Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.     

Effects of calcium, pH, and blockiness on kinetic rheological behavior and microstructure of HM pectin gels

The kinetic behavior during gel formation and the microstructure of 0.75% high methoxyl (HM) pectin gels in 60% sucrose have been investigated by oscillatory measurements and transmission electron microscopy for three comparable citrus pectin samples differing in their degree of blockiness (DB). Ca2+ addition at pH 3.0 resulted in faster gel formation and a lower storage modulus after 3 h for gels of the blockwise pectin A. For gels of the randomly esterified pectin B, the Ca2+ addition resulted in faster gel formation and a higher storage modulus at pH 3.0. At pH 3.5, both pectins A and B were reinforced by the addition of Ca2+. In the absence of Ca2+, the shortest gelation time was obtained for the sample with the highest DB. Microstructural characterization of the gel network, 4 and 20 h after gel preparation, showed no visible changes on a nanometer scale. The microstructure of pectins A and B without Ca2+ was similar, whereas the presence of Ca2+ in pectin A resulted in an inhomogeneous structure.     

Polyelectrolyte films based on polysaccharides of different conformations: effects on multilayer structure and mechanical properties

Ultrathin films were prepared with cationic poly(allylamine hydrochloride) (PAH) and two anionic polysaccharides, iota- and lambda-carrageenan, of similar chemical composition but different conformations using the layer-by-layer (LbL) technique. The study of aqueous solutions of carrageenans confirms that iota-carrageenan is at room temperature in helical conformation while lambda-carrageenan is in random coil conformation. Characterization of the multilayers by ellipsometry, circular dichroism, and AFM revealed that iota-carrageenan keeps its helical conformation within the films while lambda-carrageenan chains are in random coil conformation. Investigation of the mechanical properties of the films by performing nanoindentation experiments using force spectroscopy showed clear differences between the two films based on carrageenans of different conformations.     

Polyelectrolyte behavior of carrageenans in aqueous solutions

Osmotic coefficients of sodium, potassium, and calcium counterions have been determined in aqueous solutions on kappa-, iota-, and lambda-carrageenans at 25°C. The experimental results are correlated with the calculated ones from the limiting law of Manning. An orderd secondary structure exists in kappa- and iota-carrageenans. Its stability is discussed as a function of temperature, ionic strength, and the nature of the counterions.     

On the structure of kappa/iota-hybrid carrageenans

The coil-to-helix transition and temperature dependence of the viscosity of commercial kappa/iota-hybrid carrageenans produced by the red algae Sarcothalia crispata, Mazaella laminarioides, and Chondrus crispus were studied using rheometry and optical rotation. The structure of these kappa/iota-hybrid carrageenans was determined by 1H and 13C NMR spectroscopy combined with monosaccharide composition analysis. The coil-to-helix transitions, measured by polarimetry and rheometry, of the kappa/iota-hybrid carrageenans are significantly different from those of pure kappa- and iota-carrageenan, and from hand-made mixtures thereof. This provides evidence that the kappa/iota-hybrid carrageenans are mixed chains, containing both kappa- and iota-repeating units.     

Small and Large Deformation Rheology of Acid Gels from Transglutaminase Treated Milks

The purpose of this study was to evaluate the effects of microbial transglutaminase on the rheological and mechanical properties of gels from a commercial UHT milk made by acidification with d-gluconic-acid-δ-lactone. Although further heating of UHT milk, primarily carried out to inactivate the enzyme, showed a significant impact on gel firmness, we observed no enzyme-related effects at small deformation. The impact of the additional thermal treatment was also evident in large deformation measurements. In these experiments, gels from enzyme-treated milk revealed much higher elasticity and rupture force. Prolonged incubation time or increased enzyme concentration resulted in lower gel firmness (small deformation), indicating a reduced number of total bonds as a result of the restriction of proper rearrangements during acidification. Large deformation measurements revealed a heat-induced amplification of this effect, i.e., lower rupture forces at almost constant elasticity levels. In gels from milk, which was not subjected to thermal enzyme inactivation, we observed a further increase in both parameters. This is presumably caused by a combination of residual enzyme activity and of additional substrate, liberated during acidification.     

