Phase equilibria and gelation in gelatin/maltodextrin systems — Part III: phase separation in mixed gels

Co-gels of potato maltodextrin Paselli SA-6 with gelatin were prepared by rapid quenching of mixed solutions from 90°C. At fixed setting temperature and fixed concentration of gelatin, the time required to form a self-supporting network showed an initial steady decrease with increasing concentration of SA-6 (as expected from polymer exclusion), but then increased dramatically before again decreasing. The interpretation of this behaviour as phase inversion from a gelatin-continuous network with SA-6 inclusions to a (more slowly-forming) SA-6 network with gelatin inclusions was confirmed by differential scanning calorimetry (showing both components melting separately, with no evidence of specific interaction), mechanical spectroscopy (showing that the mixed gel network was destroyed completely by melting of the gelatin component at low concentrations of SA-6, but only weakened at SA-6 concentrations above the inversion point) and by light microscopy (showing the expected changes in distribution of the two polymers).

In similar studies using the faster-gelling potato maltodextrin Paselli SA-2, microscopy and gel-melting profiles again showed phase-inversion from a gelatin-continuous network at low concentrations of SA-2 to a maltodextrin-continuous network at higher concentrations. Inversion, however, occurred at a lower concentration of maltodextrin than in the gelatin/SA-6 systems, and the accompanying change in gelation rate was confined to a sharp decrease in the dependence of gel-time on SA-2 concentration.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part IV: composition-dependence of mixed-gel moduli

The storage moduli (G′) of phase-separated co-gels formed by quench-cooling mixed solutions of gelatin and potato maltodextrin (Paselli SA-2 and SA-6) have been related quantitatively to the experimentally-determined concentration-dependence of G′ for the constituent polymers. Distribution of water between the phases was examined explicitly by using polymer blending laws to derive calculated moduli for gelatin-continuous and maltodextrin-continuous networks over the entire range of solvent partition. Allowance was made for the direct contribution of polymer chains, and for density differences between the phases, in calculating relative phase-volumes. The effect of gel formation prior to phase-separation was calculated using classical theory for network de-swelling. Good agreement with observed moduli for more than 30 gelatin/maltodextrin combinations was achieved using a single adjustable parameter, p (the ratio of solvent to polymer in the gelatin phase divided by the same ratio for the maltodextrin phase), with an optimum value of p ≈ 1·8 for both SA-6 and SA-2.     

Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels

The kinetics of phase separation and gel formation of gelatin/maltodextrin mixtures have been studied using confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), stereological image analysis and rheology. The quantified microstructural parameters were the volume-weighted mean volume and the interfacial area. The temperature of phase separation was defined as the temperature where the first signs of phase separation were observed in CLSM. The gelatin concentration varied between 4 (wt.) and 5% and the maltodextrin concentration varied between 2 and 6%. Maltodextrin was labelled covalently with RITC to improve the contrast between the gelatin phase and the maltodextrin phase. The solutions were cooled from 60 to 10 degrees C, and the cooling rates used were 0.4, 1 and 3 degrees C/min. All systems were found to be gelatin continuous under the experimental conditions used. The results showed that the temperature of phase separation (TPS) increased both with the gelatin concentration and the maltodextrin concentration, but particularly with the former. The size of the maltodextrin inclusions increased with TPS, and the interfacial area decreased with increasing TPS. The diameter of the maltodextrin inclusions varied between 1.6 and 8.5 microm at 1 degrees C/min. The size of the maltodextrin inclusions was found to increase with decreasing cooling rate and was largest at 0.4 degrees C/min. The TPS was compared with the gelation temperature (Tgel) at three different concentrations of gelatin and maltodextrin (4/3, 4/5, 5/5%). CLSM micrographs and TEM micrographs showed that these three concentrations of gelatin and maltodextrin had different microstructures. At a TPS above Tgel (5/5%), the phase separation proceeded faster than the gel formation and the microstructure had few, large maltodextrin inclusions and a clean continuous gelatin phase. At a TPS comparable with Tgel (4/5%), phase separation occurred during gel formation, which led to a varying microstructure and competition between the phase separation and the gel formation. At a TPS below Tgel (4/3%), gel formation proceeded faster than the phase separation and the microstructure had many, small inclusions and a diffuse microstructure, and the phase separation was incomplete. It was established that the microstructure was determined by the relative rates of the phase separation and the gel formation. Three different zones of phase separation could be distinguished based on comparisons of TPS and Tgel, and results from CLSM, TEM and image analysis.     

