles of the aroma compounds were determined under stirring. At the same time, purge and trap measurements were performed to determine the release profiles of the aroma compounds without stirring. The comparison of the patterns obtained using these two complementary methods made it possible to determine how the structure of the matrix, the mechanical treatment, and the properties of the aroma compound affect aroma release. A far greater proportion of the aroma compounds was retained in the fruit preparation systems than in the sucrose solution. The different release profiles could be interpreted in terms of the volatility of the aroma compounds and of their diffusion through the gels.     

Multivariate analysis of the influence of pectin,white syrup,and citric acid on aroma concentration in the headspace above pectin gels

Pectin gels consist of polysaccharide networks surrounded by water. The gel networks can prevent release of aroma molecules from the gel to the gas phase above. In this study static headspace measurements were performed to correlate aroma concentration in the gas phase above pectin gels to different amounts of the gel ingredients. As a consequence, aroma concentration in the headspace in relation to gel texture, as characterized by rheology measurements, was also studied. Aroma concentration in the headspace above strong gels was low, due to entrapment of aroma molecules within the gel structure. Viscous solutions generally gave a high aroma concentration in the headspace, but owing to a complex matrix, this was lowered when large amounts of the gel ingredients were added. However, a high correlation between interaction terms and square terms of design variables and rheology parameters with aroma compounds indicated nonlinear and complex relationships.     

Monovalent salt-induced gelation of enzymatically deesterified pectin

Pectin gels were induced by monovalent salts (0.2 M) concurrently with deesterification of high methoxy pectin using a salt-independent orange pectin methylesterase (PME). Constant pH was maintained during deesterification and gelation. If salt or PME was absent, the pectin did not form a gel. The gel strength was influenced by both pH and species of monovalent cation. At pH 5.0, the pectin gel induced by KCl was significantly stronger than the NaCl-induced gel. In contrast, a much stronger gel was produced in the presence of NaCl as compared to KCl at pH 7.0. LiCl did not induce pectin gelation at either pH. Molecular weights of pectins increased from 1.38 x 10(5) to 2.26 x 10(5) during NaCl-induced gelation at pH 7. One proposal to explain these pectin molecular weight changes is a hypothetical PME transacylation mechanism. However, these pectin molecular weight changes can also be explained by metastable aggregation of the enzymatically deesterified low methoxy pectin. We postulate that gelation was induced by a slow deesterification of pectin under conditions that would normally salt out (precipitate) low methoxy pectin in the absence of PME.     

Interactions of high methoxyl pectin with whey proteins at oil/water interfaces at acid pH

The interactions between whey protein isolate (WPI) and high methoxyl pectin (HMP) at pH 3.5 were investigated in situ using ultrasound (US) and diffusing wave spectroscopy (DWS). HMP was added to 10% oil-in-water emulsions containing 1% WPI. At neutral pH, no protein-pectin interactions were observed as both molecules are negatively charged, while at pH 3.5 bridging flocculation occurred via electrostatic interactions. Four different stages were distinguished during the addition of HMP in WPI-stabilized emulsions at pH 3.5. At a concentration below a critical value, no interactions were observed. At concentrations >0.02% HMP, a change in the l factor indicated a change in the ordering of the emulsion droplets, influenced by long-range interactions. At higher concentrations (in the range between 0.04 and 0.06% HMP), attenuation showed significant changes in the surface of the oil droplets, changes which affected the droplet-droplet interactions. At pectin concentrations >0.05%, attenuation of sound and 1/l* decreased, while velocity of sound and particle size increased, as a result of bridging flocculation. These results demonstrated for the first time that methods such as US and DWS combined permit the observation of the early stages of the interactions between two biopolymers at the interface. This is significant in light of increasing efforts in engineering complex interfacial layers.     

Interactions of soy protein fractions with high-methoxyl pectin

A protein-binding technique was employed to visualize, using scanning electron microscopy, the soy protein as well as the association between HMP and soy protein fractions. Image analysis indicated that at pH 7.5 and 3.5 soy protein isolate showed a bimodal distribution of sizes with an average [ d(0.5)] of about 0.05 microm, but at pH 3.8 the proteins formed larger aggregates than at high pH. Addition of HMP at pH 3.8 changed the surface charge of the particles from +20 to -15 mV. A small addition of HMP caused bridging of the pectin between soy protein aggregates and destabilization. With sufficient HMP, the suspensions showed improved stability to precipitation. The microscopy images are the first direct evidence of the interactions between soy proteins with high-methoxyl pectin (HMP).     

