Mechanical characterization of network formation during heat-induced gelation of whey protein dispersions

The formation of gel network structures during isothermal heating of whey protein aqueous dispersions was probed by mechanical spectroscopy. It was anticipated that the pathway of the sol-to-gel transition of whey protein dispersions is quite different from that of ordinary cross-linking polymers (e.g., percolation-type transition), since aqueous solutions of native whey proteins have been shown to be highly structured even before gelation, in our previous study. At 20 degrees C, aqueous dispersions of beta-lactoglobulin, the major whey protein, and those of whey protein isolate (WPI), a mixture of whey proteins, exhibited solid-like mechanical spectra, i.e., the predominant storage modulus G' over the loss modulus G", in a certain range of the frequency omega (1-100 rad/s), regardless of the presence or absence of added NaCl. The existence of the added salt was, however, a critical factor for determining transitions in mechanical spectra during gelation at 70 degrees C. beta-Lactoglobulin dispersions in 0.1 mol/dm(3) NaCl maintained the solid-like nature during the entire gelation process and, after passing through the gelation point, satisfied parallel power laws (G' approximately G" approximately omega(n)) that have been proposed for a critical gel (i.e., the gel at the gelation point) that possesses a self-similar or fractal network structure. In contrast, beta-lactoglobulin dispersions without added salt exhibited a transition from solid-like [G'(omega) > G"(omega)] to liquid-like [G'(omega) < G"(omega)] mechanical spectra before gelation, but no parallel power law behavior was recognized at the gelation point. During extended heating time (aging), beta-lactoglobulin gels with 0.1 mol/dm(3) NaCl showed deviations from the parallel power laws, while spectra of gels without added NaCl approached the parallel power laws, suggesting that post-gelation reactions also significantly affect gel network structures. A percolation-type sol-to-gel transition was found only for WPI dispersions without added salt.     

Functional properties of hemoglobin immobilized in coacervates prepared from gelatin A and polyanionic carbohydrates

Complex coacervation is a phenomenon of phase separation that may occur in a solution of positively and negatively charged polyions. The resulting two phases are distinguished by the total concentration of both polyions, with the concentrated phase often containing vesicular structures composed of the two polyelectrolytes. We have used this phenomenon in an attempt to-prepare a hemoglobin-based red blood cell analog. Hemoglobin-containing coacervate vesicles have been prepared from gelatin A and the polyanionic carbohydrates acacia, pectin, or dextranstilfate. Hemoglobin seems to be anchored into the vesicle walls through interaction of its polyanion binding site with the negatively charged residues on the carbohydrates. Oxygen binding by the immobilized HbA is reversible and cooperative, with p50 values at 20 degrees C of 2.8, 6, and 24 mm Hg for the acacia- (pH 7.5), pectin- (pH 6.6), and dextransulfate-(pH 6.6) derived coacervates. Kinetic studies on CO binding show that the rate of CO uptake by the coacervates (t((1/2)) = 13-27 ms at 0.5 mM CO) is similar to that of human erythrocytes.The HbA-containing coacervates slowly dissolve in isotonic salt solutions (145 mM NaCl, pH 7.4), but they can be stabilized by treatment with glutaraldehyde. Oxygen binding by HbA incorporated into the stabilized coacervates derived from dextran sulfate is very similar to oxy gen binding by human red blood cells: p50 = 26 mm Hg and n = 1.89 at 37 degrees C in isotonic salt. These results show how a novel approach, based on an old concept, has led to the preparation of immobilized HbA, with functional properties similar to those of intraerythrocytic HbA.     

Microstructure and kinetic rheological behavior of amidated and nonamidated LM pectin gels

The microstructure, kinetics of gelation, and rheological properties have been investigated for gels of nonamidated pectin (C30), amidated pectin (G), and saponified pectin (sG) at different pH values, both with and without sucrose. The low-methoxyl (LM) pectin gels were characterized in the presence of Ca(2+) by oscillatory measurements and transmission electron microscopy (TEM). The appearance of the gel microstructure varied with the pH, the gel structure being sparse and aggregated at pH 3 but dense and somewhat entangled at pH 7. During gel formation of pectins G and C30 at pH 3 there was a rapid increase in G' initially followed by a small increase with time. At pH 7 G' increased very rapidly at first but then remained constant. The presence of sucrose influenced neither the kinetic behavior nor the microstructure of the gels but strongly increased the storage modulus. Pectins G and C30 showed large variations in G' at pH values 3, 4, 5, and 7 in the presence of sucrose, and the maximum in G' in the samples occurred at different pH values. Due to its high Ca(2+) sensitivity, pectin sG had a storage modulus that was about 50 times higher than that of its mother pectin G at pH 7.     