The structure of kappa/iota-hybrid carrageenans II. Coil-helix transition as a function of chain composition

This paper describes the effect of the kappa/iota-ratio on the physical properties of kappa/iota-hybrid carrageenans (synonyms: kappa-2, kappa-2, weak kappa, weak gelling kappa). To this end, a series of kappa/iota-hybrid carrageenans ranging from almost homopolymeric kappa-carrageenan (98 mol-% kappa-units) to almost homopolymeric-carrageenan (99 mol-% iota-units) have been extracted from selected species of marine red algae (Rhodophyta). The kappa/iota-ratio of these kappa/iota-hybrids was determined by NMR spectroscopy. Their rheological properties were determined by small deformation oscillatory rheology. The gel strength (storage modulus, G') of the kappa/iota-hybrids decreases with decreasing kappa-content. On the other hand, the gelation temperature of the kappa-rich kappa/iota-hybrids is independent of their composition. This allows one to control the gel strength independent of the gelation or melting temperature. The conformational order-disorder transition of the kappa/iota-hybrids was studied using optical rotation and high-sensitivity differential scanning calorimetry. High-sensitivity DSC showed that the total transition enthalpy of the kappa/iota-hybrids goes through a minimum at 60 mol-% kappa-units, whereas for the mixture of kappa- and iota-carrageenan, the total transition enthalpy is a linear function of the composition. With respect to the ordering capability, the kappa/iota-hybrid carrageenans seem to behave as random block copolymers with length sequence distributions truncated from the side of the small lengths. Intrinsic thermodynamic properties (e.g., transition temperature and enthalpy) of kappa- and iota-sequences in these copolymers are close to those of their parent homopolymers. The critical sequence length for kappa-sequences is 2-fold of that for iota-sequences.     

High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity,Stability, Milk Coagulation Profile and Gel Development

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Microstructure and rheological behavior of pure and mixed pectin gels

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca(2+) content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca(2+) ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels.     

Diffusing Wave and Ultrasonic Spectroscopy of Rennet-Induced Gelation of Milk in the Presence of High-Methoxyl Pectin

Rennet-induced aggregation was studied in milk systems containing high-methoxyl pectin (HMP) using ultrasonic and diffusing wave spectroscopy. These two techniques allow for in situ measurements of sol–gel transitions without the need for dilution. At low HMP concentrations, the casein micelles aggregation behavior was similar to that of skim milk, although changes could be noted in the microstructure of the renneted gels. At HMP concentrations between 0.1 and 0.15%, phase-separation kinetics were slower than the rennet-induced aggregation, and different microstructures formed caused by different dynamics of interactions between casein micelles present in HMP-depleted flocs. Higher amounts of HMP failed to create a continuous gel, as phase separation occurred at a faster rate than rennet aggregation. These results highlight the importance of non-invasive techniques in the study of concentration-dependent phase separating and aggregating systems, as only with observations in situ is it possible to determine new ways to control the structuring of protein–polysaccharide mixed systems.     

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat-induced gelation of ovalbumin (egg-white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr-spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally-induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat-set at high pH(pH～10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat-setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat-set has a much lower effect on gel properties.     

Physicochemical characterization of carrageenan fromGigartina teedii (Rooth) Lamouroux (Gigartinales,Rhodophyta)

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.     

Modification of sex pheromone blend discrimination in male Oriental fruit moths by pre-exposure to (E)-8-dodecenyl acetate

ABSTRACT. A pre-exposure regime was developed to test the hypothesis that arrestment of in-flight behaviour in Oriental fruit moths, Grapholitha molesta (Busck), to high dosage and/or high %( E )-8-dodecenyl acetate blends was due to a high proportion of E8-12: Ac in the pheromone blend. When tested in a sustained-flight tunnel to various ratios of ( Z )- and ( E )-8-dodecenyl acetate plus a constant % of Z8-12: OH at two dosages (3 and 100μg), males displayed an optimum number of completed flights to the source to the natural 6% E blend at 3 μg, with significant decreases in response levels occurring to high % E blends at both dosages. Pre-exposure of males to E8-12: Ac alone enhanced their response at each dosage to selected higher % E blends that normally elicited low numbers of completed flights. Pre-exposure enhanced all aspects of the flight response, but this was dependent on both dosage and duration of pre-exposure, with later behaviours in the sequence (landing and hairpencil display) requiring greater amounts of pre-exposure than early behaviour (taking flight). Pre-exposure did not affect high response levels to optimal blends or low response levels to the highest or lowest blends tested at each dosage. The selective nature of the pre-exposure effect implied alteration of central processes in discrimination of blend rather than disturbance of olfactory receptor function.     