Viscoelastic and Thermal Properties of Collagen–Xanthan Gum and Collagen–Maltodextrin Suspensions During Heating and Cooling

The purpose of this work was to investigate the viscoelastic properties of aqueous suspensions of crude collagen powder extracted from bovine hides and nonsubmitted to the hydrolysis reaction that leads to gelatin. The studied variables included the collagen concentration and the addition of xanthan gum or maltodextrin at varied concentrations during heating/cooling of the mixtures. Differential scanning calorimetry thermograms showed that the addition of polysaccharides decreased the endothermic peak areas observed at the denaturation temperature of collagen. The rheological properties of the pure collagen suspensions were highly dependent on concentration: 4% and 6% collagen suspensions presented a great increase in the storage modulus after heating/cooling, whereas for concentrations of 8% and 10% G′ decreased during heating and did not recover its original value after heating/cooling. The frequency sweeps showed that the thermal treatment was responsible by the strengthening of the interactions that formed the polymer network. Addition of 0.1% xanthan gum to collagen suspensions increased the gel strength, especially after heating/cooling of the system, whereas increasing gum concentration to 0.3% resulted in a weaker gel, which could indicate thermodynamic incompatibility between the biopolymers. Mixtures of collagen and maltodextrin resulted in more fluid structures than those obtained with pure collagen at the same collagen concentration and the range of temperatures in which these mixtures behaved as a gel decreased with increasing concentrations of both collagen and maltodextrin, suggesting incompatibilities between the biopolymers.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part I: gelation of individual components

As a prelude to studies of co-gelation with galatin, the gelation behaviour of Paselli maltodextrins SA-6 and SA-2 (DE ≈ 6 and 2, respectively) was mapped out over the experimentally-accessible range of temperature (T) and concentration (c), using a simple visual method to determine the time required for formation of a self-supporting network (t_g). For both samples, log t_g decreased linearly with log c and increased linearly with T. At equivalent temperatures and concentrations, SA-2 gelled between 20 and 60 times faster than SA-6.

Selected samples were monitored more rigorously by mechanical spectroscopy, taking t_g as the time at which elastic response (G′) became greater than viscous response (G″). In all cases the values of t_g obtained by this procedure were lower than those from visual inspection, by a constant factor of about 3·4.

The concentration-dependence of gel moduli (G′) for SA-2 and for gelatin (second-extract limed ossein; LO-2) fitted accurately to the form anticipated from cascade theory for normal polymer networks. For SA-6, by contrast, log G′ varied linearly with log c over the entire range at which measurements could be made, indicating a different mechanism of structure-formation (such as the agglomeration of short, aggregated helices).     

Measurement of Temperature-dependent Ice Fraction in Frozen Foods

Ice fraction was measured for solutions containing glucose, sucrose, gelatin, and egg albumin at various concentrations at temperatures from 0 to -20°C. For glucose and sucrose solutions, the ice fraction was accurately measured from phase diagram, which could be interpreted by solution thermodynamics with two parameters. The ice fractions of these sample solutions increased with decreases in both temperature and concentration. Because of the limited applicability of the phase diagram method only to systems with low molecular weight materials, the DSC method was also used for ice fraction measurement. The DSC method, corrected for temperature-dependent latent heat of ice and corrected with Pham’s equation, provided a good approximation for ice fractions with general applicability. The DSC method was used to measure the ice fractions of gelatin and egg albumin gels as a function of solute concentration. The freezing point and bound water of gelatin and egg albumin gels were described as a function of concentration. Effects of the differences in molecular structure on ice fraction were analyzed for various carbohydrate solutions at the same concentration. The ice fraction proved to be strongly dependent on the colligative properties of the solution with nonideal behavior.     