Construction of viscoelastic biocompatible films via the layer-by-layer assembly of hyaluronan and phosphorylcholine-modified chitosan

Films of hyaluronan (HA) and a phosphorylcholine-modified chitosan (PC-CH) were constructed by the polyelectrolyte multilayer (PEM) deposition technique and their buildup in 0.15 M NaCl was followed by atomic force microscopy, surface plasmon resonance spectroscopy (SPR), and dissipative quartz crystal microbalance (QCM). The HA/PC-CH films were stable over a wide pH range (3.0-12.0), exhibiting a stronger resistance against alkaline conditions as compared to HA/CH films. The loss and storage moduli, G' and G", of the films throughout the growth of eight bilayer assemblies were derived from an impedance analysis of the QCM data recorded in situ. Both G' and G" values were one order of magnitude lower than the moduli of HA/CH films. The fluid gel-like characteristics of HA/PC-CH multilayers were attributed to their high water content (50 wt %), which was estimated by comparing the surface coverage values derived from SPR and QCM measurements. Given the versatility of the PEM methodology, HA/PC-CH films are attractive tools for developing biocompatible surface coatings of controlled mechanical properties.     

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.     

Nutrition and growth of the moderately halophilic bacteria Micrococcus morrhuae K-17 and Micrococcus luteus K-15

Chemically defined media have been developed for the growth of two moderately halophilic bacteria, Micrococcus morrhuae K-17 and Micrococcus luteus K-15. M. morrhuae K-17 grows well in a synthetic medium (SM-1) which contains a number of salts, 0.21 M KCl, 2 M NaCl, D-mannose, five vitamins and ten amino acids. The synthetic medium (SM-2) for M. luteus K-15 contains a number of salts, 0.21 M KCl, 1 M NaCl, D-fructose, nine vitamins and nine amino acids. Nutritional studies show that M. morrhuae K-17 can utilize a large number of organic compounds as carbon and energy source while the ability of M. luteus K-15 in utilizing the organic compounds is rather limited. The minimum salt requirement is 0.5 M NaCl for both strains when growth at the optimum temperature of 30 degrees C. However, this requirement can be lowered to 0.2 M in M. luteus K-15 when grown at a lower temperature of 25 degrees C. It is concluded that the ability to grow in a wider range of salt concentrations in response to temperature is species specific in moderate halophiles. The salt range for growth to occur can be extended when cells of both strains are grown in complex medium which might provide the amino acids and growth factors that cannot be synthesized by these strains at high salt concentrations.     

Heat-induced gelation of bovine serum albumin/low-methoxyl pectin systems and the effect of calcium ions

Influence of low-methoxyl pectin (LM pectin) and calcium ions (3 mM) on mechanical behavior and microstructure of bovine serum albumin (BSA) gels (pH 6.8, in 0.1 M NaCl) was evaluated. Protein and LM pectin concentrations were fixed at 2, 4, and 8 wt % and 0.21, 0.43, and 0.85 wt %, respectively. Rheological measurements and confocal laser scanning microscopy coupled with texture image analysis by use of the co-occurrence method were performed. Heat treatment of BSA/LM pectin mixtures induced protein gelation and a phase separation process between the two biopolymers, which was kinetically trapped. Calcium ions induced pectin gelation and modified BSA gel properties. Depending on biopolymer concentrations, a balance between pectin and/or protein gel contribution on final gel strength exists. The microstructures of the mixed systems in the presence of calcium can be interpreted as interpenetrated structures. Texture image analysis allowed one to classify more precisely the different microstructures observed in relation with mechanical properties.     

Association of the radiosensitizers metronidazole and misonidazole (RO-07-0582) with bovine serum albumin. Effect of urea and ionic strength

The stability of association of nitroimidazole radiosensitizers (metronidazole and misonidazole) with bovine serum albumin (BSA) was examined in aqueous solutions by 1H n.m.r. spectroscopy in the presence of urea (0-8M) as denaturant, or high salt concentration (NaCl0-5M). A broadening of n.m.r. lines of the two radiosensitizers observed in the presence of BSA disappeared with increasing urea concentration. An especially large narrowing effect was observed for the lines attributed to the methylene group near to the hydroxyl in the side chain of misonidazole. The results suggest a release of both radiosensitizers from their binding sites on unfolding by urea of the polypeptide chain of BSA. The increase of ionic strength I caused a monotonic enhancement of broadening by BSA of the metronidazole lines. For misonidazole, the enhancement of broadening was observed at values of I greater than 1, but at low (less than 1 M) concentrations of NaCl the broadening disappeared. Thus, the results obtained in the systems with salt reflect quantitative changes in hydrophobic and hydrogen-bonded contributions to binding of aliphatic moieties of radiosensitizers to BSA.     