Diffusion of aroma compounds in stirred yogurts with different complex viscosities

To better understand aroma release in relation to yogurt structure and perception, the apparent diffusivity of aroma compounds within complex dairy gels was determined using an experimental diffusion cell. Apparent diffusion coefficients of four aroma compounds (diacetyl, ethyl acetate, ethyl hexanoate, and linalool) at 7 degrees C in yogurts (varying in composition and structure) ranged from 0.07 x 10 (-10) to 8.91 x 10 (-10) m (2) s (-1), depending on aroma compounds and on product structure. The strong effect of yogurt fat content on the apparent diffusivity of hydrophobic compounds was revealed (15-fold and 50-fold decreases in the apparent diffusion coefficient of linalool and ethyl hexanoate, respectively). Protein composition seemed to have a greater effect than that of mechanical treatment. However, variations in the apparent diffusion coefficient for the considered products remained limited and cannot completely explain differences in flavor release and in perception that were previously observed.     

Complex viscosity induced by protein composition variation influences the aroma release of flavored stirred yogurt

Dairy protein composition is known to influence the structure and the texture characteristics of yogurt. The objective of the present work was therefore to investigate the impact of protein composition, at a constant protein level, on the physicochemical properties of 4% fat flavored stirred yogurt and, more specifically, on the rheological properties, the microstructure, and the aroma release. The results showed that caseinate-enriched yogurt generally presented changes in their microstructure network and had a higher complex viscosity than whey protein-enriched yogurt. To a lesser extent, the release of the majority of aroma compounds was lower in caseinate-enriched yogurt. It was therefore possible to quantify physicochemical interactions between aroma compounds and proteins. The influence of gel structure on the flavor release was observed and was in agreement with sensory characteristics previously studied for these products.     

Raman spectroscopic study of changes in fish actomyosin during setting.

Actomyosins (AMs) isolated from tilapia, lemon sole, ling cod, and rock fish were heated at 40 degrees C, and structural changes in AMs were investigated using Raman spectroscopy to elucidate low-temperature gelling phenomenon, that is, "setting", of surimi. The following conformational transitions were observed in lemon sole, ling cod, and rock fish gels during setting: a slow unfolding of alpha-helix and exposure of hydrophobic amino acid residues occurring in long-time incubation at 40 degrees C, thereby forming hydrophobic interactions among AM molecules. In addition, the most frequent conformation in disulfide bonds was gauche-gauche-trans (g-g-t) form in the set gel. On the other hand, tilapia AM did not form a gel with heating at 40 degrees C, its alpha-helical structure in the myosin being far more stable than that of the other species. The heat resistance of the tight alpha-helix may prevent the gelation of tilapia AM. It is, therefore, likely that the unfolding of the alpha-helix of myosin is a prerequisite for gelation of AM during setting.     

Changes in macroscopic viscosity do not affect the release of aroma aldehydes from a pectinaceous food model system of low sucrose content

The effects of pectin and viscosity on the release of a systematic series of aldehydes (alkanals, methyl-alkanals, alkenals, and alkandienals) were studied in a food model system of low sucrose content (10% w/w). The viscosity was varied by adding different amounts of Ca(2+) (0, 13.5, and 27 mg/g pectin) to the model system of constant pectin concentration (0.4% w/w). Air-liquid partition coefficients, K (37 degrees C), of the aroma compounds were determined in aqueous and pectin-thickened solutions. Diffusivities of the aroma compounds in water and three pectin-thickened solutions were estimated from release rate constants that were obtained via timed collection of volatiles in the gas phase and quantifications by dynamic headspace-gas chromatography. The partition coefficients increased as the carbon chain increased within each homologous series. Overall, no significant difference was found between partition coefficients of aldehydes in water and in pectin solutions except for 2-methyl-propanal and butanal that showed higher K values when pectin was present. Furthermore, the diffusional properties of the model system with a constant pectin level (0.4% w/w) remained constant when the viscosity was increased from 0.001 to 150 Pa s. It was concluded that neither pectin nor alterations in macroscopic viscosity as such influenced the release of aldehydes from the pectin-thickened food model system.     

Uniaxial compression of thermal gels based on microfluidized blends of WPI and heat-denatured WPI.