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.     

Effect of co-solute and gelation temperature on milk protein and gum tragacanth interaction in acidified gels

The aim of this study was to investigate the role of process conditions and system composition on the acid-induced gelation of a mixture of milk protein and gum tragacanth. This was studied by determining the effects of co-solute (lactose) addition (3, 5 and 7%) and gelation temperature (25, 37 and 45°C) on the mixture's rheological properties and microstructure using a combination of techniques including small-deformation rheology and scanning electron microscopy. The presence of lactose played an important role in the microstructure formation of gels but did not change most rheological properties. The microstructure of gels formed in the presence of lactose was coarser and more particulate, but less interconnected; this can be explained by lactose's role in improving protein aggregation. Gels prepared at a lower temperature had a high structure strength, as indicated by their high storage modulus, τ(f) and G(f) values. Low gelation temperature also caused a more branched and homogenous microstructure.     

Antiviral activity of carrageenan on hepatitis A virus replication in cell culture

Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5. No cytotoxic effects were detected with concentrations of carrageenans up to 200 μg/ml. The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50 % (CD₅₀) to the effective dose that inhibited 50 % of viral antigen expression (ED₅₀), were > 400 with iota-carrageenan, > 222 with lambda-carrageenan and > 10 with kappa-carrageenan. The selectivity index of ribavirin (reference substance) was only 5. The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity. Iota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.     

Calorimetric and chiroptical evidence of aggregate-driven helix formation in carrageenan systems

Thermally induced, order-disorder transitions of iota- and kappa-carrageenan have been monitored by optical rotation and differential-scanning calorimetry in various ionic environments. Conformational ordering in kappa-carrageenan is observed only in the presence of cations that have been shown previously to promote helix-helix aggregation, and shows marked hysteresis between heating and cooling. Iota-carrageenan, by contrast, shows an order-disorder transition in the non-aggregating, tetramethylammonium salt form, at substantially lower temperature than for kappa-carrageenan, and without hysteresis. In the presence of potassium ions, which are known to promote aggregation, iota-carrageenan shows two distinct thermal-transitions, one without hysteresis at the same temperature as observed under non-aggregating conditions, and one with significant hysteresis close to the temperature of the kappa-carrageenan transition. We interpret these transitions as helix-to-coil and aggregated helix-to-coil, respectively. This interpretation is supported by measurements of the enthalpy changes of the transitions; ΔH values show a systematic increase with increasing aggregation and hysteresis. We conclude that the double helix of iota-carrageenan can exist as a stable entity in isolation, but may be further stabilised by aggregation, whereas the kappa-carrageenan helix is stable only when aggregated.     

Coil-helix transition of iota-carrageenan as a function of chain regularity

A series of iota-carrageenans containing different amounts of nu-carrageenan (0-23 monomer %) have been prepared from neutrally extracted carrageenan of Eucheuma denticulatum. nu-Carrageenan is the biochemical precursor of iota-carrageenan. The conformational order-disorder transition and rheological properties of these carrageenans were studied using optical rotation, rheometry, size exclusion chromatography coupled to multiangle laser light scattering, and high-sensitivity differential scanning calorimetry. The helix forming capacity of iota-carrageenan turns out to decrease monotonously with increasing amount of nu-units. In contrast, the rheological properties of iota-carrageenan are remarkably enhanced by the presence of a small amount of nu-units, yielding a maximum twofold increase in G' at 3% nu-units. It is concluded that the structure-forming capacity of iota-carrageenan, containing a small amount of nu-carrageenan, is significantly higher than that of pure iota-carrageenan. This phenomenon is explained in terms of the balance between the helical content and the number of cross-links between chains, taking into consideration the fact that nu-units introduce "kinks" in the chain conformation enabling neighboring chains to connect. Increasing amounts of nu-units increase the number of cross-links in the network, resulting in increased gel strength. On the other hand, a reduced length of the helical strands weakens the cross-links between the different chains and, consequently, the gel.