盐增强培养对弗氏链霉菌产新霉素的影响

目的:弗氏链霉菌(Streptomyces fradiae)作为氨基糖苷类抗生素新霉素的主要生产菌株,其新霉素B具有抗菌活性强、抗癌、抗HIV等作用,提高新霉素B的效价具有重要意义.方法:在满足微生物正常生长所需盐离子的条件下,通过盐增强培养的方式向培养基中添加不同种类、浓度无机盐来改变细胞壁附近的理化特性、渗透压以及...     

Enhancement of chymotrypsin-inhibitor/substrate interactions by 3 M NaCl

A series of 16 bovine pancreatic trypsin inhibitor variants mutated at the P(1) position of the binding loop and seven tetrapeptide p-nitroanilide (pNa) substrates of the general formula: suc-Ala-Ala-Pro-Aaa-pNa (where Aaa denotes either: Phe, Arg, Lys, Leu, Met, Nva, Nle) were used to investigate the influence of high salt concentration on the activity of bovine chymotrypsin. The increase of the association constant (K(a)) and the specificity index (k(cat)/K(m)) in the presence of 3 M NaCl highly depends on the chemical nature of the residue at the P(1) position. The highest increase was observed for inhibitors/substrates containing the basic side chains at this site. Surprisingly, for the remaining 13 residues the observed salt effect is not correlated with any side chain properties. In particular, there is a lack of correlation between the accessible non-polar surface area and the magnitude of the salt effect. It suggests that salt-induced increase of the K(a) and k(cat)/K(m) values is not caused by the enhancement of the hydrophobic interactions in chymotrypsin-inhibitor/substrate complex. Moreover, the increase of the K(a) and k(cat)/K(m) values occurs only in the presence of Na(+) ions, while K(+) and Li(+) ions do not change the activity of chymotrypsin. Additionally, the activities of two other proteinases: bovine trypsin and Streptomyces griseus proteinase B were tested in the presence of 3 M NaCl using their specific substrates. The activity of both enzymes was almost not affected by the presence of high NaCl concentration.     

Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Characterization of beta-lactoglobulin A gelation in the presence of sodium caprate by ultrasound spectroscopy and electron microscopy

The effect of sodium caprate (a fatty acid salt) on the formation of beta-lactoglobulin A gels was studied at constant temperature (30 or 35 degrees C) using ultrasonic spectroscopy. During incubation at these temperatures, ultrasonic attenuation increased with the addition of sodium caprate, and reached a plateau after 5-7 h of incubation. Comparing beta-lactoglobulin A with and without sodium caprate, a decrease in net ultrasonic velocity was observed. These results suggested that aggregation occurred during incubation with sodium caprate, and the sample showed an increase in compressibility. Transmission electron microscopy with negative staining showed the formation of filamentous aggregates of beta-lactoglobulin A at around 3-4.5 h of incubation with sodium caprate. These results demonstrated that sodium caprate induced the formation of structures with unique gel properties compared to those formed by heating beta-lactoglobulin in the presence of NaCl alone.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

The stabilization of beta-lactoglobulin by glycine and NaCl

Effect of glycine and NaCl on the thermal denaturation of beta-lactoglobulin was examined. The results showed that the transition temperature of beta-lactoglobulin is increased by the addition of glycine and NaCl at 0.5 and 1M. This observed stabilization by glycine and NaCl was interpreted in terms of their favorable interactions with the native state of beta-lactoglobulin and unfavorable interactions with the denatured state.     

Gelation of globular proteins: effect of pH and ionic strength on the critical concentration for gel formation. A simple model and its application to beta-lactoglobulin heat-induced gelation.

The influence of pH (2-9) and ionic strength (0-0.14 M NaCl) on the sol-gel transition of beta-lactoglobulin was investigated in order to determine the critical gel concentration (C0). The concentration necessary to form a gel near the isoelectric pH remains approximately constant (approximately 1% w/v) independently of the ionic strength. At other pH values, the higher the ionic strength is, the lower the protein concentration must be to form a gel. A theoretical model to relate the effect of the intensity and the range of electrostatic interactions on the critical concentration (C0) is proposed and fits reasonably with the experimental results.     

Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.     

Salt induced conformational transition between A and Z forms of r(CGCGCG) as revealed by a Raman spectroscopic study

Solution conformation in different conditions of r(CGCGCG) has been studied by a Raman spectroscopic method. In NaCl solution, r (CGCGCG) takes only an A-form duplex in which guanosine and cytidine have C3'endo-anti conformation even at 5M salt concentration. In much higher ionic strength condition (5M NaCl plus 1M MgCl2 or 6M NaClO4), it undergoes a transition to a left-handed Z-form. The Raman spectrum of the Z-form RNA was found to be very similar to that of Z-form DNA, suggesting that Z-RNA involves a C3'endo-syn guanosine and an in between form of C2'endo-Cl'exo-anti cytidine.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force