The effect of heat-denatured whey protein isolate (dWPI)/whey protein isolate (WPI) ratio (0-0.6), microfluidization pressure (0-1000 bar), and number of passes (1-10) on the uniaxial shear stress at 10% (sigma(10)) and 80% (sigma(80)) relative deformation of dWPI/WPI heat-induced gels (14% total protein, w/w) was studied. No correlation between the average diameter of aggregates and the dWPI/WPI ratio, microfluidization pressure, or number of passes was found. However, increasing the microfluidization pressure or the number of passes resulted in a narrower size distribution of aggregates. Increasing the dWPI/WPI ratio and the number of passes resulted in a decrease and an increase of gel hardness, respectively. The results were interpreted in terms of more random aggregation/gelation of proteins in the presence of aggregates that could result in localized heterogeneities into gels and more dissipation of the deformation energy during compression. The positive effect of the number of passes on the gel hardness was also considered to be due to a more homogeneous aggregation/gelation of proteins in the presence of smaller aggregates.     

Effects of heat treatment and pectin addition on beta-lactoglobulin allergenicity

The specific effects of heat treatment and/or addition of low/high-methylated pectin (LMP/HMP) on the allergenicity of beta-lactoglobulin (beta-Lg) and its hydrolysis products were investigated through a two-step in vitro digestion approach. beta-Lg was first hydrolyzed by pepsin and then by a trypsin/chymotrypsin (T/C) mixture done in a dialysis bag with a molecular weight cutoff of 1000. The protein digestion was followed by SDS-PAGE electrophoresis performed on each digestion product, and their in vitro allergenicity was analyzed by immunoblotting. Such procedure was applied on beta-Lg samples mixed with the two kinds of pectin before or after heating (80 degrees C, 25 min) to determine the respective impact of heat treatment and pectin addition. Heat denaturation improved significantly the susceptibility of beta-Lg against the pepsin and the T/C. This effect, which was coupled to a reduction in immunoreactivity of the digested beta-Lg, appeared to be distinctively modulated by LMP and HMP. Through nonspecific interaction with the beta-Lg, pectin could reduce the accessibility of cleavage sites and/or epitope sequences. This mechanism of action is discussed in relation to the intra- and intermolecular interactions between beta-Lg and pectin initiated under the experimental conditions.     

Heat-induced gel formation by soy proteins at neutral pH

Heat-induced gel formation by soy protein isolate at pH 7 is discussed. Different heating and cooling rates, heating times, and heating temperatures were used to elucidate the various processes that occur and to study the relative role of covalent and noncovalent protein interactions therein. Gel formation was followed by dynamic rheological measurements. Heat denaturation was a prerequisite for gel formation. The gelation temperature (84 degrees C) was just above the onset denaturation temperature of glycinin. The stiffness of the gels, measured as the elastic modulus, G', increased with the proportion of denatured protein. An increase in G' was also observed during prolonged heating at 90 degrees C. This increase is explained by the occurrence of rearrangements in the network structure and probably also by further incorporation of protein in the network. The increase in G' upon cooling was thermoreversible indicating that disulfide bond formation and rearrangements do not occur upon cooling.     

Evaluation of Compatibility of Rice Starch and Pectins by Glass Transition and Sub‐Tg Endotherms and the Effect of Compatibility on Gel Viscosity and Water Loss

Differences in molecular structure and hydrophilicity may affect the compatibility of food components in a highly concentrated solution. Mixtures of TNuS19 rice starch (RS) and pectins with three different degrees of esterification (22, 64, and 92%) were used as a model system to evaluate the components' compatibility in a low‐moisture system. When analyzed individually by differential scanning calorimetry (DSC), RS, low methoxyl pectin (LMP), intermediate methoxyl pectin (IMP), and high methoxyl pectin (HMP) showed the presence of a glass transition temperature (T_g) at 75.2, 96.2, 96.4, and 93.5°C, respectively. Among mixtures, the compatible RS‐HMP exhibited only a single T_g between the T_g values of the two components, whereas the incompatible RS‐LMP showed two T_g values that were close to those of the individual components. The sub‐T_g endotherms of all three mixtures (1:1) were lower than the means of the corresponding components. The degree of decrease was more pronounced in the RS‐HMP mixture than in the others. The above results imply that the interaction, which led to close contact between side chains of the two components, was more intense in the compatible RS‐HMP mixture than in the RS‐IMP and RS‐LMP mixtures. The decrease of the sub‐T_g endotherm can be used as an index to evaluate the degree of compatibility as well as the interaction occurring between the two molecules. The above findings were further verified by dynamic mechanical analyses. Both viscosity and water retention of the compatible RS‐HMP mixed gel were significantly higher than those of the RS‐IMP and RS‐LMP mixed gels. This evidence further suggests that RS and HMP are compatible and exhibit a strong intermolecular interaction that increases gel viscosity and decreases water loss during high‐temperature heating.     