Phase Behavior of the ι-Carrageenan/Maltodextrin/Water System at Different Potassium Chloride Concentrations and Temperatures

Equilibrium phase diagrams of the ι-carrageenan/maltodextrin/water system have been established at potassium chloride (KCl) concentrations of 0.1, 0.2, and 0.3 M and 80, 85 and 90°C. All pseudo-binary phase diagrams of ι-carrageenan/maltodextrin mixtures suggested classic segregative phase separation. The binodal was heavily skewed toward the maltodextrin axis. The high asymmetry of the ι-carrageenan/maltodextrin/water phase diagram determined by the phase-volume-ratio method was consistent with the compositional analysis of phase-separated ι-carrageenan/maltodextrin samples and can be explained in terms of the Flory–Huggins interaction parameter, reflecting a higher water-binding ability of the charged ι-carrageenan than neutral maltodextrin. Increasing the concentration of ι-carrageenan-gel-promoting KCl from 0.1 to 0.3 M at 80°C enlarged the two-phase domain, whereas increasing temperature from 80 to 90°C at 0.3 M KCl enhanced biopolymer compatibility. The effects of salt concentration and temperature have been related to the differences in the Flory–Huggins interaction parameters of the two biopolymers with water as well as the helix formation of ι-carrageenan in the presence of KCl through the changes in the slopes of tie lines of phase-separated samples.

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms

Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.     

Temperature—composition phase diagram and gel properties of the gelatin—starch—water system

The gelatin-starch-water system has been studied at different temperatures, at a total biopolymer concentration of 5.0 wt%. The weight ratios (W) of gelatin/ starch used were 9:1, 8:2.4. 2:8, 1:9, with pH values between 5.82 (at W = 9:1) and 6.50 (at W = 1:9). The systems were characterized rheologically and by turbidity measurements to construct a phase diagram in the temperature (T) and composition (W) variables. The T-W quadrant consists of three regions: a singlephase solutions region (A) and regions of complete and incomplete phase separation (B and C, respectively). The system in region C is a gel. Region B, lying between A and C, corresponds to two co-existing liquid phases. The transition from A to C (obtained by cooling the system at constant W) involves crossing region B. The properties of the resulting gels depend on the rate of this intersection. Gels formed on rapid cooling have an even distribution of turbidity, whereas slow cooling gives two gel layers of different turbidity. The gelation temperature and gel strength of the mixed systems are dominated by the gelatin component, with no indication of network formation by starch.     

Some physical and microstructural properties of genipin-crosslinked gelatin-maltodextrin hydrogels

The physical properties and microstructure of gelatin-maltodextrin hydrogels fixed with genipin (GP) were investigated as a function of pH (3-7), maltodextrin (MD) (0-9%, w/w) and GP (0-10 mM levels), at a constant gelatin (G) concentration (10%, w/w). Network strength (elastic modulus, E) and swelling behavior were characterized by large deformation testing and by swelling index (SI). In general, network strength increased and swelling decreased at higher pH, MD and GP levels, except at pH 3, where E was independent of the GP concentration until approximately 7.5 mM, above which it declined. Confocal scanning laser microscopy (CLSM) images showed phase separation to be suppressed at pH 3, whereas at pH 7, separation into a self-similar dispersed phase was apparent. Overall, the judicious use of GP to crosslink G was an appropriate means of kinetically trapping MD within the gelatin network.     

Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth

By employing serial enrichment, a derivative of Clostridium acetobutylicum ATCC 824 was obtained which grew at concentrations of butanol that prevented growth of the wild-type strain. The parent strain demonstrated a negative growth rate at 15 g of butanol/liter, whereas the SA-1 mutant was still able to grow at a rate which was 66% of the uninhibited control. SA-1 produced consistently higher concentrations of butanol (from 5 to 14%) and lower concentrations of acetone (12.5 to 40%) than the wild-type strain in 4 to 20% extruded corn broth (ECB). Although the highest concentration of butanol was produced by SA-1 and the wild-type strain in 14% ECB, the best solvent ratio with respect to optimizing butanol and decreasing acetone occurred between 4 and 8% ECB for SA-1. SA-1 demonstrated higher conversion efficiency to butanol than the wild-type strain at every concentration of ECB tested. Characterization of the wild-type and SA-1 strain in 6% ECB demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, carbohydrate utilization, pH resistance, and final butanol concentration in the fermentation broth.     

A liquid crystalline phase in spermidine-condensed DNA.

Over a large range of salt and spermidine concentrations, short DNA fragments precipitated by spermidine (a polyamine) sediment in a pellet from a dilute isotropic supernatant. We report here that the DNA-condensed phase consists of a cholesteric liquid crystal in equilibrium with a more concentrated phase. These results are discussed according to Flory's theory for the ordering of rigid polymers. The liquid crystal described here corresponds to an ordering in the presence of attractive interactions, in contrast with classical liquid crystalline DNA. Polyamines are often used in vitro to study the functional properties of DNA. We suggest that the existence of a liquid crystalline state in spermidine-condensed DNA is relevant to these studies.     

Properties of agents that effectively entrap liquid lipids.

A droplet of an oil-in-water emulsion of methyl linoleate in a saccharide or protein solution that contained with a surfactant, a stabilizer, or both was dehydrated by drying equipment for a single droplet that resembled a spray drier. The lipid exposed on the surface of dehydated samples was extracted and measured by gas chromatography. Gum arabic or gelatin without additives resulted in little lipid being exposed; they were good entrapping agents. Little lipid was exposed with a pullulan solution containing lecithin, sugar ester, carboxymethylcellulose, or sodium caseinate but much was exposed with a maltodextrin solution containing any of the surfactants tested. When both the surfactant lecithin and the stabilizer xanthan gum were added to the emulsion prepared in a maltodextrin solution, lipid was not detected. The results suggested that effective entrapping agents of liquid lipids cause much emulsification, stabilize the emulsion (that is, they cause the continuous phase to be very viscous), and create a dehydrated matrix of fine, dense network layers.     

Influence of salts and gelatin on disintegration of Saccharomyces cerevisiae by freeze-pressing

The disintegration by freeze-pressing of a low concentration of Saccharomyces cerevisiae suspended in aqueous solutions of gelatin and different salts has been studied at different temperatures. In the freeze-pressing process deionized water and salt solutions flow in pulses, whereas samples with increasing concentrations of gelatin or cells tend to flow more smoothly. This smooth flow enhances the disruption efficiency particularly at lower temperatures, which seems to be of great practical importance. The addition of salts also promotes disintegration. The presence of both gelatin and salts works antagonistically on disintegration presumably because of different modes of action at disruption of cells.     

Studies on the physical state of water in living cells and model systems. X. The dependence of the equilibrium distribution coefficient of a solute in polarized water on the molecular weights of the solute: experimental confirmation of the "size rule" in model studies

The equilibrium distribution of 14 sugars, sugar alcohols, and other nonelectrolytes in solutions of polyethylene oxide (PEO) and of native and alkali-denatured bovine hemoglobin were studied over wide concentration ranges. The results show that the equilibrium concentrations of all the solutes studies are rectilinearly related to their external concentrations. This straight-line relationship demonstrates the existence of these solutes entirely or almost entirely in the aqueous phase of these systems. Therefore the slope of each of these straight lines equals the equilibrium distribution coefficient or q-value of the solute involved. In general, the q-values decrease with increasing molecule weights (M.W.) of the solutes in 15% solutions of PEO, 20% solutions of alkali-denatured hemoglobin (and in 18% gelatin) but not in 39% solution of native hemoglobin. In solutions of PEO, of alkali-denatured hemoglobin studied (and of gelatin) a fraction of the water (20% to 30%) appears to have solvency similar to that of normal liquid water. The experimental findings of M.W.-dependent solute exclusion were discussed in the light of four alternative theories that have been offered to explain this type of phenomena. Among these four theories only the polarized multilayer theory agrees with most, if not all the facts known.     