Hydrolytic and oxidative stability of L-(+)-ascorbic acid supported in pectin films: influence of the macromolecular structure and calcium presence

The hydrolytic and oxidative stability of L-(+)-ascorbic acid (AA) into plasticized pectin films were separately studied in view of preserving vitamin C activity and/or to achieve localized antioxidant activity at pharmaceutical and food interfaces. Films were made with each one of the enzymatically tailored pectins (50%, 70%, and 80% DM; Cameron et al. Carbohydr. Polym.2008, 71, 287-299) or commercial high methoxyl pectin (HMP; 72% DM). Since AA stability was dependent on water availability in the network, pectin nanostructure affected the AA kinetics. Higher AA retention and lower browning rates were achieved in HMP films, and calcium presence in them stabilized AA because of higher water immobilization. Air storage did not change AA decay and browning rates in HMP films, but they significantly increased in Ca-HMP films. It was concluded that the ability of the polymeric network to immobilize water seems to be the main factor to consider in order to succeed in retaining AA into film materials.     

Physical and chemical interactions in cold gelation of food proteins

pH-Induced cold gelation of whey proteins is a two-step process. After protein aggregates have been prepared by heat treatment, gelation is established at ambient temperature by gradually lowering the pH. To demonstrate the importance of electrostatic interactions between aggregates during this latter process, beta-lactoglobulin aggregates with a decreased iso-electric point were prepared via succinylation of primary amino groups. The kinetics of pH-induced gelation was affected significantly, with the pH gelation curves shifting to lower pH after succinylation. With increasing modification, the pH of gelation decreased to about 2.5. In contrast, unmodified aggregates gel around pH 5. Increasing the iso-electric point of beta-lactoglobulin via methylation of carboxylic acid groups resulted in gelation at more alkaline pH values. Comparable results were obtained with whey protein isolate. At low pH disulfide cross-links between modified aggregates were not formed after gelation and the gels displayed both syneresis and spontaneous gel fracture, in this way resembling the morphology of previously characterized thiol-blocked whey protein isolate gels (Alting, et al., J. Agric. Food Chem. 2000, 48, 5001-5007). Our results clearly demonstrate the importance of the net electric charge of the aggregates during pH-induced gelation. In addition, the absence of disulfide bond formation between aggregates during low-pH gelation was demonstrated with the modified aggregates.     

Effects of sugars on whey protein isolate gelation

Whey protein isolate (WPI) gels were prepared from solutions containing ribose or lactose at pH values ranging from 6 to 9. The gels with added lactose had no color development, whereas the gels with added ribose were orange/brown. Lactose stabilized the WPI to denaturation, which increased the time and temperature required for gelation, thus decreasing the fracture modulus of the gel compared to the gels with added ribose and the gels with no sugar added. Ribose, however, favored the Maillard reaction and covalent cross-linking of proteins, which increased gel fracture modulus. The decreased pH caused by the Maillard reaction in the gels containing ribose occurred after protein denaturation and gelation, thus having little if any effect on the gelation process.     

Controlled proteolysis and the properties of milk gels

Milk gels induced by partial proteolysis of the kappa-casein followed by acidification were studied, and their gelation behavior was compared to that of milk gels induced by simultaneous acidification and renneting, using dynamic rheology. There were generally two stages (at pH values below and above 5.0) in the gelation of the milk whose kappa-casein had been partially proteolyzed and acidified. The onset of gelation was at higher pH as the degree of kappa-casein proteolysis increased. The development of G' immediately after the onset of gelation was faster in the milk gels induced by simultaneous acidification and renneting, because of the continuing kappa-casein proteolysis. Preheat treatment caused the onset of gelation to occur at higher pH than for unheated milk. However, the maximum tan delta during gelation always occurred at the same pH (for a given concentration of acidulant), and its value and position were independent of the extent of renneting and whether the milks had been heat treated. The results are discussed in terms of the interactions between casein micelles occurring during gelation.