Phase separation induced molecular fractionation of gum arabic—Sugar beet pectin systems

This paper investigates the phase separation and phase separation-induced fractionation of gum arabic (GA)/sugar beet pectin (SBP) mixed solutions. A phase diagram, including cloud and binodal curves, was established by visual observation and phase composition analysis. The deviation of the binodal curve from the cloud curve was a result of phase separation-induced fractionation of polydisperse GA and SBP molecules. Fractionation of GA increased the content of arabinogalactan-protein complex (AGP) from ca. 13% to 27%. The fractionated GA (FGA) showed improved emulsifying functionality, whereas the fractionated SBP (FSBP) had a reduced emulsifying functionality. The changes in emulsifying efficiency can be explained by interfacial adsorption behaviors at the oil–water interface as indicated by interfacial tension measurements.     

A TEST FOR DIFFUSIBLE IONS : I. THE IONIC NATURE OF TRYPSIN.

1. The Donnan equilibrium furnishes a test for the ionic nature of any diffusible substance, since the ratio of the concentration of any ion on the two sides of a membrane must be equal to the ratio of the concentrations of any other ion of the same sign and valence, whereas a non-ionic substance would be equally distributed on both sides. 2. The distribution of trypsin inside and outside of gelatin particles has been compared to the distribution of hydrogen and chloride ions under the same conditions. 3. The ratio of the trypsin concentration in the gelatin to the concentration in the outside liquid is equal to the ratio of the hydrogen ion under the same conditions and to the reciprocal of the chloride ion ratio. 4. This result was obtained between pH 2.0 and 10.2. At pH 10.2 the trypsin is equally distributed and on the akaline side of 10.2 the ratio is directly equal to the chloride ratio. 5. Trypsin is therefore a positive monovalent ion in solutions of pH 10 to 2. It is probably isoelectric at 10.2 and a monovalent negative ion on the alkaline side of 10.2 6. Trypsin must also be a strong base since there is no evidence of any undissociated form on the acid side of pH 10.2.     

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van''t Hoff''s law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H₂O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin.     

Mechanism and kinetics of phase separation in a gelatin/maltodextrin mixture studied by small-angle light scattering

Phase separation mechanisms and kinetics were studied using small-angle light scattering in a gelatin/maltodextrin system where phase separation could be studied in both liquid and gelled states. Nucleation and growth or spinodal decomposition occurred, depending on the quench depth. The transition between the two mechanisms occurred relatively sharply. The different mechanisms were distinguishable by the different behavior of the scattering function even though a peak was observed in both cases. Particular differences were the different evolution of the peak intensity and position, the absence of dynamic scaling of the nucleation and growth scattering function, and the final coarsening exponent of 1/3 that was measured when spinodal decomposition occurred but not for nucleation and growth. Gelation severely reduced the coarsening rate and initially placed the phase compositions far from their equilibrium values. Despite the loss of molecular mobility caused by gelation, the gelled systems did continue to evolve, albeit much more slowly than in the liquid case. Multiple coarsening rates were observed for some of the gelled samples, which were ascribed to the gradual movement of these systems toward the equilibrium compositions.     

Critical helix content in gelatin gels

Aqueous gelatin solutions of different concentrations have been investigated at various quench temperatures by viscosity measurements to determine the gel times and by optical rotation measurements to derive the evolution of the helix content by reference to native collagen. As a result, it appears that the gelation of the different aqueous gelatin solutions tested takes place at a common helix concentration independent of the initial gelatin concentration and quench temperature. Further, for each concentration, the dependence of gel time as a function of quench temperature has revealed the existence of two domains: a higher temperature domain where gel times increase strongly with quench temperature and a lower temperature domain where gel times are short and only slightly dependent on quench temperature.